Estimation of autophagy in HTR8/SVneo cells, when CoCl₂ induced HIF1α expression.

<p>a) Western blots in HTR8/SVneo cells under 250 µM CoCl₂, 2% oxygen tension, or DMSO (control) for 24 h were shown as follows: HIF1α and α-tubulin. b) The expression of MAP1LC3B-I and -II (LC3-I and LC3-II) in HTR8/SVneo cells was examined in the presence of E64d and pepstatin under 250 µM CoCl₂ for the indicated times. The expression of α-tubulin was used as an internal control. c) Representative panels show anti-LC3 staining in HTR8/SVneo cells under DMSO (control) or 250 µM CoCl₂ for 24 h. Negative control (Nega cont) was treated with rabbit serum instead of anti-LC3 antibody and stained with DAPI (4′, 6-diamidino-2-phenylindole). Scale bar: 30 µm. d) The graph indicates the number of LC3 puncta in HTR8/SVneo cells under DMSO (control), rapamycin (500 nM), 250 µM CoCl₂, or 250 µM CoCl₂ with 5 mM 3-MA for 24 h. These experiments were independently performed at least three times.