Fig. 6: ROS can stabilize PIASxα to promote p38α-SUMOylation.

a HEK293T cells were transfected with HA-tagged PIASs for 48 h, and cell lysates were incubated with purified GST-p38α protein overnight at 4° C and subjected to immunoblotting analysis. b Immunoprecipitation was conducted to detect the endogenous interaction between PIASxα and p38α in HGC27 cells. c HEK293T cells were co-transfected with FLAG-SUMO1, Myc-Ubc9, HA-p38α-WT, and gradient HAPIASxα for 48 h; cell lysates were immunoprecipitated with anti-FLAG antibody for the SUMOylation assay. d HEK293T cells were transfected with FLAG-SUMO1, Myc-Ubc9, HA-p38α-WT with or without PIASxα-siRNA, and cell lysates were immunoprecipitated for the SUMOylation assay. e, f HGC27 cells were treated with a gradient concentration of H₂O₂ (e) or NAC (f) for 6 h before harvesting, and the cells were lysed for Western blotting. g HGC27 cells stimulated by H₂O₂ or vehicle were treated with 50 μM CHX for indicated times and cell lysates were subjected to immunoblotting (upper panel). The degradation rate is represented as a line diagram (lower panel). h HGC27 cells were treated with or without H₂O₂ (0.8 mM) for 6 h and were harvested for detecting the SUMOylation level of endogenous p38α by immunoprecipitation. i GEPIA online database was used to show the mRNA level of PIASx in GC tissue and normal gastric tissues (T: tumor; N: normal). j Immunoblotting for determination of PIASxα, pp38, and pMK2 levels in 5 pairs of GC tissues and adjacent normal tissues (A: adjacent normal gastric tissues; T: GC tissues). k–m The survival probabilities were predicted by the online database. OS: overall survival (k), PFS: progression-free survival (l), or PPS: post-progression survival (m). Patients with high or low PIASx (PIASxα and PIASxβ) expression are indicated in red or black line, respectively. HR hazard ratio. The p-value was calculated by a log-rank test.