Fig. 6.

HMA induces autophagy. (A) ARPE 19 cells were untreated or treated with rapamycin for 2 hrs, with HMA or etoposide for 24 hrs and then probed with anti-LC3 antibody. Scale bar 25 μm. (B) ARPE-19 cells were treated as before and analysed by Western blot using anti-beclin 1, ATG-7, AGT5-12 and LC3 antibodies. γ tubulin was used as a loading control (left panel). On middle and right panels, cells were treated as before and analysed using anti-ERK and phospho-ERK (*) antibodies (middle panel) or with anti JNK1 or phosphorylated JNK1 (*) antibodies (right panel). Under the western images the quantification of the bands is reported showing a significant increase of Beclin 1 (Means are different from each other as calculated from a one-way anova test (P < 0.0001). ATG7 did not change (P = 0.165, one-way anova). ATG 12 is increased in treated samples (Means are different from each other as calculated from a-one way anova test (P < 0.05) The lipidated form of LC3 was also significantly increased as compared with the control (P < 0.0001) for all treatments. The same holds true for the phosphorylated form of ERK (P < 0.0001, except for etoposide, anova test). JNK was significantly increased at 120 μM HMA and in the presence of rapamycin (anova test P < 0.0001). (C) RT real-time PCR experiments were performed on untreated and HMA or rapamycin-treated ARPE-19 cells. HMA was used at 40 μM concentration for the indicated times, whereas rapamycin was used at 1 μM concentration for 4 hrs. GAPDH expression level was used as internal standard. Results are expressed as the mean ± SD of three independent experiments. Relative expression levels for each gene were calculated using the ΔΔCt method. Only the 40 μM HMA results are significantly different from the others P < 0.05.