Figure 3. Induction of autophagosomes in colon epithelial cells by H₂S.

(A) Treating the cells with NaHS for 24 h or 48 h prominently enhanced the formation of autophagic vacuoles as determined by immunofluorescent staining for LC3B. (B) Ultrastructural analysis by electron microscopy revealed the formation of autophagosome or secondary lysosomes with the residual digested material in NaHS-treated HT-29 (1 mmol/L; 48 h). (C) NaHS increased LC3-II levels in HT-29, SW1116 and YAMC cells after treatment with NaHS (1 mmol/L) for 48 h. (D) NaHS dose-dependently increased LC3B-II level in HT-29 cells at 48 h. The expression of Beclin-1 was not altered. (E) Increased autophagic flux was confirmed by co-treating SW1116 cells with NaHS and bafilomycin A₁. Treatment with bafilomycin A₁ (10 nmol/L) did not prevent the upregulation of LC3B-II in cells incubated with NaHS (1 mmol/L; 48 h).