Fig 2: Downregulation of GSTA4 and TXN2 by UHRF1 depletion contributes to enhanced sensitivity to HDAC inhibitors in RB cells. (A) Detection of basal intracellular ROS levels by reactivity with a fluorescent probe in Y79 shCTL and shUHRF1 cells. ROS (green fluorescence)‐positive cells are shown along with phase contrast images of the cells on the left. Scale bars: 50 µm. (B) Quantification of ROS‐positive cells shown in (A). Over 900 total cells from five randomly selected fields were evaluated for ROS positivity. The data represent the mean ± SD from two replicates. (C) Immunoblots for indicated proteins in Y79 control (−) and UHRF1‐knockdown (+) cells subjected to a single or combined treatment with 10 mm NAC and 1 µm MS‐275 for 48 h. (D) Immunoblots in Y79 shGSTA4 (clones #654 and #839) cells treated with 1 µm MS‐275 for 24 h. (E) Immunoblots in Weri‐Rb1 shGSTA4 cells treated with 1 µm MS‐275 or 1 mm NaBu for 20 h. (F) Immunoblots in Y79 shTXN2 (clones #200 and #202) cells treated with 1 µm MS‐275 for 48 h. (G) Immunoblots in Weri‐Rb1 shTXN2 cells treated as in (E). (H) Expression of GSTA4 and TXN2 in human RB tumor lysates. Relative abundance of proteins determined by densitometry is shown below each panel of the blots, in comparison with the first human RB sample on each panel. RPE: retinal pigment epithelium.

Fig 3: Representative fluorescence micrographs show (a) peroxiredoxin 5 (PRX5); (b) peroxiredoxin 6 (PRX6); (c) sulfonylated peroxiredoxin peptide (PRX-SO3) positivity in the cornu ammonis 1 (CA1), CA3, and gyrus dentatus (GD) of the hippocampus and the primary motor cortex (M1) area of rat brains (5 samples per each group) subjected to the IR and IPC. White arrows indicate positive cells and red arrows are PRX6 positive astrocytes. Nuclei were labeled with blue DAPI. Scale bar = 50 μm; (d) Western blots for PRX5, PRX6, PRX-SO3, and thioredoxin 2 (TRX2) detected in IR and IPC groups. Results in the bar graphs are presented as the relative protein content normalized to β-actin and compared to the basal control value. Graphs show the mean ± SEM for at least 3 replicates. A one-way ANOVA with post hoc comparisons by the Student–Neuman–Keuls test was carried out to test for significant differences when compared to the control. CON (control), IR (ischemia-reperfusion), and IPC (ischemic preconditioning).

Fig 4: A schematic illustration depicting the mechanism by which disulfiram(DSF) exerts its inhibitory effect on BRAFi-resistant cells by inducing mitochondrial dysfunction.DSF disulfiram, TXNIP thioredoxin-interacting protein, TRX2 thioredoxin 2, MT-ROS mitochondrial reactive oxygen species, OCR, oxygen consumption rate, ΔΨm mitochondrial membrane potential, BRAFi BRAF inhibitors.

Fig 5: TXNIP provokes mitochondrial oxidative stress in vemurafenib-resistant cells via the interaction with TRX2.A Interactome of TXNIP by the STRING database. B The volcano plot of proteins interacting with TXNIP by Data-Independent Acquisition (DIA) quantitative proteomics. C The heatmap of the proteins interacting with TXNIP by Data-Independent Acquisition (DIA) quantitative proteomics. D CCK-8 assay of TXNIP-in-1 pretreated vemurafenib-resistant cells exposed to Vem or Vem+DSF. E Colony formation of TXNIP-in-1 pretreated vemurafenib-resistant cells exposed to Vem or Vem+DSF. F TEM observation of TXNIP-in-1 pretreated vemurafenib-resistant cells exposed to Vem or Vem+DSF. G Immunofluorescence to determine MMP of TXNIP-in-1 pretreated vemurafenib-resistant cells exposed to Vem or Vem+DSF. H Seahorse assay of TXNIP-in-1 pretreated vemurafenib-resistant cells exposed to Vem or Vem+DSF. I ATP levels of TXNIP-in-1 pretreated vemurafenib-resistant cells exposed to Vem or Vem+DSF. J Flow cytometry to detect mt-ROS of TXNIP-in-1 pretreated vemurafenib-resistant cells exposed to Vem or Vem+DSF. Data represent the mean ± SD of triplicate. The differences were analyzed using one-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001. ns non-significant.