Fig 1: ZG inhibits the mTORC1/S6K1 signaling pathway to promote osteoblast differentiation in vitro. (A) Q-PCR analysis for mRNA expression of osteogenic differentiation marker genes including osteocalcin (OCN), collagen 1, RUNX2, and osteopontin (OPN) in preosteoblast treated with vehicle (ctr) and ZG at 90 or 180 μg/ml concentration, respectively. (B) The representative images of ALP staining for preosteoblast at day 7 after treatment with vehicle (Ctrl) and ZG at 90 or 180 μg/ml concentration, respectively. (C) Quantification for the percentage of ALP staining area relative to total area in indicated groups. (D) Western blot analysis for protein levels of osteoblastic differentiation markers (collagen1, RUNX2, and β-actin) and mTORC1/S6K1 signaling pathway markers (p-70S6K1, p-mTOR, and mTOR) in preosteoblast treated with vehicle (ctr) and ZG at 90 or 180 μg/ml concentration, respectively. (E) Q-PCR analysis for mRNA expression of osteogenic differentiation marker genes including OCN, collagen 1, RUNX2, and OPN in preosteoblast treated with ZG in combination with or without MHY1485. (F) The representative images of ALP staining for preosteoblast treated with ZG in combination with or without MHY1485. (G) The representative images of alizarin red staining for preosteoblast treated with ZG in combination with or without MHY1485. (H) Quantification for the percentage of ALP staining area relative to total area in indicated groups. (I) Quantification for the percentage of Alizarin red staining area relative to total area in indicated groups. (J) Western blot analysis for protein levels of osteoblastic differentiation markers (collagen1, RUNX2, and β-catenin) and mTORC1/S6K1 signaling pathway markers (p-70S6K1, p-mTOR and mTOR) in preosteoblast treated with ZG in combination with or without MHY1485. Notes: Scale bar = 100 μm. Data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s HSD post hoc test. Each experiment was performed in triplicate and repeated three times independently.