Fig. 3.

K1 promotes cell growth in 3D spheroid culture system. (A) Representative pictures and size quantification of 3D spheroids of mock-, K1 WT-, or mutant-expressing TIME and HFF cells cocultured in ultralow attachment condition. (Scale bars: 200 μm.) *P < 0.05, by one-way ANOVA. (B) A 3D confocal microscopy image of R-TIME-cultured hydrogel scaffold. Bottom shows the average of total fluorescence intensity at day 9 normalized to day 2 (seeding amount). *P < 0.05 and **P < 0.01, by one-way ANOVA. The 3D reconstruction and analysis of fluorescence intensity from multiple z-series images were performed using ImageJ. (C) Representative pictures and size quantification of 3D spheroids of mock-, K1 WT-, or mutant-expressing MCF10A cells. (D) MDA-MB-231 cells cultured in ultralow attachment condition. (Scale bars: 200 μm.) *P < 0.05; **P < 0.01; ****P = 0.0001, by one-way ANOVA. (E) ATP production of MCF10A cells expressing mock, K1 WT, or mutant. *P < 0.05, by one-way ANOVA. (F) Representative pictures (Upper) and size quantification (Lower) of 3D spheroids of uninfected, KSHV WT-infected, or KSHV K1 ΔC-infected or KSHV K1 TYF-infected TIME cells cultured in Matrigel for 12 d. (Scale bars: 100 μm.) The number of biological replicates for each experiment was n ≥ 3. **P = 0.0001, by one-way ANOVA. (G) Representative images of uninfected, KSHV WT-infected, or KSHV K1 ΔC-infected or KSHV K1 TYF-infected MCF10A cells cultured on Matrigel for 21 d. (Upper) Acini were stained with the nuclei counterstained with Hoechst 33342 (blue) and Ki67 antibody (red). GFP is from the BAC16 KSHV genome. (Scale bars: 100 μm.) (Lower) Quantification of Ki67 positive cells in acini. The number of biological replicates for each experiment was n ≥ 3. **P < 0.01 and ****P = 0.0001; ns, not significant, by one-way ANOVA.