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ROS level measured by flow cytometry using H 2 DCFDA dye in embryonic stem cells (ESCs) and their differentiated progenies (difESCs): effect of the cell size. (A) Flow cytometry histograms of ESCs and difESCs treated with the ROS-sensitive H 2 DCFDA (left panel) and the ROS-insensitive DCFDA (right panel) fluorescein-based dyes. (B) Scheme demonstrating intracellular multistep conversion of the ROS-sensitive (H 2 DCFDA) and ROS-insensitive (DCFDA) dyes. (C) Confocal microscopy image of H 2 DCFDA-treated ESCs grown upon difESC feeder layer. Scale bar, 100 µm. (D) Oxidized H 2 DCFDA fluorescence signal quantified per cell (DCF/cell and mean DCF) or per cell area/volume/protein (DCF/area, DCF/volume, DCF/ protein,) in ESCs and difESCs. Signal was measured either by flow cytometry (right panel), or by processing of the confocal microscopy images of the cells (left panel). Data are normalized to the ESC fluorescence and presented as mean ± SD (n=3, * P < 0.01). (E) Cell size distribution for ESCs and difESCs obtained by measuring the diameter of the suspended cells in the counting chamber. Abbreviations: H 2 DCFDA, 2',7'-dichlorodihydrofluorescein diacetate; DCFDA, 2',7'-dichlorofluorescein diacetate; DCF, 2',7'-dichlorodihydrofluorescein, FC, flow cytometry; MIC, microscopy.
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Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with the...
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... find out whether the weak fluorescence of the oxidized H 2 DCFDA in ESCs suggests a highly specific intracellular redox environment in stem cells, we used 2',7'-dichlorofluorescein diacetate (DCFDA), ROS-insensitive modification of the dichlorofluorescein dye. Both H 2 DCFDA and DCFDA probes are non-fluorescent in their initial form but they undergo multistep conversion inside the cell (Fig. 2B) that results in the formation of fluorescent product dichlorofluorescein (DCF). The only difference between these two probes is that the conversion of H 2 DCFDA involves oxidation. Therefore, fluorescence of the H 2 DCFDA-treated cells depends on the intracellular ROS level, in contrast to fluorescence associated with the DCFDA probe. Surprisingly, flow cytometry analysis showed that the difference between fluores- cence levels of ESCs and difESCs loaded with H 2 DCFDA ( Fig. 2A, left panel) is close to the difference between the signals from DCFDA- treated cells (Fig. 2A, right panel). The latter indicates a ROS- independent reason for low fluorescent signal of oxidized H 2 DCFDA in ...
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... find out whether the weak fluorescence of the oxidized H 2 DCFDA in ESCs suggests a highly specific intracellular redox environment in stem cells, we used 2',7'-dichlorofluorescein diacetate (DCFDA), ROS-insensitive modification of the dichlorofluorescein dye. Both H 2 DCFDA and DCFDA probes are non-fluorescent in their initial form but they undergo multistep conversion inside the cell (Fig. 2B) that results in the formation of fluorescent product dichlorofluorescein (DCF). The only difference between these two probes is that the conversion of H 2 DCFDA involves oxidation. Therefore, fluorescence of the H 2 DCFDA-treated cells depends on the intracellular ROS level, in contrast to fluorescence associated with the DCFDA probe. Surprisingly, flow cytometry analysis showed that the difference between fluores- cence levels of ESCs and difESCs loaded with H 2 DCFDA ( Fig. 2A, left panel) is close to the difference between the signals from DCFDA- treated cells (Fig. 2A, right panel). The latter indicates a ROS- independent reason for low fluorescent signal of oxidized H 2 DCFDA in ...
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... find out whether the weak fluorescence of the oxidized H 2 DCFDA in ESCs suggests a highly specific intracellular redox environment in stem cells, we used 2',7'-dichlorofluorescein diacetate (DCFDA), ROS-insensitive modification of the dichlorofluorescein dye. Both H 2 DCFDA and DCFDA probes are non-fluorescent in their initial form but they undergo multistep conversion inside the cell (Fig. 2B) that results in the formation of fluorescent product dichlorofluorescein (DCF). The only difference between these two probes is that the conversion of H 2 DCFDA involves oxidation. Therefore, fluorescence of the H 2 DCFDA-treated cells depends on the intracellular ROS level, in contrast to fluorescence associated with the DCFDA probe. Surprisingly, flow cytometry analysis showed that the difference between fluores- cence levels of ESCs and difESCs loaded with H 2 DCFDA ( Fig. 2A, left panel) is close to the difference between the signals from DCFDA- treated cells (Fig. 2A, right panel). The latter indicates a ROS- independent reason for low fluorescent signal of oxidized H 2 DCFDA in ...
