Research Paper Volume 12, Issue 7 pp 6129—6150

Figure 6. Acidic conditions stimulate shFOXK1 to induce autophagy and inhibit EMT through the downregulation of MAZ. (A) Transwell assays were performed to assess the migration and invasion of MGC803 and AGS cells following MAZ knockdown at pH 6.5. Scale bar, 500 μm. (B) Western blotting was performed to detect the protein expression of MAZ, autophagy-related proteins and EMT-related proteins in GC cells treated with ctrl or shMAZ in an acidic microenvironment. (C) Immunofluorescence staining was used to detect the autophagic flux in acidic MGC803 cells treated with ctrl or shMAZ, and the quantified results are shown in (D). Scale bar, 10 μm. (E) qRT-PCR analysis of EMT-related mRNA expression in MGC803 and AGS cells expressing ctrl, shFOXK1-1, shFOXK1-2 and shMAZ. (F) Western blotting was performed to detect the expression of MAZ, autophagy-related proteins and EMT-related proteins in ctrl (or shFOXK1-1)-transfected acidic GC cells cotransfected with MAZ plasmids. (G) MGC803 and AGS cells cultured at pH 6.5 were pretreated with 2 mM 3-MA or PBS (control) and then incubated with LV-ctrl, shFOXK1-1 and shFOXK1-2 for 24 h. The expression levels of autophagy-related proteins and EMT-related proteins were verified by Western blotting and quantified. The data are presented as the means ± S.D.s from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.