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. 2016 Jun 15;12(9):1507–1520. doi: 10.1080/15548627.2016.1191857

Figure 5.

Figure 5.

PRKAA activity is required for autophagic flux. (A) Immunoblot analysis of total protein extracts from cells treated with DMSO (0.1%), or CC (10 μM) in the absence or presence of E-64d (E, 10 µg/mL) and pepstatin A (P, 10 µg/mL) for 24 h. (B) HepG2.2.15 or HepAD38 cells were transfected with siScramble or siPRKAA1/2 for 48 h, and then treated with E-64d (E, 10 µg/mL) and pepstatin A (P, 10 µg/mL) for 24 h. The total protein extracts were subjected to immunoblot assay. Relative intensity of LC3B-II was quantified by normalization to ACTB by ImageJ software. Values were means ± SD (n = 3). (C) Immunofluorescence analysis of LC3B puncta in cells that were incubated with DMSO (0.1%), CC (10 µM), or CC in combination with E-64d and pepstatin A (E+P, 10 µg/mL each) for another 24 h. (D) Immunofluorescence analysis of LC3B puncta in cells that were transfected with siScramble or siPRKAA1/2, followed by incubation with E-64d and pepstatin A (E+P, 10 µg/mL each) for another 24 h. The fluorescent signal was visualized using a Leica DM2500 microscope. The number of LC3B puncta (mean ± SD) was quantified by ImageJ software. Values were means ± SD (n = 30). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (in HepG2.2.15); #, p < 0.01; ##, p < 0.01; ###, p < 0.001 (in HepAD38); NS, non-significant. Scale bar: 10 μm.