ros detection assay kit dcf-da; cellrox ® green reagent  (Thermo Fisher)

Journal: Cell Death & Disease

Article Title: Autophagy caused by oxidative stress promotes TGF-β1-induced epithelial-to-mesenchymal transition in human peritoneal mesothelial cells

doi: 10.1038/s41419-024-06753-z

Figure Lengend Snippet: A TGF-β1 treatment (2 [T2] and 5 [T5] ng/mL) increased mRNA expression of the profibrotic mesenchymal markers (E-cadherin, fibronectin, and α-SMA) in HPMCs. B , C TGF-β1 treatment (2 and 5 ng/mL) increased protein levels of the profibrotic mesenchymal markers and activated the phosphorylation of Smad2/3 signaling in HPMCs. D TGF-β1 treatment (2 and 5 ng/mL) increased the mRNA expression of NOX2/4 and P22phox in HPMCs after 48 h. E TGF-β1 induced ROS generation, which was measured using DCF-DA 1 h after TGF-β1 treatment (2 and 5 ng/mL). F TGF-β1 induced H 2 O 2 generation 24 h after treatment (2 and 5 ng/mL). The data are presented as mean ± standard error. n = 4 per group. * P < 0.05 vs. control ( C ); ** P < 0.01 vs. control; and *** P < 0.001 vs. control.

Article Snippet: The concentrations of intracellular ROS in the HPMCs were measured using a 2′,7′-dichlorofluorescin diacetate (DCF-DA)-Cellular ROS Assay Kit (Abcam, Cambridge, MA, USA), according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Autophagy caused by oxidative stress promotes TGF-β1-induced epithelial-to-mesenchymal transition in human peritoneal mesothelial cells

doi: 10.1038/s41419-024-06753-z

Figure Lengend Snippet: A The determination of ROS production using DCF-DA ( n = 4). B Mitochondrial superoxide production in live cells was measured using fluorescence microscopy with MitoSOX Red dye. Representative fluorescence images showing the localization of MitoSOX Red fluorescence. Scale bar = 40 μm. C , D The level of MitoSOX Red fluorescence per cell was quantified using ImageJ software. Image data from 51–60 cells per treatment condition were averaged ( n = 3). E , F The measurement of mitochondrial oxygen consumption ratio (OCR) and extracellular acidification rates (ECAR) under NOX4 inhibition with GKT137831-treated HPMCs. The parametric indices of OCR and ECAR, mitochondrial respiration, and glycolysis are shown ( n = 4–6). G , H The measurement of mitochondrial OCR and extracellular acidification rates (ECAR) under autophagy inhibition by 3-MA treatment. The parametric indices of OCR and ECAR, mitochondrial respiration, and glycolysis are shown. ( n = 4–7). The data are presented as mean ± standard error (SE). * P < 0.05 vs. control, *** P < 0.001 vs. control; # P < 0.05 vs. TGF-β1 2 ng/mL; ## P < 0.01 vs. TGF-β1 2 ng/mL; ### P < 0.001 vs. TGF-β1 2 ng/mL; + P < 0.05 vs. TGF-β1 5 ng/mL; ++ P < 0.01 vs. TGF-β1 5 ng/mL; and +++ P < 0.001 vs. TGF-β1 5 ng/mL.

Article Snippet: The concentrations of intracellular ROS in the HPMCs were measured using a 2′,7′-dichlorofluorescin diacetate (DCF-DA)-Cellular ROS Assay Kit (Abcam, Cambridge, MA, USA), according to the manufacturer’s instructions.

Techniques: Fluorescence, Microscopy, Software, Inhibition