Fig. 4: Arsenic destabilizes TXNL1 protein by downregulating USP10 in human bronchial epithelial cells.

a BEAS-2B cells were exposed to 1 μM arsenic for 0, 12, and 24 h. TXNL1 protein levels were then assessed via Western blotting. b QPCR was conducted to evaluate the effects of 1 μM arsenic exposure on TXNL1 mRNA levels at 0, 12, and 24 h. Results are shown as mean ± s.d. Results are shown as mean ± s.d. n = 3 biologically independent samples. c BEAS-2B cells were treated with the CHX (50 µg/mL) in the presence or absence of arsenic (1 μM). TXNL1 protein stability was assessed by western blotting. d BEAS-2B cells were treated with arsenic (1 μM) alone or in combination with the proteasome inhibitor MG132 (2.5 μM) for 0 or 12 h. TXNL1 protein levels were analyzed by western blotting. e BEAS-2B cells were transfected with Vector plasmid or Myc-Ub plasmid in combination with TXNL1-HA plasmid for 36 h and then treated with MG132 (2.5 μM) alone or in combination with arsenic (1 μM) for 12 h. TXNL1 ubiquitination was analyzed by immunoprecipitation (IP) with an anti-HA antibody followed by western blotting. f Venn diagram illustrating the intersecting proteins between TXNL1-binding proteins (IP-MS), arsenic-regulated proteins (iTRAQ), and ubiquitin-proteasome pathway-related proteins. g Co-immunoprecipitation of endogenous USP10 with TXNL1-HA from BEAS-2B cell lysates using an anti-HA antibody. h Western blot analysis of TXNL1 and USP10 protein levels in BEAS-2B cells after exposed to 1 μM arsenic for 0, 12, and 24 h. i Validation of USP10-overexpressing BEAS-2B cells (USP10-OE) and its effect on endogenous TXNL1 protein levels by western blotting. j BEAS-2B (Vector) and BEAS-2B (USP10-OE) were exposed to 1 μM arsenic for 24 h. TXNL1 protein levels were assessed by western blotting. k BEAS-2B (Vector) and BEAS-2B (USP10-OE) cells were pretreated with MG132 (2.5 μM) for 8 h, followed by co-treatment with CHX (50 µg/mL) and arsenic (1 μM) for indicated times. TXNL1 protein decay was monitored by western blotting. Numbers indicate relative TXNL1/β-actin ratios. l 293 T cells were co-transfected with Myc-Ub and the indicated combinations of TXNL1-HA and USP10 plasmids. Cells were treated with MG132 (2.5 μM) for 12 h before harvesting. TXNL1 ubiquitination was analyzed by HA immunoprecipitation and western blotting. An asterisk (*) indicates a significant difference at P < 0.05.