Fig 2: Analyses of Atg3 expression in leukemia cells.(A-C) Atg3 expression was analyzed by qRT-PCR and Western blotting in healthy bone marrow cells and four leukemia cell lines. Representative results from triplicate experiments are shown as the mean±SD. (D) Atg3 mRNA expression in healthy people (n = 10) and MDS patients (n = 10) was detected by qRT-PCR and plotted as mean ± SD of three independent experiments. *p<0.05.

Fig 3: Autophagy inhibition promotes oncogenic RAS-induced EMT via SQSTM1. (a) Immunofluorescence staining of SQSTM1 (red) and ZEB1 (green), and differential interference contrast (DIC) images from 4-OHT-treated HKe3 ER:HRAS V12 cells with the indicated treatments. Scale bar: 20 μm. (b) Protein expression of ZEB1, SQSTM1, ATG3, ATG12–ATG5 and phospho-AKT (p-AKT) in HKe3 ER:HRAS V12 cells with the indicated treatments. TUBB was used as a loading control. (c) 4-OHT-treated HKe3 ER:HRAS V12 cells were transfected with Cherry-SQSTM1 (red) followed by immunofluorescence staining of ZEB1 (green). TO-PRO-3 (blue) was used to stain nucleic acids. Scale bar: 20 μm. (d) Protein expression of ZEB1, exogenously expressed Cherry-SQSTM1 and endogenous SQSTM1 in 4-OHT-treated HKe3 ER:HRAS V12 cells transfected with control vector or Cherry-SQSTM1. TUBB was used as a loading control. (E) Fold change in mRNA levels of ZEB1 and SNAI2 in HKe3 ER:HRAS V12 cells with the indicated treatments. mRNA levels normalized to GAPDH in control cells were used to set the baseline value at unity. Data are mean ± s.d. n = 3 samples per group. * P < 0.05. ** P < 0.01. *** P < 0.001.

Fig 4: SH3BGRL drives autophagy through PIK3C3 translation by interaction ribosome. (A) GST affinity isolation of SH3BGRL with ribosomal subunits. GST affinity isolation was performed with lysates of MCF-7 and MDA-MB-453 cells, followed by MS peptide sequencing. (B) Heatmap of RNA-seq and Polyribosome-profile analyses of ULK1, PIK3C3, BECN1, ATG7, ATG5, ATG3, MAP1LC3B and SQSTM1. (C) Immunoblots of SH3BGRL and PIK3C3 in indicated cells. ACTB was used as an internal loading control. Statistical analysis of PIK3C3 expression in indicated cells by four independent immunoblots. (D) Immunoblots of PIK3C3 protein in the indicated cells treated with or without 50 µg/ml cycloheximide (CHX) for 6 h. PIK3C3 expression was statistically analyzed based on three independent experiments. (E,F) Polyribosome profiling of PIK3C3 mRNA in MCF-7 (E) or MDA-MB-453 (F) cells. ACTB was used as an internal negative loading control. The marked numbers are equivalent to the collected fractions after gradient centrifuge. Fraction 1 represents the top (10%) of the gradient, fraction 10 represents the bottom (50%) of the gradient. (G) Polyribosome profiling of ATG12 mRNA in indicated cells as (E). All experiments were independently repeated at least three times. (H) Immunoblots of ULK1, BECN1, ATG7, ATG5 and ATG3 in the indicated cells. ACTB was used as a loading control. All significant differences are shown as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: Effects of interfering with the 14G2a mAb-stimulated autophagy on the 14G2a/CQ-induced apoptosis. a Cells were pre-treated with 10 μM CQ for 1.5 h and subsequently treated with the 14G2a mAb or PBS (control) for 72 h. Relative autophagy and apoptosis-associated protein expression levels (P62, Bcl-xl, cleaved caspase 3, cleaved PARP, LC3A/B-I, LC3A/B-II, ATG7, ATG3, and Bax) were measured in control and the 14G2a mAb and/or CQ treated IMR-32 cells by western blot and normalized to α-tubulin. b Representative blots are presented. c Relative caspase 3/7 level was measured in control and 14G2a mAb and/or CQ-treated IMR-32 cells and was divided by ATP level signals of the respective groups of cells. d IMR-32 cells were transfected with ATG7 siRNA (S) or control siRNA (Mock). The ATG7 gene mRNA and ATG7 protein expression levels were analyzed at 24 h by RT-qPCR and western blot, respectively and compared to non-transfected cells (WT). RPS13 cDNA and α-tubulin protein were used respectively, as the references. e IMR-32 cells were treated 24 h after siRNA transfection with the 14G2a mAb (40 μg/ml) or PBS (control) for 24 h. LC3A/B expression was analyzed by western blot, α-tubulin served as the loading control. f Relative expression levels of cleaved caspase 3 and g cleaved PARP were measured at 24 h after transfection in the 14G2a mAb, and/or CQ-treated ATG7 silenced (S), the control cells (Mock). h Representative blots are presented. i LC3A/B expression was determined by western blot in IMR-32 cells treated with 10 μM CQ or 16 nM Baf for 24 h. j The relative caspase 3/7 level was measured in IMR-32 cells treated with 14G2a mAb (40 μg/ml), and/or indicated concentrations of Baf or its solvent—DMSO, and divided by ATP level signals of the respective groups of cells. C—control, solvent-treated cells (PBS for the 14G2a mAb, water for CQ or DMSO for Baf); 14G2a—the 14G2a mAb-treated cells; CQ—chloroquine-treated cells; Baf—bafilomycin-treated cells. P-values for t test were as follow: *p < 0.05, **p < 0.01