Fig 2: ALKBH5-regulated autophagy acts upstream of ULK1 and the PIK3C3-BECN1 complex. a SKOV3 cells were infected with LVRU6P-01, LVRU6P-02, or LVRU6P-NC. A2780 cells were infected with LV121-NC or LV121-ALKBH5. After 48 h, puromycin was added at a concentration of 2.5 mg/mL. After 72 h, the expression of ATG5, ATG7, Beclin1, Ulk1, and PI3KC3 was detected by WB. b SKOV3 cells were infected with LVRU6P-01 or LVRU6P-NC. After 48 h, puromycin was added at a concentration of 2.5 mg/mL. After 24 h, the LVRU6P-01-infected cells were transfected with ATG5 siRNAs. After 48 h, the expression of ATG5 and LC3-II was detected using WB. c SKOV3 cells were infected with LVRU6P-01 or LVRU6P-NC. After 48 h, puromycin was added at a concentration of 2.5 mg/mL. After 24 h, the LVRU6P-01 infected cells were transfected with Beclin1 siRNAs. After 48 h, the expression of Beclin1 and LC3-II was detected using WB. d SKOV3 cells were infected with LVRU6P-01 or LVRU6P-NC. After 48 h, puromycin was added at a concentration of 2.5 mg/mL. After 24 h, the LVRU6P-01 infected cells were transfected with ATG7 siRNAs. After 48 h, the expression of ATG7 and LC3-II was detected using WB. e SKOV3 cells were infected with LVRU6P-01 or LVRU6P-NC. After 48 h, puromycin was added at a concentration of 2.5 mg/mL. After 24 h, the LVRU6P-01 infected cells were transfected with Ulk1 siRNAs. After 48 h, the expression of Ulk1 and LC3-II was detected by WB. f SKOV3 cells were infected with LVRU6P-01 or LVRU6P-NC. After 48 h, puromycin was added at a concentration of 2.5 mg/mL. After 24 h, the LVRU6P-01-infected cells were transfected with PI3KC3 siRNAs. After 48 h, the expression of PI3KC3 and LC3-II was detected using WB

Fig 3: Augmentation of the stemness of human lung CSCs requires cooperation of Zeb1 in A549-oncosphere and analysis of clinical relevance of ATG5 and Zeb1.A–D Analysis of mRNA expression and protein level of Zeb1 in A549-oncosphere treated by DMSO, rapa, 3-MA, and BafA1 A, B; and upon silencing of ATG5 C, D; Tbp and GAPDH were used as reference controls, *p < 0.05. The concentrations of 3-MA, BafA1, and rapa were 3 mM, 100 nM, and 5 µM, respectively, and cells in Fig. 6B were treated for 24 h. E, F Analysis of mRNA expression and protein level of Zeb1 in A549-oncosphere-shATG5 after overexpression of Zeb1 (A549-oncosphere-shATG5-OE-ZEB1); Tbp and GAPDH were used as reference controls, ***p < 0.01. G Analysis of mRNA expression of Oct4, Nanog, Aldh1, and CD133 in A549-oncosphere-shN.C.-OE-vector, A549-oncosphere-shATG5-OE-vector, and A549-oncosphere-shATG5-OE-ZEB1; Tbp was used as a reference control, *p < 0.05, ***p < 0.01. Colony formation assay (H bar = 120 µm) and single-cell cloning assay (I different superscripts represent significant differences, p < 0.05), using A549-oncosphere-shN.C.-OE-vector, A549-oncosphere-shATG5-OE-vector, and A549-oncosphere-shATG5-OE-ZEB1 cells. J, K Analysis of the tumors obtained from mice receiving A549-oncosphere-shATG5-OE-vector and A549-oncosphere-shATG5-OE-ZEB1, ***p < 0.01. Bar = 1 cm. L mRNA expression of ATG5 and ZEB1 in normal tissue and different stages of human lung adenocarcinoma using TCGA data sets, *p < 0.05. M Analysis of the correlation of ATG5 and ZEB1 in human lung adenocarcinoma using TCGA datasets; R2 was the correlation coefficient. N Survival analysis of ATG5 and ZEB1 in human lung adenocarcinoma using TCGA datasets by OncoLnc.

