FIGURE 4

Mixed cultures of COG4^p.G516R and COG4-KO cells with WT cells and conditioned medium supplement testing. (A) Representative confocal images on Day 0 and Day 2 of mixed cultures of COG4^p.G516R and COG4-KO cells with WT cells. Left column, WT cells expressing RFP only. Middle panels, mixed culture of COG4^p.G516R (GFP labeled) and WT (RFP labeled). Right column, COG4^p.G516R cells expressing GFP only. Bar, 200 µm. (B) Left panels, mixed culture of COG4-KO (GFP labeled) and WT (RFP labeled). Right column, COG4-KO cells only. (C) Top panel, PI staining of COG4^p.G516R (GFP labeled) only. Middle panel, PI staining of mixed culture of COG4^p.G516R (GFP labeled) and WT (RFP labeled). Bottom panel, PI staining of mixed culture of WT-RFP and WT-GFP. Bar 50 µm. (D) Statistical analysis of (C) (n = 3). The dashed grey line indicates predicted PI staining cells if no interaction presents between WT and G516R cells. (E) Conditioned medium of WT as a supplement for spheroid formation in ULA plate. Top panel, WT, COG4^p.G516R, and COG4-KO cells only. Bottom panel, WT cells supplemented with conditioned medium of COG4^p.G516R and COG4^p.G516R and COG4-KO cells supplemented with conditioned medium of WT on Day 3. Bar, 200 µm. (F) Statistical analysis of middle panel (E), area measurements. All experiments were performed in duplicate with similar results. Unpaired two-tailed t-test was used. **, p < 0.01; *, p < 0.05.