Figure 3.

Enhanced autophagic flux on combined treatment with morphine and Tat. HPMECs untransfected (A, C, D) or transfected with pBABE-puro mCherry-EGFP-LC3B plasmid (B) were treated with morphine and/or HIV-Tat in the absence or presence of BAF. ((A)and B) For MAP1LC3B immunostaining (A) and visualization of GFP or mCherry LC3B puncta (B) cells were fixed with 4% paraformaldehyde at 24 h post-treatment and viewed using a confocal microscope. ((C)and D) Western blot analysis of MAP1LC3B-II (C) and SQSTM1 (D) at 24 h and 48 h post treatment, respectively. The graphs represent the densitometry analysis of western blots from 2 or 3 independent experiments. Mean +SEM, *P < 0.05, **P < 0.01, ***P < 0.001 vs. control, ##P < 0.01, ###P < 0.001 vs. morphine, $P < 0.05 vs. Tat, @P < 0.05 vs. combined morphine and Tat (M+T), θP < 0.05, θθP < 0.01, θθθP < 0.001 vs BAF. Note: In order to visualize autophagosomes (yellow puncta) more clearly in M+T+BAF, we lowered the green and increased the red fluorescence intensities compared to the images captured for Figure 2.