Research Paper Volume 10, Issue 5 pp 951—972

Figure 7. Overexpression of HyLMN lacking CaaX-box does not affect stem cell activity. (A) Construct used for overexpression of the truncated HyLMN (HyLMNΔCaaX) fused to GFP. C-terminal CaaX-motif (red) is deleted. (B) A representative polyp from a line B3 overexpressing HyLMNΔCaaX-GFP fusion protein in all endodermal cells, stained with anti-GFP antibodies (GFP, green) and Phalloidin (Actin, magenta). Inset - empty control polyp. Scale bar: 500 μm. (C) hyLMN mRNA expression levels in two HyLMNΔCaaX-OE lines (B3 and C11) and respective controls, analyzed by qRT-PCR (B3 n=3, C1 n=4; mean±S.D.). Asterisks indicate significant changes in expression levels (Mann-Whitney test); P values: B3 line = 0.001, C11 line = 0.003. (D) Aggregation of the HyLMNΔCaaX protein in the nucleoplasm of transgenic epithelial cells (white arrowhead), as evidenced by immunostaining with anti-HyLMN (red) and anti-GFP (green) antibodies. Chromatin (TO-PRO staining, blue) is displaced to the nuclei periphery. Endogenous HyLMN protein forms nuclear lamina in both, control and transgenic cells (empty arrowhead). Scale bar: 10 μm. (E) BrdU-labeling index of endodermal epithelial cells in the HyLMNΔCaaX-OE (B3 and C11 lines) and control polyps after 3 h exposure to BrdU (B3 n=10, C11 n=13, 494.2±99.65 cells per replicate, mean±S.D.). Inset: Transgenic cells are able to incorporate BrDU in spite of HyLMNΔCaaX aggregation. BrDU-positive (pos.) and BrDU-negative (neg.) nuclei of transgenic cells. (F) BrdU-labeling index of endodermal epithelial cells in the HyLMNΔCaaX-OE (B3 and C11 lines) and control polyps after 72 h exposure to BrdU (B3 n=10, C11 n=10, 470.7±29.9 cells per replicate, mean±S.D.). (G) Growth curves for HyLMNΔCaaX-OE line B3 and control polyps (n=6 replicates, each five polyps on day 0; mean±S.D., linear regression lines with 95% CI corridors and goodness of fit R²). (H) Population doubling time (mean±95% CI) for HyLMNΔCaaX-OE line B3 and control polyps derived from the plot on G.