HCQ effects on cell proliferation and apoptosis.

<p><b>(A)</b> Three different human RCC derived cell lines were seeded on 96-well plates at a concentration of 1x10⁴ cells/well in triplicate and were treated with HCQ from 0 to 125 μM. After 48 hours, cell growth was measured using an MTT assay. Error bars show standard deviation, and the letter at top indicates statistically significant differences between columns with different letters (P<0.05, ANOVA with Tukey post-hoc test). <b>(B)</b> Indicated cell lines were incubated with either nothing, RAD001 (10 μM), HCQ (75 μM) or combination of RAD001 and HCQ for two days. Growth was measured as in (A). <b>(C)</b> RCC cells were cultured in the absence (C) or presence of either 10 μM RAD001 (R) or 75 μM HCQ (H) for two days, and Western analyses was used to measure the levels of autophagy (p62, LC3) and apoptosis (PARP) markers. The different forms of LC3 and PARP are shown. The relative intensities of each protein are assigned as Arbitrary Units obtained by calculating the ratio of test sample divided by a control. <b>(D)</b> Indicated cell lines were treated with either nothing or 75 μM HCQ for 48 hours and then assessed by staining and cell sorting for the apoptotic markers Annexin V-PE and 7-AAD. Bars represent the percentage of early apoptotic cells in each sample.