Fig 2: TMEM74-induced autophagy is associated with ATG5-ATG12 complex, independent with BECN1-PIK3C3 complex. HeLa cells were firstly treated with the indicated siRNAs (siATG5 (a), siATG7 (b), siATG16L1 (c), siATG3 (d), siPI3KC3 (e), siATG10 (f), siBECN1 (g), siULK1 (h)) for 24 h, then transfected with GFP-TMEM74 or GFP for 24 h,meanwhile treated with or without bafilomycin A1 (100 nM) for at least 12 h. The levels of LC3B-II were detected by western blotting. (i) HeLa cells were transfected with GFP-TMEM74 or GFP (control) for 24 h respectively, treated with or without LY294002 (10 µM) for at least 8 h. The levels of LC3B-II were detected by western blotting

Fig 3: YQHXR promoted VPS34 expression in the formation of Beclin1-VPS34 complex. (A) The effect of YQHXR on the formation of Beclin1-VPS34 complex in the absence or presence of 3-MA was analyzed by western blotting and immunoprecipitation analysis. (B) The effect of YQHXR on the pathological changes of IVDD rats in the absence or presence of 3-MA. The data represent the mean ± SD. Significant differences among different groups are shown as ## p < 0.01 vs Sham + NC group; *p < 0.05 vs IVDD + NC group; && p < 0.01 vs IVDD + YQHXR-H + NC group.

Fig 4: Inhibition of KDM5B activates PIK3C3 expression by promoting H3K4me3 methylation modification of the PIK3C3 promoter. (A) UCSC prediction of PIK3C3 histone methylation activation. (B) Detection of PIK3C3 expression in ESCC tissues by RT-qPCR. (C) Detection of mRNA expression of PIK3C3 in cells after transfection by RT-qPCR. (D) The protein expression of H3K4me3 in cells after transfection using Western blot. (E) Detection of PIK3C3 promoter enrichment by Anti-KDM5B and Anti-H3K4me3 examined using ChIP-qPCR. **P < 0.01, ***P < 0.01 compared with adjacent tissues or sh-NC. The results were measurement data, which were expressed as the mean ± SD (n = 30). Comparisons between two groups were conducted using paired t-test (B), and comparisons between multiple groups analyzed by two-way ANOVA (C, D, E) with Tukey’s post hoc test. The experiment was independently repeated three times.

Fig 5: YQHXR increased the protein level of the ubiquitinase USP13 for the inhibition of Beclin1 degradation. (A) The effect of YQHXR on stabilization of Beclin1-VPS34 complex in the absence or presence of Spautin-1 was analyzed by western blotting and immunoprecipitation analysis. (B) The effect of YQHXR on the pathological changes of IVDD rats in the absence or presence of Spautin-1. The data represent the mean ± SD. Significant differences among different groups are shown as ## p < 0.01 vs Sham + NC group; *p < 0.05, **p < 0.01 vs IVDD + NC group; && p < 0.01 vs IVDD + YQHXR-H + NC group.