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... is now well-established [14,19,23,24] that oxidation of H 2 DCFDA is catalyst-and context-dependent. It is influenced by the variety of intracellular parameters such as acidity, activity of esterases that provide the probe trapping inside the cells, availability of catalysts and so on. In this context, the best way to validate our H 2 DCFDA-based measurements is to use an alternative methodology for the assessment of the ROS balance in ESCs and difESCs. For this purpose, we chose HyPer, genetically encoded fluorescent probe for hydrogen peroxide [35]. This probe allows monitoring the intracellular H 2 O 2 levels with a high degree of sensitivity and specificity and is widely used for the ratiometric imaging of the cells. Microscopic techniques provide an opportunity to visualize the H 2 O 2 fluxes in intact cells [54,55], and thus is a method of choice for single cell analysis. In contrast, flow cytometry is suitable for large massive data collection, so we use multi-color flow cytometry ratiometric analysis for the comparative study of the ROS balance in ESCs and difESCs. Ratiometric method is a perfect way to eliminate analysis ambiguity arising from the difference of stem and differentiated cells in their morphology. Fig. 4 shows the histograms of the transfected cells, as well as the corresponding microscopy images of ESCs and difESCs expressing HyPer. Data were collected 36 h after transfection, when the fraction of HyPer+ cells was about 5-8% in the case of ESCs (Fig. 4A, B) and 15-30% in the case of difESCs (Fig. 4C, D). In ESC cultures, HyPer+ cells were positive for pluripotency marker SSEA3, in contrast to DifESC cells (Fig. 4E). In spite of the huge diversity in the fluorescence intensity of transfected cells within each sample, the ratio of EX488/FL530 and EX405/FL520 signals, denoted here and after as 488/405 ratio, was almost the same, and the corresponding histograms, which depict ratio within the fraction of HyPer+ cells, had small dispersions ( Fig. 4F and Fig. 2S in the Supplement). Cell treatments with H 2 O 2 and dithiothrei- tol (DTT) shifted the ROS balance (see, for example, the ratiometric microscopy images in Fig. 4G), and we used total reduction and total oxidation of HyPer with 30 mM DTT and 1 mM H 2 O 2 correspondingly for the calibration of our measurements. We designated the shift of 488/405 ratio from the totally reduced state (defined as 0%) towards totally oxidized state (defined as 100%) as HyPer-index H quantified in %% [55]. HyPer-indexes derived from the measurements of 488/405 ratio in HyPer+ ESCs, difESCs and eMSCs occurred to be the same (about 6 ± 1%, see Fig. 4H), that confirms the results of H 2 DCFDA- based analysis and supports the hypothesis about the similar ROS status of tested ...
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... of H 2 DCFDA probe oxidation by means of flow cytometry technique showed that the basal level of ROS in difESCs is about 6-7 times larger than that in ESCs ( Fig. 2A, left panel). This observation is in accord with previously published data obtained with the same fluor- escent probe in the studies of stem and differentiated cells [26][27][28]. Our experiments revealed that the signal from the oxidized dye in ESCs was sensitive to the variation of the intracellular ROS level caused by pro- and antioxidants, was stable during at least one hour after cell staining and did not depend on the substrate for growing ESCs (matrigel, mouse embryonic fibroblast feeder layer, or human endometrial mesenchymal stem cell feeder layer) (see Supplement, Fig. ...
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... difESC and eMSC cells were transfected with HyPer expression vector using commercially available plasmid pHyPer-cyto (Evrogen, Russia) and FuGene 6 (Promega, USA) transfection reagent [45]. Transfection mixtures were prepared according to the manufacturer's instructions at a 7:2 ratio of FuGene to DNA. 36-48 h after transfection, cells were harvested with 0.05% trypsin-EDTA solution, suspended in a fresh medium, incubated in suspension for 30 min in standard growth conditions (37 °C, 5%CO 2 ) for adaptation to a new environment, and analyzed with flow cytometer (CytoFLEX, Beckman Coulter, USA; 405/ 488 nm laser). Before the analysis, suspension was split into 3 probes: the first one was analyzed immediately, the second one was analyzed after 5 min incubation with 1 mM of H 2 O 2 , while the rest part of suspension was incubated for 10 min with 30 mM of dithiothreitol (DTT) and then analyzed. During the analysis, cells were gated for HyPer expression (see Supplement, Fig. 2S), and within this gate the mean ratio of EX488/FL530 and EX405/FL520 signals (denoted here and after as 488/405 ratio) was determined. Intracellular peroxide concentration was assessed using HyPer-index (H), which was quanti- fied in %% as ...
