Kaposin B increases autophagic flux.

Primary human umbilical vein endothelial cells (HUVECs) were transduced with recombinant lentiviruses expressing either Kaposin B (KapB) or an empty vector control and selected with blasticidin (5 μg/mL). A: Cells were treated with Bafilomycin A1 (BafA1, 10 nM) or a vehicle control (DMSO) for the indicated times prior to harvest in 2X Laemmli buffer. Protein lysates were resolved by SDS-PAGE and immunoblot was performed for p62 and LC3. Samples were quantified using Image Lab (BioRad) software and then normalized, first to total protein and then to their respective starting time points (0 h). Results were plotted in GraphPad and a linear regression statistical test was performed, ±SEM; n = 3, * = P<0.05, *** = P<0.001. B: Cells were treated with BafA1 for 30 min prior to fixation in methanol. Immunofluorescence was performed for LC3 (white) and DAPI (nuclei, blue). Scale bar = 20 μm. Total LC3 area per field was quantified by identifying LC3-positive puncta using CellProfiler and normalizing to the number of nuclei and the vector DMSO control. Results were plotted in GraphPad, a 2-way ANOVA was performed with a Šidák’s multiple comparison test, matched data from each replicate was paired for comparison, ±SEM; n = 3, * = P<0.05. C: HUVECs were transduced with recombinant lentiviruses expressing either KapB or an empty vector control and selected with blasticidin (5 μg/mL). Cells were treated with sodium arsenite (0.25 mM) or a vehicle control for 30 min prior to fixation in 4% paraformaldehyde and permeabilization in 0.1% Triton X-100. Scale bar = 20 μm. Hedls puncta were quantified using CellProfiler and presented as number of Hedls puncta per cell, all cells counted are displayed. Results were plotted in GraphPad and a 2-way ANOVA was performed on the main column effects with a Tukey’s multiple comparison test, bar represents the mean; n = 3, ** = P<0.01, **** = P<0.0001.