RIPK1 Coordinates Bone Marrow Mesenchymal Stem Cell Survival by Maintaining Mitochondrial Homeostasis via p53

Figure 1

Biological function of RIPK1 on proliferation, differentiation, and migration of bone marrow MSCs. (a) The bone marrow MSCs were transfected with siControl or siRIPK1 for 24 h; then, knockdown efficiency of RIPK1 was assessed by western blotting. Representative western blots of the expression of RIPK1. Histogram analysis showing the relative protein levels of RIPK1. Data are presented as the from three independent experiments (, vs. siControl; ). siRIPK1(#1) showed the highest efficiency among three siRIPK1. (b) Morphologic changes in bone marrow MSCs after 72 h of siRNA transfection. In the siRIPK1 group, more cells detached from culture plates with a morphology transition ( μm). (c) Proliferation of bone marrow MSCs was estimated by CCK-8 assay at 24, 48, and 72 h after transfection with siRNA. Knockdown of RIPK1 resulted in inhibition of cell proliferation, compared with the control group. Values are expressed as the from three independent experiments ( vs. siControl; ). (d) Quantification of EDU-positive cells in two groups, showing that knockdown of RIPK1 led to decrease of EDU incorporation. Values are expressed as the from three independent experiments (, vs. siControl; ). (e) Representative images of EDU incorporation in bone marrow MSCs, and nuclei were stained with Hoechst ( μm). (f, g) Effect of RIPK1 on osteogenic differentiation in bone marrow MSCs. The Alizarin Red staining of bone marrow MSCs after incubation with osteogenic medium for 14 d ( μm) (f). Representative western blots of the expression of Runx2. Histogram analysis showing the relative protein levels of Runx2. Data are presented as the from three independent experiments (, vs. siControl; ) (PM: proliferative medium; OM: osteogenic medium) (g). (h, i) Effect of RIPK1 on adipogenic differentiation in bone marrow MSCs. The Oil Red O staining of bone marrow MSCs after incubation with adipogenic medium for 14 d ( μm) (h). Representative western blots of the expression of PPARγ. Quantitative data showing the relative protein levels of PPARγ. Data are presented as the from three independent experiments (, vs. siControl; ) (PM: proliferative medium; AM: adipogenic medium) (i). (j, k) Effect of RIPK1 on chondrogenic differentiation in bone marrow MSCs. The Alcian Blue staining of bone marrow MSCs after incubation with chondrogenic medium for 14 d ( μm) (j). Representative western blots of the expression of Sox9. Quantitative data showing the relative protein levels of Sox9. Data are presented as the from three independent experiments (, vs. siControl; ) (PM: proliferative medium; CM: chondrogenic medium) (k). (l, m) Effect of RIPK1 on migration of bone marrow MSCs. Transwell invasion assay (l) and quantitative data (m) showed significant decrease of invasive cells in siRIPK1 group. Values are expressed as the from three independent experiments (, vs. siControl; ).

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