Fig 2: Downregulation of circSKIL suppresses osteogenic differentiation and inhibits the JNK/STAT3 pathway in OPLL cells. OPLL cells were transfected with either a circSKIL-targeting siRNA or a negative control siRNA (si-NC). (A) mRNA expression levels of RUNX2, ALP, COL I, OCN, and OPN (n=3). (B) Western blot analysis of the protein expression levels of RUNX2, ALP, COL1A1, OCN, and OPN (n=3). (C) Western blot analysis of the protein levels of p-JNK, JNK, p-STAT3, and STAT3 (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. si-NC. circ, circular RNA; OPLL, ossification of the posterior longitudinal ligament; si, small interfering; NC, negative control; RUNX2, runt-related transcription factor 2; ALP, alkaline phosphatase; COL I, collagen I; OCN, osteocalcin; OPN, osteopontin; COL1A1, collagen type I a 1 chain; p-, phosphorylated.

Fig 3: GLS1-mediated osteogenesis of VICs via histone.(A) Representative immunoblots and densitometric data showed that GLS1 inhibitor BPTES markedly reduced Yoda1-induced osteogenic markers in VICs of normal aortic valves; n = 5. (B) Alizarin red staining of mineralization nodules showed that BPTES treatment significantly reduced Yoda1-induced mineralization nodule formation of normal VICs; n = 5; scale bar, 500 µm. (C) ALP staining showed that BPTES treatment significantly reduced Yoda1-induced ALP activity of normal VICs; n = 5; scale bar, 500 µm. (D) Acetyl-CoA concentration in VICs treated with Yoda1 and BPTES; n = 5. (E) Representative immunoblots and densitometric data showed that GLS1 inhibitor BPTES markedly reduced Yoda1-induced H3K27ac expression; n = 5. (F) ChIP-PCR showed histone acetylation of RUNX2 promoter regions; n = 3. (G) Immunoblot of H3K27ac levels in control and BPTES-treated aortic VICs, with or without acetate supplementation; n = 5. (H) Representative immunoblots and densitometric data showed that GLS1 knockdown markedly reduced Yoda1-induced osteogenic markers; n = 5. (I) Immunofluorescence staining of RUNX2 in VICs; n = 5; scale bar, 50 µm. (J) Representative immunoblots and densitometric data showed that, under Yoda1 treatment, aortic VICs with GLS1 knockdown showed reduced expression of H3K27ac compared with scrambled siRNA–transfected VICs of normal aortic valves; n = 5. (K) ChIP-PCR showed histone acetylation of RUNX2 promoter regions; n = 3. (L) Alizarin red staining of mineralization nodules showed that GLS1 knockdown significantly reduced Yoda1-induced mineralization nodule formation of VICs of normal aortic valves; n = 5; scale bar, 500 µm. (M) ALP staining showed that GLS1 knockdown significantly reduced Yoda1-induced ALP activity of normal VICs; n = 5; scale bar, 500 µm. Data are means ± SEM. ***P < 0.001; ****P < 0.0001; ns, P > 0.05.

Fig 4: Piezo1 activated YAP to control osteogenic differentiation of VICs.(A) Representative immunoblots and densitometric data showed YAP and its phosphorylation expression in VICs from normal and CAVD aortic valves under Yoda1 stimulation; n = 5. (B) Representative immunoblots and densitometric data showed that verteporfin markedly reduced Yoda1-induced osteogenic responses; n = 5. (C) Alizarin red staining of mineralization nodules showed that verteporfin treatment significantly reduced Yoda1-induced mineralization nodule formation of VICs; n = 5; scale bar, 500 µm. (D) ALP staining showed that verteporfin treatment significantly reduced Yoda1-induced ALP activity of VICs; n = 5; scale bar, 500 µm. (E) Representative immunoblots and densitometric data showed that knockdown of YAP reduced Yoda1-induced osteogenic responses in normal VICs; n = 5. (F) Immunofluorescence staining of RUNX2 in VICs; n = 5; scale bar, 50 µm. (G) Alizarin red staining of mineralization nodules showed that knockdown of YAP significantly reduced Yoda1-induced mineralization nodule formation of VICs; n = 5; scale bar, 500 µm. (H) ALP staining showed that knockdown of YAP significantly reduced Yoda1-induced ALP activity of VICs; n = 5; scale bar, 500 µm. Data are means ± SEM. ****P < 0.0001; ns, P > 0.05.

Fig 5: Activation of Piezo1 by Yoda1 induced osteogenic differentiation of VICs.(A) Human aortic VICs from normal and CAVD aortic valves were treated with Yoda1 (0, 2.5, 5, and 10 µM) for 24 hours. Data showed ALP, RUNX2, and BMP2 protein levels; n = 5. (B) VICs from normal and CAVD aortic valves were pretreated with the Piezo1 inhibitor Dooku1. Representative immunoblots and densitometric data showed the protein level of ALP, RUNX2, and BMP2; n = 5. (C) Alizarin red staining of mineralization nodules showed that Dooku1 treatment significantly reduced Yoda1-induced mineralization nodule formation of normal VICs; n = 5; scale bar, 500 µm. (D) ALP staining showed that Dooku1 treatment significantly reduced Yoda1-induced ALP activity of VICs; n = 5; scale bar, 500 µm. (E) Representative immunoblots and densitometric data showed that knockdown of Piezo1 in normal VICs reduced Yoda1-induced osteogenic responses; n = 5. (F) Immunofluorescence staining of RUNX2 in VICs; n = 5; scale bar, 50 µm. (G) Alizarin red staining of mineralization nodules showed that knockdown of Piezo1 significantly reduced Yoda1-induced mineralization nodule formation of VICs; n = 5; scale bar, 500 µm. (H) ALP staining showed that knockdown of Piezo1 significantly reduced Yoda1-induced ALP activity of VICs; n = 5; scale bar, 500 µm. Data are means ± SEM. ****P < 0.0001; ns, P > 0.05.