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... of the microscopy images using the ImageJ software confirms these observations. Measurements of the mean value of fluorescence intensity within the cells (i.e. signal collected per unit area of the cell) show that the difference in the mean values (Fig. 2D, DCF/area) for ESCs and DifESCs is negligible. At the same time, comparison of integrated density of the cell fluorescence (i.e. signal collected from the whole area of the cell) shows the 8-fold difference between ESCs and difESCs in favor of difESC (Fig. 2D, DCF/cell), that is very close to the results obtained with flow cytometry. This means that concentra- tion of the oxidized H 2 DCFDA is about the same in ESCs and DifESCs, whereas the amount of oxidized dye per cell is different mainly due to the different cell sizes. Fig. 2E shows histograms of the cell size distribution for ESCs and difESCs, obtained by measuring the diameter of the suspended cells in the counting chamber. Mean values of ESCs and difESCs diameters differ nearly twice (Table 1). Accordingly, the difference in the volumes of two cell types turned out to be much more obvious. Our measurements of the mean cell volume with the usage of the Scepter™ Cell Counter revealed about the 6-fold difference in the volumes of ESCs and DifESCs (Table 1). In order to find out whether the difference in ESCs' and difESCs' cell sizes is the main reason for the difference in the ROS levels revealed by flow cytometry assay of H 2 DCFDA-loaded ESCs and difESCs (Fig. 2D, mean DCF), we assessed the oxidized dye concentration in these cells, using the normalization of the cytometric signal from the H 2 DCFDA-treated cells to the measured cell volume, or, alternatively, to the cell protein content (Table 1) determined in the same probes (Fig. 2D, DCF/volume and DCF/ protein). Similarly to the microscopy-based estimations, concentration of the oxidized H 2 DCFDA dye in ESCs probed by flow cytometry occurred to be very close to that in difESCs. Thus, our experiments showed that the ability of the cell's unit volume to oxidize H 2 DCFDA in ESCs and difESCs is quite similar, which challenges the hypothesis about the highly specific redox environment in stem ...
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... of the microscopy images using the ImageJ software confirms these observations. Measurements of the mean value of fluorescence intensity within the cells (i.e. signal collected per unit area of the cell) show that the difference in the mean values (Fig. 2D, DCF/area) for ESCs and DifESCs is negligible. At the same time, comparison of integrated density of the cell fluorescence (i.e. signal collected from the whole area of the cell) shows the 8-fold difference between ESCs and difESCs in favor of difESC (Fig. 2D, DCF/cell), that is very close to the results obtained with flow cytometry. This means that concentra- tion of the oxidized H 2 DCFDA is about the same in ESCs and DifESCs, whereas the amount of oxidized dye per cell is different mainly due to the different cell sizes. Fig. 2E shows histograms of the cell size distribution for ESCs and difESCs, obtained by measuring the diameter of the suspended cells in the counting chamber. Mean values of ESCs and difESCs diameters differ nearly twice (Table 1). Accordingly, the difference in the volumes of two cell types turned out to be much more obvious. Our measurements of the mean cell volume with the usage of the Scepter™ Cell Counter revealed about the 6-fold difference in the volumes of ESCs and DifESCs (Table 1). In order to find out whether the difference in ESCs' and difESCs' cell sizes is the main reason for the difference in the ROS levels revealed by flow cytometry assay of H 2 DCFDA-loaded ESCs and difESCs (Fig. 2D, mean DCF), we assessed the oxidized dye concentration in these cells, using the normalization of the cytometric signal from the H 2 DCFDA-treated cells to the measured cell volume, or, alternatively, to the cell protein content (Table 1) determined in the same probes (Fig. 2D, DCF/volume and DCF/ protein). Similarly to the microscopy-based estimations, concentration of the oxidized H 2 DCFDA dye in ESCs probed by flow cytometry occurred to be very close to that in difESCs. Thus, our experiments showed that the ability of the cell's unit volume to oxidize H 2 DCFDA in ESCs and difESCs is quite similar, which challenges the hypothesis about the highly specific redox environment in stem ...
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... of the microscopy images using the ImageJ software confirms these observations. Measurements of the mean value of fluorescence intensity within the cells (i.e. signal collected per unit area of the cell) show that the difference in the mean values (Fig. 2D, DCF/area) for ESCs and DifESCs is negligible. At the same time, comparison of integrated density of the cell fluorescence (i.e. signal collected from the whole area of the cell) shows the 8-fold difference between ESCs and difESCs in favor of difESC (Fig. 2D, DCF/cell), that is very close to the results obtained with flow cytometry. This means that concentra- tion of the oxidized H 2 DCFDA is about the same in ESCs and DifESCs, whereas the amount of oxidized dye per cell is different mainly due to the different cell sizes. Fig. 2E shows histograms of the cell size distribution for ESCs and difESCs, obtained by measuring the diameter of the suspended cells in the counting chamber. Mean values of ESCs and difESCs diameters differ nearly twice (Table 1). Accordingly, the difference in the volumes of two cell types turned out to be much more obvious. Our measurements of the mean cell volume with the usage of the Scepter™ Cell Counter revealed about the 6-fold difference in the volumes of ESCs and DifESCs (Table 1). In order to find out whether the difference in ESCs' and difESCs' cell sizes is the main reason for the difference in the ROS levels revealed by flow cytometry assay of H 2 DCFDA-loaded ESCs and difESCs (Fig. 2D, mean DCF), we assessed the oxidized dye concentration in these cells, using the normalization of the cytometric signal from the H 2 DCFDA-treated cells to the measured cell volume, or, alternatively, to the cell protein content (Table 1) determined in the same probes (Fig. 2D, DCF/volume and DCF/ protein). Similarly to the microscopy-based estimations, concentration of the oxidized H 2 DCFDA dye in ESCs probed by flow cytometry occurred to be very close to that in difESCs. Thus, our experiments showed that the ability of the cell's unit volume to oxidize H 2 DCFDA in ESCs and difESCs is quite similar, which challenges the hypothesis about the highly specific redox environment in stem ...
