FIG 3.

iNOS is essential for KSHV-induced cell proliferation and cellular transformation. (A) Examination of eNOS, iNOS, or nNOS expression by RT-qPCR in MM and KMM cells. (B and C) Analysis of iNOS expression in MM and KMM cells following iNOS knockdown by RT-qPCR (B) and Western blotting (C). (D and E) iNOS knockdown inhibits cell proliferation and cellular transformation of KMM cells. Cell proliferation (D) and colony formation in soft agar (E) were determined following depletion of iNOS expression with 3 shRNAs (sh1, sh2, or sh3) or a scrambled shRNA (ctl). Representative pictures at a ×40 magnification from soft agar assays are shown (E, left). Colonies with diameters of >50 μm were counted, and the results were graphed (E, right). (F and G) iNOS knockdown induces cell cycle arrest and apoptosis. (F) The cell cycle was analyzed by flow cytometry 48 h following transduction with iNOS shRNAs. (G) Apoptotic cells were detected by annexin V staining 72 h following transduction with ASS1 shRNAs. *, **, and *** represent P values of <0.05, <0.01, and <0.001, respectively, while NS indicates “not significant.”