Figure 6.

Kras-p53 IHCC exhibits high basal levels of autophagy and is growth inhibited by chloroquine. A) IHCC cells were infected with a retrovirus expressing GFP-LC3 and grown in complete media with serum and fixed for confocal fluorescence microscopy. These were analyzed for the presence of LC3 dots, representing autophagic vesicles, and quantified. The % of autophagic cells (out of total number of 100 counted) with > 5 foci are shown in the upper right corners of each picture. Below is shown a western blot demonstrating high levels of LC3-II as compared with LC3-I, also consistent with a high level of basal autophagy. B) IHCC cells were cultured under normal growth conditions where the total number of LC3 foci was markedly increased in the presence of CQ (error bars are shown, students t-test is used for comparison, p< 0.001 for all cell lines treated). C & D) Growth of IHCC cell lines is inhibited in the presence of CQ p= <0.001 for samples 335, 518, and 339 and p= <0.01 for samples 449 (two-way ANOVA, error bars are shown for all data points, those not evident on the graphs fall within the area encompassed by the triangle or circle data-point). Consistent with a block in autophagic flux induced by CQ treatment we observed that treated lines had an accumulation of LC3-II and induction of p62 as compared with Day 0 control, with the exception of #518 where p62 expression was absent.