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... of the microscopy images using the ImageJ software confirms these observations. Measurements of the mean value of fluorescence intensity within the cells (i.e. signal collected per unit area of the cell) show that the difference in the mean values (Fig. 2D, DCF/area) for ESCs and DifESCs is negligible. At the same time, comparison of integrated density of the cell fluorescence (i.e. signal collected from the whole area of the cell) shows the 8-fold difference between ESCs and difESCs in favor of difESC (Fig. 2D, DCF/cell), that is very close to the results obtained with flow cytometry. This means that concentra- tion of the oxidized H 2 DCFDA is about the same in ESCs and DifESCs, whereas the amount of oxidized dye per cell is different mainly due to the different cell sizes. Fig. 2E shows histograms of the cell size distribution for ESCs and difESCs, obtained by measuring the diameter of the suspended cells in the counting chamber. Mean values of ESCs and difESCs diameters differ nearly twice (Table 1). Accordingly, the difference in the volumes of two cell types turned out to be much more obvious. Our measurements of the mean cell volume with the usage of the Scepter™ Cell Counter revealed about the 6-fold difference in the volumes of ESCs and DifESCs (Table 1). In order to find out whether the difference in ESCs' and difESCs' cell sizes is the main reason for the difference in the ROS levels revealed by flow cytometry assay of H 2 DCFDA-loaded ESCs and difESCs (Fig. 2D, mean DCF), we assessed the oxidized dye concentration in these cells, using the normalization of the cytometric signal from the H 2 DCFDA-treated cells to the measured cell volume, or, alternatively, to the cell protein content (Table 1) determined in the same probes (Fig. 2D, DCF/volume and DCF/ protein). Similarly to the microscopy-based estimations, concentration of the oxidized H 2 DCFDA dye in ESCs probed by flow cytometry occurred to be very close to that in difESCs. Thus, our experiments showed that the ability of the cell's unit volume to oxidize H 2 DCFDA in ESCs and difESCs is quite similar, which challenges the hypothesis about the highly specific redox environment in stem ...
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... of the microscopy images using the ImageJ software confirms these observations. Measurements of the mean value of fluorescence intensity within the cells (i.e. signal collected per unit area of the cell) show that the difference in the mean values (Fig. 2D, DCF/area) for ESCs and DifESCs is negligible. At the same time, comparison of integrated density of the cell fluorescence (i.e. signal collected from the whole area of the cell) shows the 8-fold difference between ESCs and difESCs in favor of difESC (Fig. 2D, DCF/cell), that is very close to the results obtained with flow cytometry. This means that concentra- tion of the oxidized H 2 DCFDA is about the same in ESCs and DifESCs, whereas the amount of oxidized dye per cell is different mainly due to the different cell sizes. Fig. 2E shows histograms of the cell size distribution for ESCs and difESCs, obtained by measuring the diameter of the suspended cells in the counting chamber. Mean values of ESCs and difESCs diameters differ nearly twice (Table 1). Accordingly, the difference in the volumes of two cell types turned out to be much more obvious. Our measurements of the mean cell volume with the usage of the Scepter™ Cell Counter revealed about the 6-fold difference in the volumes of ESCs and DifESCs (Table 1). In order to find out whether the difference in ESCs' and difESCs' cell sizes is the main reason for the difference in the ROS levels revealed by flow cytometry assay of H 2 DCFDA-loaded ESCs and difESCs (Fig. 2D, mean DCF), we assessed the oxidized dye concentration in these cells, using the normalization of the cytometric signal from the H 2 DCFDA-treated cells to the measured cell volume, or, alternatively, to the cell protein content (Table 1) determined in the same probes (Fig. 2D, DCF/volume and DCF/ protein). Similarly to the microscopy-based estimations, concentration of the oxidized H 2 DCFDA dye in ESCs probed by flow cytometry occurred to be very close to that in difESCs. Thus, our experiments showed that the ability of the cell's unit volume to oxidize H 2 DCFDA in ESCs and difESCs is quite similar, which challenges the hypothesis about the highly specific redox environment in stem ...
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... we employed confocal laser scanning microscopy for the comparative visualization of the fluorescence from ESCs and difESCs treated with H 2 DCFDA. ESCs were grown on the coverslips upon the difESCs used as a feeder layer so that both cell types can be treated with H 2 DCFDA and analyzed simultaneously. Visually estimated intensity of fluorescence from ESCs and difESCs (Fig. 2C) was about the ...
