Fig 2: Autophagy inhibition promotes oncogenic RAS-induced EMT. (a) Immunofluorescence staining of LC3 (green), CDH1 (green), ZEB1 (red) or F-actin in HKe3 ER:HRAS V12 cells with the indicated treatments. Rhodamine-phalloidin was used to stain F-actin. TO-PRO-3 (blue) was used to stain nucleic acids. Scale bar: 20μm. (b) Protein expression of CDH1, ZEB1, SNAI2, SNAI1, ZEB2, TWIST1, ATG12–ATG5 and phospho-MAPK/ERK (p-MAPK) in HKe3 ER:HRAS V12 cells with the indicated treatments. TUBB was used as a loading control. (c) Fold change in mRNA levels of CDH1, ZEB1 and SNAI2 in HKe3 ER:HRAS V12 cells with the indicated treatments. mRNA levels normalized to ACTB/β-actin in control cells were used to set the baseline value at unity. Data are mean ± s.d. n = 3 samples per group. n.s. P > 0.05, *** P < 0.001. (d) Protein expression of CDH1, ZEB1, SNAI2 and ATG12–ATG5 in 4-OHT-treated HKe3 ER:HRAS V12 cells with the indicated treatments. TUBB was used as a loading control.

Fig 3: Elevated KDM3A expression in hypoxia upregulates Slug, which suppresses CDH1 expression. (A and B) KDM3A knockdown impairs CDH1 (E-cadherin) downregulation as well as SNAI2 (Slug) upregulation in hypoxia. MCF7 cells transfected with either si-CTL (−) or si-KDM3A (+) were cultured for 48 h in normoxia. Next, the cells were divided for culture under normoxic (21% O2) or hypoxic conditions (1% O2) for 24 h. The expression of mRNA specific to each target gene, as determined by RT-PCR, is displayed in panel A, while the protein expression levels measured by IB using specific antibodies against the proteins indicated in the figure are shown in panel B. (C and D) KDM3A overexpression decreases CDH1 expression while increasing SNAI2 expression in normoxia. MCF7 cells transfected with either empty vector (−) or Flag-tagged KDM3A-encoding vector (+) were cultured for 48 h, and CDH1 and SNAI2 expression at the (C) mRNA and (D) protein level was examined using RT-PCR and IB, respectively. (E) KDM3A knockdown in MCF7 cells decreases Twist upregulation in hypoxia while increasing the hypoxic induction of Snail expression. (F) KDM3A overexpression increases Twist expression in MCF7 cells cultured under normoxic conditions. The experiments were performed as described in (D). si, small interfering; RT-PCR, reverse transcription-PCR; KDM, lysine demethylase; IB, immunoblotting.

Fig 4: ASPP1 deficiency promotes oncogenic RAS-induced EMT.a Immunofluorescence staining of E-cadherin (green) in HKe3 ER:HRAS V12 cells with indicated treatments. TO-PRO-3 (blue) was used to stain nuclei. Scale bars: 10 μm. b Fold change in mRNA levels of SNAI2 (Snail2), CDH1 (E-cadherin) and VIM (Vimentin) in HKe3 ER:HRAS V12 cells with indicated treatments. **P < 0.01. ***P < 0.001. Scores above the bars are relative levels when compared with control cells. Protein expression of Snail1, Snail2, E-cadherin, phospho-AKT (p-AKT), AKT and ASPP1 in HKe3 ER:HRAS V12 cells with indicated treatment. β-tubulin was used as a loading control.

Fig 5: Autophagy inhibition promotes EMT in RAS-mutated cells. (a) Protein expression of CDH1 and ATG12–ATG5 in the indicated pancreatic cancer cell lines transfected with control siRNA or ATG5 siRNA. TUBB/β1-tubulin was used as a loading control. For protein expression of CDH1 and ATG12–ATG5 in pancreatic cancer cell lines with mutant KRAS, both short and long exposures (respectively) are shown. KRAS mutation status is indicated under the blots. (b) Fold change in mRNA levels of CDH1, SNAI1, SNAI2, TWIST1, ZEB1 and ZEB2 in the indicated pancreatic cancer cell lines transfected with control siRNA or ATG5 siRNA. GAPDH-normalized mRNA levels 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. (c) Immunofluorescence staining of CDH1 (green) in HRas V12-expressing Atg7+/+ tumors or HRas V12-expressing atg7−/- tumors. TO-PRO-3 (blue) was used to stain nucleic acids. Scale bar: 20 μm. Atg7+/+ or atg7−/- iBMK cells transduced with HRas V12 were subcutaneously injected in nude mice to form tumors. The graph shows the average relative intensity of CDH1 per cell evaluated using ImageJ, and data are mean ± s.d. n = 4 random fields. *** P < 0.001.