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Cancers 2020, 12, 2371 7 of 14 Cancers 2020, 12, x 7 of 14 Figure 3. CQ inhibits PI3Ki-induced autophagy in HNSCC cell lines. (A) SCC-9 cells were incubated Figure 3. CQ inhibits PI3Ki-induced autophagy in HNSCC cell lines. (A) SCC-9 cells were incubated with vehicle DMSO (CTL), 1 nM Omi, 10 μM CQ or both inhibitors for one day and stained for 15 min. with vehicle DMSO (CTL), 1 nM Omi, 10 μM CQ or both inhibitors for one day and stained for 15 with acridine orange. Nuclei stain green and acidic organelles stain orange. Images are representative min. with acridine orange. Nuclei stain green and acidic organelles stain orange. Images are of three independent experiments. Scale bar = 100 μm. (B) Top: Western blot of autophagy markers representative of three independent experiments. Scale bar = 100 μm. (B) Top: Western blot of SQSTM1 and MAP1LC3B, using alpha-tubuline as a loading control. FaDu cells were incubated for autophagy markers SQSTM1 and MAP1LC3B, using alpha-tubuline as a loading control. FaDu cells 3 days with vehicle, 0.5 μM Bup or 1 nM Omi and ± 10 μM CQ. Bottom: Densitometric analysis of were incubated for 3 days with vehicle, 0.5 μM Bup or 1 nM Omi and ± 10 μM CQ. Bottom: SQSTM1 protein levels relative to TUBA and of MAP1LC3B-II protein levels relative to MAP1LC3B-I. Densitometric analysis of SQSTM1 protein levels relative to TUBA and of MAP1LC3B-II protein levels relative to MAP1LC3B-I. Data are presented as mean ± SD, n = 3 independent experiments and analyzed by ANOVA test. * p < 0.05 for normalized SQSTM1 Bup vs CTL and for relative MAP1LC3B Bup + CQ vs CTL and Omi + CQ vs CTL. (C) Autophagy flux analysis with tandem sensor RFP-GFP- LC3B. Top: GFP is sensitive to acidic pH of AL. Red = AL, yellow = AV. Bottom: Confocal images andPDF Image | Dual Inhibition of Autophagy Pathway as a Therapeutic Strategy
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