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... experiments confirmed that the measurement of ROS levels with flow cytometry by using H 2 DCFDA probe in stem and differen- tiated cells depends not only on the amount of intracellular ROS and other biochemical parameters which affect the oxidation of the probe [14,19,23,24], but also on the cell size (Fig. 2). The diameter of ESCs in suspension turned out to be twice as small as that of difESCs and therefore ESCs are several times smaller in volume. Fluorescence signal of H 2 DCFDA detected by flow cytometry reflects the oxidized dye content in the cell and is proportional to cell volume. Thus, low level of ROS detected in ESCs by using H 2 DCFDA is caused mainly by small geometric size of these cells. Flow cytometry measurements with signal normalization to the cell volume or cell protein, showed that the intracellular concentration of oxidized H 2 DCFDA was about the same in the ESC and difESC. These results challenge the widespread hypothesis that stem cells have a highly specific redox status. Furthermore, our H 2 DCFDA-based assay showed that intracellular concentration of oxidized H 2 DCFDA in ESCs was similar to that in other primary-and non-primary human cell cultures: mesenchymal stem cells, adult lymphocytes, and HeLa cells (Fig. 3). Our measurements proved that the intracellular unit volume in all tested cells had essentially the same ability to oxidize the probe that indicates the same intracellular ROS concentration. These findings allowed us to advance the provocative hypothesis that intracellular ROS concentration in normal cells appears to be some kind of physiological constant. We suggest that interplay between the pro-and antioxidant systems raises and lowers ROS concentration in cells, depending on their current physiological status, but these oscillations occur near some regular value, which is almost the same in all normal cells. Of course, ROS concentration is a nominal parameter composed from contributions of different chemical sub- stances and averaged over the whole cell volume. Local concentration of the redox-active compounds may substantially differ from the value averaged over the whole cell [54], and we suggest using the concept of ROS concentration solely for the rough estimation of the cellular redox status in comparative cell ...
Citations
... Intracellular ROS levels were assessed via DCFH-DA (MCE, Monmouth Junction, NJ, USA) assay as previously outlined [28]. Briefly, adherent cells treated with varying agent concentrations and durations were stained with DCFH-DA (10 µM) for 30 min. ...
Simple Summary
The objective of drug repurposing is to discover novel therapeutic uses for established pharmaceuticals. Paroxetine (PX), a commonly prescribed antidepressant, has demonstrated promising anticancer properties in experimental studies. Nevertheless, its precise role and mechanisms of action in triple-negative breast cancer (TNBC) remain incompletely elucidated. Our research findings propose that PX may impact TNBC by modulating critical molecular pathways, providing significant implications for enhancing chemosensitivity and identifying potential therapeutic synergies in clinical practice. These findings provide a basis for the creation of individualized treatment plans and the identification of the most effective therapeutic interventions to enhance treatment precision and effectiveness in patients with TNBC.
Abstract
The strategy of drug repurposing has gained traction in the field of cancer therapy as a means of discovering novel therapeutic uses for established pharmaceuticals. Paroxetine (PX), a selective serotonin reuptake inhibitor typically utilized in the treatment of depression, has demonstrated promise as an agent for combating cancer. Nevertheless, the specific functions and mechanisms by which PX operates in the context of triple-negative breast cancer (TNBC) remain ambiguous. This study aimed to examine the impact of PX on TNBC cells in vitro as both a standalone treatment and in conjunction with other pharmaceutical agents. Cell viability was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, apoptosis was assessed through flow cytometry, and the effects on signaling pathways were analyzed using RNA sequencing and Western blot techniques. Furthermore, a subcutaneous tumor model was utilized to assess the in vivo efficacy of combination therapy on tumor growth. The results of our study suggest that PX may activate the Ca²⁺-dependent mitochondria-mediated intrinsic apoptosis pathway in TNBC by potentially influencing the PI3K/AKT/mTOR pathway as well as by inducing cytoprotective autophagy. Additionally, the combination of PX and chemotherapeutic agents demonstrated moderate inhibitory effects on 4T1 tumor growth in an in vivo model. These findings indicate that PX may exert its effects on TNBC through modulation of critical molecular pathways, offering important implications for improving chemosensitivity and identifying potential therapeutic combinations for clinical use.
... For detection of the intracellular ROS level, ROS-sensitive probe H 2 DCFDA (MCE, Princeton, NJ, USA) was used [41]. Peel the epidermis of Chinese cabbage leaves using tweezers, and immerse them in an MES-KCl buffer (MES 10 mM, KCl 5 mM, CaCl 2 50 mM, pH 6.15) for 30 min. ...
Chinese cabbage is the most widely consumed vegetable crop due to its high nutritional value and rock-bottom price. Notably, the presence of the physiological disease petiole spot significantly impacts the appearance quality and marketability of Chinese cabbage. It is well known that excessive nitrogen fertilizer is a crucial factor in the occurrence of petiole spots; however, the mechanism by which excessive nitrogen triggers the formation of petiole spots is not yet clear. In this study, we found that petiole spots initially gather in the intercellular or extracellular regions, then gradually extend into intracellular regions, and finally affect adjacent cells, accompanied by cell death. Transcriptomic and proteomic as well as physiology analyses revealed that the genes/proteins involved in nitrogen metabolism exhibited different expression patterns in resistant and susceptible Chinese cabbage lines. The resistant Chinese cabbage line has high assimilation ability of NH4+, whereas the susceptible one accumulates excessive NH4+, thus inducing a burst of reactive oxygen species (ROS). These results introduce a novel perspective to the investigation of petiole spot induced by the nitrogen metabolism pathway, offering a theoretical foundation for the development of resistant strains in the control of petiole spot.
... Carboxy-DCFDA was used as a control [18]. When the H2DCFDA method was used, tissue sections and HUVECs were labeled with both H2DCFDA and carboxy-DCFDA (insensitive to oxidation; C369, Invitrogen). ...
