Fig. 3

TP deficiency suppresses adipocyte but not osteoblast differentiation of ASC. Data were obtained in ASC, ASC with a CRISPR-Cas9-mediated TP-knockout (KO), and ASC transduced with a Cas9/scramble gRNA plasmid corresponding to control (CTL) cells. A Timeline representation of the ASC differentiation process using a hormonal cocktail. IBMX: 3-isobutyl-1-methylxanthine; D0: day 0; D10: day 10; D20: day 20. B Thymidine phosphorylase (TP) expression in ASC during adipocyte differentiation and validation of TP KO in ASC at D0 and at D20. Numbers on the left correspond to molecular weight markers (kDa). Western blot images are representative of three independent experiments. C Adipocyte differentiation assessed by Oil Red-O lipid staining. ASC pre-adipocytes were studied during adipocyte differentiation for 20 days. First and second lines: representative pictures of cell dishes by optical microscopy. Images are representative of five independent experiments. Third and fourth lines: representative images of fluorescence microscopy after staining of intracellular lipids (Oil Red-O, red) and nuclei (DAPI, blue). Images are representative of five independent experiments. D Quantification of Oil Red-O fluorescence normalized to DNA content (DAPI). Results are expressed as means ± SEM of five independent experiments. E Evolution of intracellular triglyceride content during in vitro adipocyte differentiation. Triglycerides were measured at D0, D10, and D20 in ASC, CTL, and TP KO cells. Measurements are representative of three independent experiments. F Timeline representation of the ASC differentiation process in osteoblasts using a hormonal cocktail. G Pictures of dishes stained by Alizarin-red S. Images are representative of three independent experiments. The ratio of Alizarin/protein was calculated after cell lysis and protein extraction. ****p < 0.0001. p-values were determined by analysis of variance (ANOVA) with Kruskal-Wallis post hoc multiple comparison test; n.s., non-significant