Fig. 3.

Citrate metabonegenic regulation and concerted action between citrate and PSer. (A) Protein synthesis of hMSCs with 200 µM citrate, Chx, or Torin 1 supplementation in GM for 1 and 4 d. (B) Western blot of SLC13a5 expression in hMSCs during osteogenic differentiation. (C) ALP production of hMSCs after 14 d of differentiation in OG medium supplemented with citrate, the SLC13a5 inhibitor PF06761281, or both. In A–C, n = 3–5 biological replicates per group. (D) Intracellular ATP determination. (E) OCR study. (F) Extracellular acidification rate (ECAR) study. (G) Lactate production of undifferentiated hMSCs with 1 d of treatment with citrate and SLC13a5 in GM. In D–G, n ≥ 8 biological replicates per group. (H and I) Intracellular ATP determination of hMSCs (H) and differentiating hMSCs (I) supplemented with citrate, PSer, or both in GM or OG medium, respectively. In H and I, n = 6–8 biological replicates per group. (J) ALP production of differentiating hMSCs after 7, 14, and 21 d of differentiation in OG medium supplemented with citrate or with citrate and PSer. n = 4 biological replicates per group; all data are presented as mean ± SD; *P < 0.05. (K) Immunofluorescent staining of OPN (red) with DAPI nuclear counterstain (blue) expression after 21 d of differentiation in OG medium supplemented with citrate, PSer, or both. The plus (+) and minus (−) symbols denote the presence and absence, respectively, of citrate and other specific chemicals in GM/OG medium. (Scale bars: 100 µm.)