Oxidative stress and iron accumulation-induced ferroptosis occurs in injured vascular cells and can promote thrombogenesis. Transferrin receptor 1 (encoded by the TFRC gene) is an initial element involved in iron transport and ferroptosis and is highly expressed in injured vascular tissues, but its role in thrombosis has not been determined. To explore the potential mechanism and therapeutic effect of TFRC on thrombogenesis, a DVT model of femoral veins (FVs) was established in rats, and weighted correlation network analysis (WGCNA) was used to identify TFRC as a hub protein that is associated with thrombus formation. TFRC was knocked down by adeno-associated virus (AAV) or lentivirus transduction in FVs or human umbilical vein endothelial cells (HUVECs), respectively. Thrombus characteristics and ferroptosis biomarkers were evaluated. Colocalization analysis, molecular docking and coimmunoprecipitation (co-IP) were used to evaluate protein interactions. Tissue-specific TFRC knockdown alleviated iron overload and redox stress, thereby preventing ferroptosis in injured FVs. Loss of TFRC in injured veins could alleviate thrombogenesis, reduce thrombus size and attenuate hypercoagulability. The protein level of thrombospondin-1 (THBS1) was increased in DVT tissues, and silencing TFRC decreased the protein level of THBS1. In vitro experiments further showed that TFRC and THBS1 were sensitive to erastin-induced ferroptosis and that TFRC knockdown reversed this effect. TFRC can interact with THBS1 in the domain spanning from TSR1-2 to TSR1-3 of THBS1. Amino acid sites, including GLN320 of TFRC and ASP502 of THBS1, could be potential pharmacological targets. Erastin induced ferroptosis affected extracellular THBS1 levels and weakened the interaction between TFRC and THBS1 both in vivo and in vitro, and promoted the interaction between THBS1 and CD47. This study revealed a linked relationship between venous ferroptosis and coagulation cascades. Controlling TFRC and ferroptosis in endothelial cells can be an efficient approach for preventing and treating thrombogenesis.
... DCFH-DA probe was used to monitor the oxidation-reduction process in cells and detect intracellular ROS production, especially H 2 O 2 , with the intensity of green fluorescence (Lyublinskaya et al., 2017). The roots of Arabidopsis treated with DSF for 0, 1, 3, 6, 12, 24, 36, and 48 h were stained by DCFH-DA. ...
Background
Many Gram-negative bacteria use quorum sensing (QS) signal molecules to monitor their local population density and to coordinate their collective behaviors. The diffusible signal factor (DSF) family represents an intriguing type of QS signal to mediate intraspecies and interspecies communication. Recently, accumulating evidence demonstrates the role of DSF in mediating inter-kingdom communication between DSF-producing bacteria and plants. However, the regulatory mechanism of DSF during the Xanthomonas-plant interactions remain unclear.
Methods
Plants were pretreated with different concentration of DSF and subsequent inoculated with pathogen Xanthomonas campestris pv. campestris (Xcc). Pathogenicity, phynotypic analysis, transcriptome combined with metabolome analysis, genetic analysis and gene expression analysis were used to evaluate the priming effects of DSF on plant disease resistance.
Results
We found that the low concentration of DSF could prime plant immunity against Xcc in both Brassica oleracea and Arabidopsis thaliana. Pretreatment with DSF and subsequent pathogen invasion triggered an augmented burst of ROS by DCFH-DA and DAB staining. CAT application could attenuate the level of ROS induced by DSF. The expression of RBOHD and RBOHF were up-regulated and the activities of antioxidases POD increased after DSF treatment followed by Xcc inoculation. Transcriptome combined with metabolome analysis showed that plant hormone jasmonic acid (JA) signaling involved in DSF-primed resistance to Xcc in Arabidopsis. The expression of JA synthesis genes (AOC2, AOS, LOX2, OPR3 and JAR1), transportor gene (JAT1), regulator genes (JAZ1 and MYC2) and responsive genes (VSP2, PDF1.2 and Thi2.1) were up-regulated significantly by DSF upon Xcc challenge. The primed effects were not observed in JA relevant mutant coi1-1 and jar1-1.
Conclusion
These results indicated that DSF-primed resistance against Xcc was dependent on the JA pathway. Our findings advanced the understanding of QS signal-mediated communication and provide a new strategy for the control of black rot in Brassica oleracea.
... The treated cells fluoresced brilliantly in contrast to the untreated control cells' modest fluorescence. The drug-treated cells, significantly increased intracellular ROS (Lyublinskaya et al., 2017). ...
