Figure 5.

Decrease in autophagic flux and cell survival in cervical adenocarcinoma HeLa cells by ECDD-S27. (a–c) ECDD-S27 inhibits autophagic flux in HeLa cells. Cells were treated with DMSO with or without bafilomycin A1 or ECDD-S27 at the indicated concentrations for 4 h. The expression levels of p62/Actin and LC3-II/Actin were then assessed using immunoblotting followed by the densitometric analysis using ImageJ. Representative images cropped from the same blot are shown and full images are included in the supplementary information. ECDD-S27 inhibits autophagic flux with the IC₅₀ of 0.016–0.080 µM. Data are mean ± SEM from three independent experiments. (d–e) ECDD-S27 autophagic flux inhibition in HeLa cells was confirmed by RFP-GFP-LC3B puncta assay. RFP-GFP-LC3B expressing HeLa cells were treated with DMSO, starvation, or ECDD-S27 at the indicated concentrations for 4 h and analyzed by confocal microscopy. The number of LC3B puncta/cell was then quantified. Only puncta ≥0.3 µm in size were counted. Data are the means ± SEM from at least three independent experiments. At least 30 cells per condition per independent experiment were quantified; *p < 0.05, **p < 0.01, and ***p < 0.001, all relative to the DMSO control, were determined by one-way ANOVA with a Tukey’s multiple comparison test. Bar 5 µm. (f) HeLa cell survival is restricted by ECDD-S27. Cells were treated with DMSO (negative control), ECDD-S27, or HCQ at the indicated concentrations for 72 h and their viability was measured by the MTS assay. Data are mean ± SD from three independent experiments; the results were expressed relative to the DMSO control, defined as 100%. ***p < 0.001, ECDD-S27 vs HCQ treatment at the same concentration, was determined by one-way ANOVA with a Tukey’s multiple comparison test.