Figure 2. Impairment of autophagy resulted in EVT invasion failure with the CoCl₂ treatment via the suppression of MMP9.

a) Invasion assays were performed with HTR8/SVneo cells in the presence or absence of three methyladenine (3-MA, 5 mM) under DMSO (control: white bars) or 250 µM CoCl₂ (black bars) for 24 h. The Y-axis indicates the number of invading cells. b) Invasion assays were performed with HTR8/SVneo cells in the presence of 100 nM or 500 nM rapamycin under DMSO (control: white bars) or 250 µM CoCl₂ (black bars) for 48 h. c) Invasion assays were performed with HTR8-Atg4B^C74A mutant cells, an autophagy-deficient EVT cell line, or HTR8-mStrawberry cells, the control cell line, under DMSO (control: white bars) or 250 µM CoCl₂ (black bars) for 48 h. The Y-axis indicates the number of invading cells. d) Migration assays were performed with HTR8-Atg4B^C74A mutant cells or HTR8-mStrawberry cells under DMSO (control: white bars) or 250 µM CoCl₂ (black bars) for 48 h. The Y-axis indicates the number of migrating cells. e) Cell proliferation rates were estimated by the WST-1 assay in HTR8-Atg4B^C74A mutant cells or HTR8-mStrawberry cells under DMSO (control: white bars) or 250 µM CoCl₂ (black bars) for 48 h. f) Cell death rates were estimated by 5 µg/ml propidium iodide (PI) staining in HTR8-Atg4B^C74A mutant cells or HTR8-mStrawberry cells under DMSO (control: white bars) or 250 µM CoCl₂ (black bars) for 48 h. g) Representative gelatin zymograms showed MMP9 secreted from HTR8-Atg4B^C74A mutant cells or HTR8-mStrawberry cells after 48 h culture in DMSO or 250 µM CoCl₂. For all samples, 20 mg total protein was loaded per well. The bars indicate the median values of MMP9 in each group. Data were normalized to the protein concentrations of the culture media for each sample. (c-g) Fold inductions were calculated in comparison to the number of HTR8 cells treated with DMSO as one. Data were shown as the mean ± S.E. of three independent experiments. N.S.: not significant