Figure 5
From: Germline variants of ATG7 in familial cholangiocarcinoma alter autophagy and p62
Impaired ATG5-12 complex formation and elevated p62 levels in cholangiocytes expressing ATG7 R659*. (a) Cholangiocytes expressing ATG7 R659* unable to form ATG5-12 complex. The ATG7 R659* and C572S variants fail to rescue the ATG5-12 complex formation defect observed in MMNK-1 ATG7-/- cholangiocytes. Protein lysates were obtained from cell lines under basal conditions. Free ATG12 and the ATG5-12 complex can be detected at 15 kDa and 55 kDa, respectively, on the western blot. (b) Top: Elevated p62 expression due to autophagy defects in cholangiocytes expressing ATG7 mutants. Cholangiocytes expressing ATG7 WT, R659*, D522E, and C572S were cultured using normal growth media. Expression of ATG7, p62, LC3B, and GAPDH were detected on the western blot. Bottom left: Bar plot with y-axis showing normalized p62/GAPDH ratio and x-axis showing the distinct cell line genotypes. Normalized refers to the normalization of all genotypes in relation to the wild-type cell line. P-values are shown for statistical t-test comparisons. Bottom right: Bar plot with y-axis showing LC3B-II/GAPDH ratio and x-axis showing the distinct cell line genotypes. (c) Top: Cholangiocytes expressing ATG7 WT, R659*, D522E, and C572S starved with HBSS for 3 h. Expression of ATG7, p62, LC3B, and GAPDH were detected on the western blot. Bottom: Bar plot with y-axis showing LC3B-II/GAPDH ratio and x-axis showing the distinct cell line genotypes. Original blots are presented in Supplementary Fig. S9 online. Quantifications reflect the mean of triplicates experiments for conditions and genotypes. P values are provided from t-tests performed.
