Research Paper Volume 16, Issue 16 pp 11776—11795

Figure 4. CORO1C analysis. (A) mRNA CORO1C expression (RT-qPCR) of fibroblasts in quiescence or after 24h of TGF-β-1 stimulation (Mean±SEM, n= 6 for young and old cells); (B) one representative western blot of CORO1C and GAPDH expression and (C) mean (±SD) quantification of CORO1C/GAPDH ratio expression in quiescence or after a 24h-TGF-β-1 stimulation of young or old fibroblasts (n=3 per type); (D) mRNA expression quantified by RT-qPCR after transfection with specific siRNA for CORO1C (siCORO1C) or with Scramble siRNA (siScramble), in quiescence or after a 24h-TGF-β-1 stimulation (Mean±SD, n=6 per type); (E) after transfection of young fibroblasts with specific siCORO1C or with siScramble, one representative western blot of CORO1C and GAPDH expression and (F) mean WB quantification of CORO1C/GAPDH ratio expression (Mean±SD, n=3); (G) one representative immunofluorescence staining after transfection with specific siCORO1C or with siScramble of one representative young and one old fibroblast population, either in quiescence or after a 24h-TGF-β-1 stimulation and (H) in the same conditions, mean quantification of immunofluorescence staining from young or old fibroblasts (Mean±SD, n=6); (I) collagen gel contraction (lattice) of old fibroblasts either from control cultures or after transfection with specific siCORO1C or with siScramble during 120h (Mean±SD, n=6); (J) wound closure over 96h as measured by % of cell confluency for analysis of migration of old controls fibroblasts (CT) or after transfection with siCORO1C or siScramble siRNA (Mean ±SD, n=6). p-value *<0.05, **<0.01, ***<0.001. (J) Schematic representation of the actin dynamic polymerization between CFL1, CORO1C and ARP2/3, regulators, adapted from Mol Biol Cell. 2010; 21:3529-39. Hypothetically, the low level of ARP2/3 in old cells might favor a potent deficiency in actin polymerization with as consequences, alteration of cell motility and capacity to interact with the ECM components. This could be reinforced by the low levels of CFL1. Moreover, the increase of CORO1C in old fibroblasts should induce a higher strength of the actin filaments, leading to cell rigidification with no renewal in the actin complex as a consequence of loss in cell contractility and effective migration.