This study emphasizes the explorations of binding of Prima-1MET with two targets, p53 a
tumor suppressor protein, and tyrosine kinase of epidermal growth factor receptor. In-silico
investigations reveal that Prima-1MET showed robust binding with both targets. Molecular
docking simulations demonstrated the binding affinity of Prima-1MET with p53 and tyrosine
kinase was found to be -38.601 kJ/mol and -38.976 kJ/mol. In addition, the stability of Prima-
1
MET was explored by molecular dynamics simulation. Prima-1MET attains stability in the
binding site of the respective protein till the simulation period is over. Moreover, the free
binding energy ΔGbind was calculated by the MM-PBSA method. The ΔGbind of Prima-1MET
with tyrosine kinase was found to be -58.585±0.327 kJ/mol and with p53 it was -
35.910±0.335 kJ/mol. Next, cytotoxicity of the Prima-1MET was evaluated using multiple
cancer cell lines and the IC50 value were ranging between 4.5 - 30 μM. The cell death was
identified by apoptosis assay. Further, the p53 and tyrosine kinase expression was monitored
using immunofluorescence techniques, it was found Prima-1MET induces the expression of
p53 protein and mimics the level of tyrosine kinase oncogenic target. Also, reactive oxygen
species and membrane potential activity of Prima-1MET was evaluated by using a lung cancer
cell line. The significant decrease in intracellular reactive oxygen species was observed and
resulted in disruption of mitochondrial transmembrane potential. This study uncovers the
underlying mechanism of Prima-1MET and could be helpful to design further leads against
lung cancers.
... We used 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA, HY-D0940, MCE), a cellpermeable non-fluorescent (but fluorescein-containing) probe for detecting oxidants, to detect the ROS levels (94). Detection of ROS levels in mouse oocytes has been described previously (95). ...
Resveratrol is an antiaging, antioxidant and anti-inflammatory natural polyphenolic compound. Growing evidence indicates that resveratrol has potential therapeutic effects for improving aging ovarian function. However, the mechanisms underlying prolonged reproductive longevity remain elusive. We found that resveratrol ameliorates ovarian aging transcriptome, some of which are associated with specific changes in methylome. In addition to known aging transcriptome of oocytes and granulosa cells such as decline in oxidoreductase activity, metabolism and mitochondria function and elevated DNA damage and apoptosis, actin cytoskeleton is notably downregulated with age, and these defects are mostly rescued by resveratrol. Moreover, the aging-associated hypermethylation of actin cytoskeleton is decreased by resveratrol. In contrast, deletion of Tet2, involved in DNA demethylation, abrogates resveratrol-reprogrammed ovarian aging transcriptome. Consistently, Tet2 deficiency results in additional altered pathways as shown by increased mTOR and Wnt signaling, as well as reduced DNA repair and actin cytoskeleton with mouse age. Moreover, genes associated with oxidoreductase activity and oxidation-reduction process were hypermethylated in Tet2-deficient oocytes from middle-age mice treated with resveratrol, indicating that loss of Tet2 abolishes the antioxidant effect of resveratrol. Taking together, our finding provides a comprehensive landscape of transcriptome and epigenetic changes associated with ovarian aging that can be reprogrammed by resveratrol administration, and suggests that aberrantly increased DNA methylation by Tet2 deficiency promotes additional aging epigenome that cannot be effectively restored to younger state by resveratrol.
... Cells were stimulated with 3% hydrogen peroxide to keep the peroxide level stable and were set as a positive control. H2DCFDA (MCE, China) is a cell-permeable probe for the detection of intracellular reactive oxygen species (ROS) [20]. H2DCFDA was dissolved to a specified concentration according to the manufacturer's instructions. ...
Dysregulation of osteoclast-osteoblast balance, resulting in abnormal bone remodeling, is responsible for postmenopausal osteoporosis (PMOP) or other secondary forms of osteoporosis. We demonstrated that dictamnine (DIC), a novel RANKL-targeted furoquinoline alkaloid, inhibits osteoclastogenesis by facilitating the activities of reactive oxygen species (ROS), NF-κB, and NFATc1 in vitro and prevents the development of OVX-induced osteoporosis mouse models in vivo. Methods. The docking mechanism of DIC and RANKL was initially identified by protein–ligand molecular docking. RNA sequencing was performed and analyzed to reveal the potential mechanism and signaling pathway of the antiosteoporosis effects of DIC. To verify the sequencing results, we examined the impact of DIC on RANKL-induced osteoclast differentiation, bone resorption, F-actin ring production, ROS generation, and NF-κB activation in osteoclasts in vitro. Moreover, a luciferase assay was performed to determine the binding and transcriptional activity of Nrf2 and NF-κB. The in vivo efficacy of DIC was assessed with an ovariectomy- (OVX-) induced osteoporosis model, which was analyzed using micro-CT and bone histomorphometry. Results. The molecular docking results indicated that DIC could bind particularly to RANKL. RNA-seq confirmed that DIC could regulate the osteoclast-related pathway. DIC suppressed osteoclastogenesis, bone resorption, F-actin belt formation, osteoclast-specific gene expression, and ROS activity by preventing NFATc1 expression and affecting NF-κB signaling pathways in vitro. The luciferase assay showed that DIC not only suppressed the activity of Nrf2 but also contributed to the combination of Nrf2 and NF-κB. Our in vivo study indicated that DIC protects against OVX-induced osteoporosis and preserves bone volume by inhibiting osteoclast activity and function. Conclusions. DIC can ameliorate osteoclast formation and OVX-induced osteoporosis and therefore is a potential therapeutic treatment for osteoporosis.
