Fig. 4

Deprivation of glutamine inhibits odontogenic differentiation of human dental pulp stem cells (hDPSCs) and matrix mineralization. (A) Alizarin red S (ARS) staining of hDPSCs cultured in odontogenic medium (DM) with or without glutamine for 14 and 21 days. (B and E) Alkaline phosphatase (ALP) staining and quantifying ALP activity of hDPSCs (n = 6) after 7 days of differentiation induction in DM with or without glutamine. (C) Protein expression levels of DMP1, RUNX2, DSPP, COL1A1, and ACTIN in hDPSCs after 7 days of induction. Full-length blots/gels are presented in Supplementary Figure S8. (D) Relative mRNA expression of genes related with odontoblastic differentiation, including ALP, DMP1, OCN, OSX, COL1A1, COL1A2, RUNX2, DSPP, and BSP, in hDPSCs after 7 days of differentiation induction in DM with or without glutamine (n = 3). (F) Relative citrate content in the cytoplasm determined by a citrate detection kit after 7 days of differentiation induction (n = 5). (G) Relative citrate content in the extracellular mineralized matrix of hDPSCs after culturing in DM with or without glutamine for 21 days (determined by LC-MS/MS) (n = 3). (H) Relative Ca content in the extracellular mineralized matrix of hDPSCs after culturing in DM with or without glutamine for 21 days (determined by ICP-MS) (n = 3). Data were analyzed using Student’s t-test (C, D, F, and H) and Mann–Whitney U test (G). * P < 0.05, ** P < 0.01, and *** P < 0.001