Fig. 4: BCAT1 regulates autophagy during hypoxia by activating ERs via the IRE1-XBP1-RIDD axis.

a Western blot analysis of BECN1 and Atg5 in PASMCs cotransfected with BCAT1 and IRE1 siRNA (n = 5). b Autophagic flux was monitored in PASMCs cotransfected with eGFP-mRFP-LC3 plasmid and control siRNA or IRE1 siRNA that were then exposed to HYP for 24 h. Scale bar = 50 μm (n = 3). c, d RT-PCR analysis of the mRNA levels of XBP1-s, sparc, pmp2, and Scara3 with rat β-actin serving as the standard (n = 5). e The formation of autophagosomes was detected, and autophagic activity was estimated in cells in which the expression of XBP1 was knocked down with XBP1 siRNA under HYP for 24 h. Scale bar = 50 µm (n = 5). Nor normoxia, Hyp hypoxia, H + G hypoxia plus gabapentin, H + NC hypoxia plus control siRNA, H + SI hypoxia plus BCAT1 siRNA, H + SI-IRE1 hypoxia plus IRE1 siRNA, H + SI-XBP1 hypoxia plus XBP1 siRNA, H + Con hypoxia plus control vector, H + B hypoxia plus BCAT1 plasmid, H + Con+NC hypoxia plus control vector plus control siRNA, H + B + Si-IRE hypoxia plus BCAT1 plasmid plus IRE1 siRNA. Statistical analysis was performed with one-way ANOVA. All values are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.