Kaposi’s sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies
Fig 2
Kaposin B requires Atg5 and Atg14 autophagy to disassemble PBs.
A & B: HUVECs were sequentially transduced: first with recombinant lentiviruses expressing either shRNAs targeting Atg5 (A) or Atg14 (B) (shAtg5, shAtg14) or a non-targeting control (NS) and selected with puromycin (1 μg/mL), and second with either KapB or an empty vector control and selected with blasticidin (5 μg/mL). Coverslips were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and immunostained for the PB-resident protein Hedls (white) and nuclei (DAPI). 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 = 4 (A), n = 3 (B), **** = P<0.0001. 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 BafA1 for 30 min, fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and immunostained for the PB-resident protein Hedls (white) and nuclei (DAPI). 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.