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. 2021 Sep 27;9:716610. doi: 10.3389/fcell.2021.716610

FIGURE 2.

FIGURE 2

Sonic hedgehog (SHH) promoted osteoarthritis mesenchymal stromal cell (OA-MSC) proliferation and gene expression of the markers for chondrogenesis, hypertrophy, catabolism, senescence, and senescence-associated secretory phenotype (SASP). (A) Micrographs of primary OA-MSCs and their treatment with human SHH recombinant protein for 24 and 48 h. Images are representative of cell culture samples in triplicates. Scale bar = 125 μm. (B) Cell number change of OA-MSC in response to human recombinant SHH incubation for 24 and 48 h. n ≥ 4 individual biological samples. (C) Cell proliferation rate of OA-MSC in response to human recombinant SHH incubation for 24 and 48 h. Cell proliferation rate was quantified by the CCK-8 proliferation assay. n = 5 biological replicates. Real-time RT-PCR analysis of transcripts encoding markers of chondrogenesis and hypertrophy (D), cell senescence, and SASP (E), hedgehog (HH) signaling (F) in primary OA-MSC. 18S RNA was used as a reference gene for RT-PCR amplification. p-values were indicated n = 3. Real-time RT-PCR analysis of transcripts encoding markers of chondrogenesis and hypertrophy (G), cell senescence, and SASP (H), in OA-MSC cell line. 18S RNA was used as a reference gene for reverse transcriptase-polymerase chain reaction (RT-PCR) amplification. p-values were indicated (n = 3). (I) Western blot analysis of protein levels of SHH, SOX9, RUNX2, and MMP13 in OA-MSC cell line in response to human recombinant SHH treatment. β-actin was used as a protein loading control. Data shown are representative of three biological samples (n = 3).