... Reactive oxygen species (ROS) are known to have an important role in intracellular mechanisms, such as the delivery of cell signals, apoptosis, and gene expression, in addition to being a stress marker. H 2 DCFDA penetrates the cell membrane and infiltrates the cell 53,54 . Here, to examine the nucleic acids, the plant cells were dyed with Hoechst 33342. ...
Biohybrid micro/nanorobots that integrate biological entities with artificial nanomaterials have shown great potential in the field of biotechnology. However, commonly used physical hybridization approaches can lead to blockages and damage to biological interfaces, impeding the optimal exploitation of natural abilities. Here, we show that magnetically propelled plant biobots (MPBs), employing tomato-callus cultivation engineering in the presence of Fe3O4 nanoparticles (NPs), are capable of active movement and directional guidance under a transversal rotating magnetic field. The Fe3O4 NPs were transported through the cell growth media and then taken up into the plant tissue cells (PTCs), imparting the plant biobot with magnetic function. Moreover, Fe ions support the growth of callus cells, resulting in nanoparticle incorporation and enabling faster growth and structurally compact texture. The magnetic plant biobots demonstrated rapid and efficient removal of chlorpyrifos (approximately 80%), a hazardous nerve gas agent that causes severe acute toxicity, and recovery using an external magnetic field. The eco-friendly plant biobots described here demonstrate their potential in biomedical and environmental applications.
... Reactive oxygen species (ROS) belong to the stimuli identified [3]. It is interesting to note, that stem cells reside in a low ROS environment [4,5] and are characterized by high antioxidant enzyme expression [4] and low ROS formation. In the course of stem cell differentiation, ROS formation increases, which has been largely attributed to mitochondrial expansion [6]. ...
... Reactive oxygen species (ROS) belong to the stimuli identified [3]. It is interesting to note, that stem cells reside in a low ROS environment [4,5] and are characterized by high antioxidant enzyme expression [4] and low ROS formation. In the course of stem cell differentiation, ROS formation increases, which has been largely attributed to mitochondrial expansion [6]. ...
Rationale
Nox4 is a constitutively active NADPH oxidase that constantly produces low levels of H2O2. Thereby, Nox4 contributes to cell homeostasis and long-term processes, such as differentiation. The high expression of Nox4 seen in endothelial cells contrasts with the low abundance of Nox4 in stem cells, which are accordingly characterized by low levels of H2O2. We hypothesize that Nox4 is a major contributor to endothelial differentiation, is induced during the process of differentiation, and facilitates homeostasis of the resulting endothelial cells.
Objective
To determine the role of No×4 in differentiation of murine inducible pluripotent stem cells (miPSC) into endothelial cells (ECs).
Methods and results
miPSC, generated from mouse embryonic wildtype (WT) and Nox4−/− fibroblasts, were differentiated into endothelial cells (miPSC-EC) by stimulation with BMP4 and VEGF. During this process, Nox4 expression increased and knockout of Nox4 prolonged the abundance of pluripotency markers, while expression of endothelial markers was delayed in differentiating Nox4-depleted iPSCs. Eventually, angiogenic capacity of iPSC-ECs is reduced in Nox4 deficient cells, indicating that an absence of Nox4 diminishes stability of the reached phenotype. As an underlying mechanism, we identified JmjD3 as a redox target of Nox4. iPSC-ECs lacking Nox4 display a lower nuclear abundance of the histone demethylase JmjD3, resulting in an increased triple methylation of histone 3 (H3K27me3), which serves as a repressive mark for several genes involved in differentiation.
Conclusions
Nox4 promotes differentiation of miPSCs into ECs by oxidation of JmjD3 and subsequent demethylation of H3K27me3, which forced endothelial differentiation and stability.
... according to multiple lines of evidence [157,158]. Besides, the maintenance of stem-like status also requires the activation of various signaling pathways, including Wnt, Notch, and Hedgehog [7,[159][160][161]. ROS mediates nuclear translocation of forkhead box O (FOXO) transcription factor through c-Jun N-terminal kinase (JNK) or macrophage stimulating 1 (MST1)-mediated phosphorylation [162,163]. ...
Dormancy occurs when cells preserve viability but stop proliferating, which is considered an important cause of tumor relapse, which may occur many years after clinical remission. Since the life cycle of dormant cancer cells is affected by both intracellular and extracellular factors, gene mutation or epigenetic regulation of tumor cells may not fully explain the mechanisms involved. Recent studies have indicated that redox signaling regulates the formation, maintenance, and reactivation of dormant cancer cells by modulating intracellular signaling pathways and the extracellular environment, which provides a molecular explanation for the life cycle of dormant tumor cells. Indeed, redox signaling regulates the onset of dormancy by balancing the intrinsic pathways, the extrinsic environment, and the response to therapy. In addition, redox signaling sustains dormancy by managing stress homeostasis, maintaining stemness and immunogenic equilibrium. However, studies on dormancy reactivation are still limited, partly explained by redox-mediated activation of lipid metabolism and the transition from the tumor microenvironment to inflammation. Encouragingly, several drug combination strategies based on redox biology are currently under clinical evaluation. Continuing to gain an in-depth understanding of redox regulation and develop specific methods targeting redox modification holds the promise to accelerate the development of strategies to treat dormant tumors and benefit cancer patients.
