Fig. 3

AIB5P promotes osteogenic differentiation of BMSCs. a Color assignment of ARS staining for oligopeptide candidate output by our deep learning model. b qPCR analysis of Runx2, Alpl, Osx, and Ocn expression levels in osteogenic-induced mBMSCs and hBMSCs after 1 week of AIB5P (0.25 μg·mL⁻¹, 0.5 μg·mL⁻¹) or BMP2 treatment (0.1 μg·mL⁻¹). n = 3. c Alkaline phosphatase (ALP) staining of osteogenic-induced mouse BMSCs and human BMSCs after one week of AIB5P (0.25 μg·mL⁻¹, 0.5 μg·mL⁻¹) or BMP2 treatment (0.1 μg·mL⁻¹). Bar, 100 μm. d Alizarin red (ARS) staining of osteogenic-induced mouse BMSCs and human BMSCs after 1 week of AIB5P (0.25 μg·mL⁻¹, 0.5 μg·mL⁻¹) or BMP2 treatment (0.1 μg·mL⁻¹). Bar, 100 μm. e Cell lineage tracing of the LepR⁺ positive cells in newly formed cortical bone (representing LepR⁺-derived osteocytes). Calcein stained the bone deposition line when AIB5P administration started, and the double-headed arrow areas indicate red-positive osteocytes embedded within cortical bone. Bar, 100 μm. f The normalized number of deposited LepR⁺-derived osteocytes after 2 weeks of AIB5P treatment at a dose of 100 μg·kg⁻¹ every 3 days. n = 6. The red cells in the calcein-labeled medial cortical bone are osteocytes derived from LepR⁺ cells. Bar, 25 μm. g Runx2, Osx, Alpl, and Ocn expression in LepR⁺ BMSCs sorted by flow cytometry. n = 6