Figure 4. Cell proliferation analysis in Wt and Rab23^-/- lambdoid sutural cells and MEF cells.

(A–H) EdU pulsed assay in the Wt and Rab23^-/- lambdoid sutures at E17.5 shows proliferating cells in red color (A–F). Proliferating cells show co-localization with osteoprogenitor and osteoblast marker RUNX2 (green) in the osteogenic front (inset, white arrow) in both Wt and Rab23^-/- lambdoid suture (A, B). Analysis of EdU pulsed cells (C–F) together with nuclear staining (E, F) in the Wt and Rab23^-/- lambdoid suture and subsequent quantification revealed that Rab23^-/- sutures show higher cell proliferation as percentage of EdU-positive cells compare to total cells (G) and percentage of EdU-positive cells only (H) in those sutures are higher compare to Wt samples (n = 4 for each genotype). Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a P-value <0.005 (**). pb: parietal bone, ipb: interparietal bone, ls: lambdoid suture, of: osteogenic front. Scale bar: 100 µm. (I) EdU incorporation in the cultured Wt and Rab23^-/- MEF cells isolated from E13.5 embryos show 8–15% more cell proliferation (DNA duplication) in Rab23^-/- samples compare to corresponding Wt samples (n = 3 for each genotype). Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a p-value <0.05 (*). (J) Represents passaging (P¹ to P³ ) of Wt and Rab23^-/- calvaria derived primary cells in the culture show the total number of Rab23^-/- cells (ml⁻¹) in each passage increases more rapidly than Wt cells, while the cell viability (determined by trypan blue) and cell size were similar in all cell lines. (n = 3 for each genotype). Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a p-value <0.005 (**), p-value <0.001 (***).