Fig 4: Augmentation of the stemness of human lung CSCs by autophagy is dependent on degradation of ubiquitinated p53.A Analysis of mRNA expression of TP53 in A549-oncosphere, and H1299-oncosphere; Tbp was used as a reference control. B Analysis of mRNA expression (B Tbp was used as a reference control) and protein level of p53 (C GAPDH was used as a reference control) in A549-oncosphere treated with DMSO, 3-MA, BafA1, rapa, and knockdown of ATG5. D Analysis of mRNA expression and protein levels of p53 in A549-oncosphere-shATG5 after siRNA knockdown of TP53 (A549-oncosphere-shATG5-shTP53); Tbp and GAPDH were used as a reference controls, ***p < 0.01. Colony formation assay (E bar = 120 µm) and single-cell cloning assay (F ***p < 0.01) using A549-oncosphere-shATG5 and H1299-oncosphere-shATG5 in which ATG5 cells treated with 3-MA, BafA1 followed knockdown of TP53, DMSO was the control group. G, H Subcutaneous tumors excised (G, images; H: tumor weight, p < 0.01) from nude mice receiving A549-oncosphere-shATG5-shTP53, A549-oncosphere-shATG5-shN.C cells. Bar = 1 cm. I, J Analysis of mRNA expression and protein levels of p53 in A549-oncosphere and H1299-oncosphere after overexpression of TP53 (A549-oncosphere-OE-TP53 and H1299-oncosphere-OE-TP53); Tbp and GAPDH were used as reference controls, ***p < 0.01. Colony formation assay (K bar = 120 µm) and single-cell cloning assay (L ***p < 0.01.) using A549-oncosphere cells overexpressing TP53 (A549-oncosphere-OE-TP53 and H1299-oncosphere-OE-TP53). M, N Subcutaneous tumors excised (M, images; N: tumor weight, ***p < 0.01) from nude mice receiving A549-oncosphere cells overexpressing TP53 (A549-oncosphere-OE-TP53 and H1299-oncosphere-OE-TP53). Bar = 1 cm. O, P Analysis of protein levels of p53 in A549-oncosphere treated with DMSO, Mdivi-1, CCCP O, MG132 and MG132 in combination with rapa (P) by WB; GAPDH was used as a reference control. Q Analysis of protein level of p53 in A549-oncosphere upon HDM2 silencing (di-HDM2) or NVP, followed by treatment with MG132 in combination with rapa; GAPDH was used as a reference control. The concentration of 3-MA, BafA1, rapa, Mdivi-1, CCCP, MG132, and NVP was 3 mM, 100 nM, 5 µM, 75 µM, 50 µM, 40 µM, and 300 nM, respectively, and cells used in this figure were treated for 24 h.

Fig 5: LRP6 deletion regulates ferroptosis in cardiomyocytes through autophagy. (a) The presented blots were obtained from the expression of autophagy-related proteins LC3-A/B, ATG, and p62 after LRP6 interference in the cardiomyocytes treated with hypoxia and erastin. Top: typical blots. Pooled data: biological replication indicated five mice in each group. (b) Effect of ATG5 siRNA mediated the expression of ATG5 in cardiomyocytes. Pooled data: biological replication indicated five mice in each group. Data were means ± s.e.m.*P < 0.05 and **P < 0.01 compared with ctrl by one-way ANOVA with Bonferroni's post hoc test. (c) Analysis of the cardiomyocytes with LRP6 siRNA and ATG5 siRNA influence on survival rate in cardiomyocytes treated with hypoxia and erastin. Statistical analysis was obtained from five mice in each group. (d, e) In this case, the presented Fe2+ and MDA levels were induced by LRP6 deletion. Pooled data: biological replication indicated five mice in each group. Data were means ± s.e.m.*P < 0.05 and **P < 0.01 compared with ctrl or si-LRP6 by one-way ANOVA with Bonferroni's post hoc test.