Fig. 2

BMP4 activates autophagic flux in HCC cell lines: a HCC cell lines were pre-treated with autophagy inhibitor 3-MA for 1 h before BMP4 administration to prevent autophagy activated by BMP4. Western blot was applied to detect the expression levels of LC3-II, p62 and BECN1 in both HepG2 and HCCLM3 cells. b GAPDH was used as the internal control. Quantification analysis of LC3-II / GAPDH in Fig. 3 (a) was normalized to the control (blank) groups. n = 3, one-way ANOVA with post-hoc Tukey’s test. c Representative fluorescent images of mRFP-GFP-LC3 transfection. mRFP-GFP-LC3-HepG2 / HCCLM3 cells were exposed to BMP4, BMP4 with 3-MA and Noggin for 24 h. The fluorescent images were obtained from the Operetta automated microscope (Original magnification: × 200). The yellow puncta indicated autophagosomes and red puncta represent autolysosomes. d Quantification autophagic flux was calculated by red puncta in the merged images with Perkin-Elmer Columbus 2.3 software. n = 3, one-way ANOVA with post-hoc Tukey’s test