Kaposi’s sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies
Fig 6
KapB-mediated PB disassembly and ARE-mRNA reporter expression require the selective autophagy receptor NDP52.
A: HUVECs were sequentially transduced, first with recombinant lentivirus expressing KapB or an empty vector control and selected with blasticidin (5 μg/mL), and second with recombinant lentivirus expressing shRNAs targeting NDP52 or a non-targeting control (NS) and selected with puromycin (1 μg/mL). Coverslips were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 and immunostained for Hedls (PBs; white), DAPI (nuclei, blue). 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<0.05. B: HUVECs were transduced with recombinant lentivirus expressing an shRNA targeting NDP52 and selected with puromycin (1 μg/mL). Cells were treated with Torin (250 nM) or a DMSO control for 2 h prior to fixation in 4% paraformaldehyde and permeabilization in 0.1% Triton X-100. Samples were immunostained for Hedls (PBs; white), DAPI (nuclei, blue). 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<0.0001. C: HUVECs were transduced with recombinant lentiviruses expressing either an shRNA targeting NDP52 or a non-targeting control (NS) and selected with puromycin (1 μg/mL). Cells were treated with DMSO or Torin (250 nM) for 4 h prior to harvest in 2X Laemmli buffer. Samples were resolved by SDS-PAGE and immunoblot was performed for Dcp1a, Pat1b or DDX6. Samples were quantified by normalizing Dcp1a or Pat1b protein levels to the total protein in each lane using Image Lab (BioRad) and then to the NS DMSO control. Results were plotted in GraphPad and a 2-way ANOVA was performed, ±SEM; n = 3 (Dcp1a) n = 4 (Pat1b), * = P<0.05. D: HeLa Tet-Off cells were transduced with recombinant lentivirus expressing shRNAs targeting NDP52, OPTN, p62 or a NS control and selected with puromycin (1 μg/mL) then cells were co-transfected, treated with Dox and luciferase activity was recorded and analyzed as in Fig 4. Data were plotted in GraphPad as the mean fold change in the relative luciferase activity of each condition compared to vector NS or KapB NS; n = 4. An unpaired t-test was performed; * = P<0.05 ** = P<0.01. E: HUVECs were sequentially transduced first with recombinant lentivirus expressing shNDP52 targeting the 3’-UTR of NDP52 or a NS control and selected with blasticidin (5 μg/mL), and second, with recombinant lentivirus expressing KapB and either mCherry control (mCh) or RFP-NDP52. Coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and immunostained with Hedls (PBs, green). Scale bar = 20 μm. F: Samples from E were quantified; 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<0.0001.