Fig 1: SCRG1 regulated chondrogenesis of UCMSCs through Wnt5a signaling pathway. (a) Regulating pluripotency of stem cell signaling pathway was the most significantly enriched KEGG pathway based on GSEA, wnt5a was negatively correlated with this pathway after SCRG1 overexpression. (b, c) Western blot analysis showed that Wnt5a decreased after shSCRG1 transfection and was restored after administration of rhSCRG1 (450 ng/mL); protein levels of β-catenin exhibited the opposite trend to Wnt5a. (d, e) Protein levels of COL2A1 and ACAN were detected in UCMSCs by IHC. The results showed that the protein levels of COL2A1 and ACAN were reduced following SCRG1 knockdown and restored after treated with rhWnt5a (300 ng/mL) and rhSCRG1 (450 ng/mL) protein at 24 h. Scale bar = 100 μm. Experiments were repeated three times, ∗P < 0.05 (t-test).
Fig 2: SCRG1 knockdown inhibited chondrogenic differentiation potential of UCMSCs. (a–c) mRNA and protein expression levels of SCRG1 were decreased after transfection with SCRG1 shRNA, detected by qRT-PCR and IHC. Scale bar = 100 μm. (d) SOX9, COL2A1, and ACAN mRNA levels decreased following SCRG1 knockdown. (e) Alcian Blue staining at day 21. Results showed that glycosaminoglycan synthesis decreased significantly after SCRG1 knockdown. (f, g) Protein levels of COL2A1 and ACAN detected by IHC at day 21 decreased after SCRG1 knockdown. Scale bar = 200 μm. Experiments were repeated three times; ∗P < 0.05 (t-test).
Fig 3: SCRG1 was upregulated during chondrogenic differentiation. (a) Alcian Blue staining of UCMSCs chondrogenic differentiation at day 0 and day 21; results showed accumulation of glycosaminoglycans at day 21 after chondrogenic differentiation, indicating chondrogenesis of UCMSCs. Scale bar = 100 μm. (b) mRNA expression levels of chondrogenic genes (SOX9, COL2A1, and ACAN) were upregulated during induction. (c) mRNA expression level of SCRG1 was upregulated during induction. (d, e) Protein level of SCRG1 was upregulated after 21 days chondrogenic differentiation, as detected by IHC. Scale bar = 100 μm. Experiments were repeated three times; ∗P < 0.05 (t-test).
Fig 4: SCRG1 overexpression promoted chondrogenic differentiation of UCMSCs. (a–c) mRNA and protein levels of SCRG1 were detected by qRT-PCR and IHC. Results showed that mRNA and protein levels of SCRG1 increased after overexpression of SCRG1. Scale bar = 100 μm. (d) SOX9, COL2A1, and ACAN mRNA levels increased after overexpression of SCRG1. (e) Alcian Blue staining showed an increase in glycosaminoglycans at day 21 after overexpression of SCRG1. Scale bar = 200 μm. (f, g) Protein levels of COL2A1 and ACAN detected by IHC at day 21 increased after SCRG1 knockdown. Scale bar = 200 μm. Experiments were repeated three times; ∗P < 0.05 (t-test).
Fig 5: UCMSCOE-SCRG1 promoted cartilage defects repair in vivo. (a) Schematic of cartilage defect model and UCMSC administration. (b) Photographs of cartilage defects at 12 weeks after UCMSC administration. Obvious defects were observed in the blank group, whereas the surface was smooth and regenerated tissue was tightly connected to surrounding cartilage in the OE-SCRG1 group. (c) H&E, Alcian Blue, and Safranin O/Fast Green staining at 12 weeks after surgery. Results showed that defects in the blank group contained necrotic and fibrous tissue, while the OE-SCRG1 group showed considerable chondrocyte regeneration and tissue morphologies similar to normal surrounding cartilage. (d) ICRS macroscopic scores at 12 weeks after surgery. Scores in the OE-SCRG1group were significantly higher than those in the OE-nc group (P < 0.05). (e) Modified ICRS histological scores at 12 weeks after surgery. Scores were significantly higher in the OE-SCRG1 group than in the OE-nc group (P < 0.05). Scale bar = 200 μm. N = 6; ∗P < 0.05 (t-test).
Supplier Page from Abcam for Recombinant Human SCRG1 protein (GST tag N-Terminus)