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LC3B targets this fluorescence construct to the AV, and differences in pH sensitivity between Green Fluorescent Protein (GFP) (sensitive to acidic pH) and Red Fluorescent Protein RFP track the progression from AV (green + red LC3B = yellow) to AL (red only). Autophagic flux is assessed by the number and color of the vesicles. The number of red LC3B vesicles increased with PI3Ki Bup or Omi (Figure 1C bottom). Altogether, our results indicate that Bup and Omi are associated with a high Cancers 2020, 12, 2371 3 of 14 autophagic flux and autophagy activation in both cell lines. Figure 1. PI3Ki induce autophagy flux in head and neck squamous cell carcinoma (HNSCC) cell lines. (A) PI3Ki increase the number of acidic vacuoles. HNSCC cell lines SCC-9 and FaDu were incubated with vehicle dimethyl sulfoxide (DMSO (CTL)) or 1 nM Omi for one day and stained for 15 min. with acridine orange. Nuclei stain green and acidic organelles stain orange. Images are representative of three independent experiments. Scale bar = 50 μm. (B) Western blot analysis of autophagy markers and AKTser473 phosphorylation. Top: Autophagy flux is indicated by SQSTM1 degradation and MAP1LC3B conversion, and inhibition of phosphatidylinositol 3-kinase (PI3K) signaling pathway is indicated by decreased AKTser473 phosphorylation. Equal loading of proteins is shown with a stain-free blot. SCC-9 and FaDu were incubated for three days with vehicle, 0.5 μM Bup or 1 nM Omi.PDF Image | Dual Inhibition of Autophagy Pathway as a Therapeutic Strategy
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