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. 2021 Jan 4;12(1):29. doi: 10.1038/s41419-020-03303-1

Fig. 6. p62 knockdown reverses the inhibitory effect of XPO1 inhibitor KPT-8602 on KSHV lytic replication but has no effect on virus infectivity, and entry and trafficking during primary infection.

Fig. 6

A HUVEC were infected with lentiviruses expressing shRNAs targeting p62 (shp62-1 and shp62-2) or a control shRNA (shCtrl). Cells were selected with puromycin (1 µg/ml) for 48 h and analyzed by Western-blotting to monitor the knockdown efficiencies. β-actin served as the loading control. B HUVEC with p62 knockdown were pretreated with 0.5 µM KPT-8602. Cells infected with KSHV for 48 h in the presence of KPT-8602 (0.5 µM) were fixed and stained for LANA (red). The expression level and staining pattern of LANA protein are shown. Nuclei were stained with DAPI. Images were taken with a confocal microscopy (magnification ×600). C Quantification of LANA-positive cells using images taken as described in (B). D HUVEC with p62 knockdown were pretreated with 0.5 µM KPT-8602 for 1 h, and then infected with KSHV in the presence of KPT-8602 (0.5 µM) for 6 h. Cells were fixed and stained for nuclei with DAPI (blue) and KSHV particles with an antibody to ORF65 (red). Images were taken with a confocal microscopy (magnification ×1000). E The total number of KSHV particles successfully docked at the perinuclear region of each nucleus was quantified and analyzed based on images taken as described in (D). F–I HUVEC with p62 knockdown was pretreated with 0.5 µM KPT-8602 for 1 h and then infected with KSHV in the presence of KPT-8602 (0.5 µM). Cells were collected for RNA extraction at 48 hpi and examined for the expression of KSHV genes including RTA (F), ORF57 (G), ORF65 (H), and LANA (I) by RT-qPCR. J HUVEC with p62 knockdown were pretreated with 0.5 µM KPT-8602 for 1 h and then infected with KSHV for 48 h in the presence of KPT-8602 (0.5 µM). Cell lysate was collected and analyzed for the expression of p62 and KSHV proteins by Western-blotting. β-actin served as the loading control. *, **, and *** indicate P values of < 0.05, < 0.01, and < 0.001, respectively; NS not significant.