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. 2022 Dec 1;132(1):52–71. doi: 10.1161/CIRCRESAHA.122.321538

Figure 7.

Figure 7.

Txnip (thioredoxin-interacting protein) knockdown in vascular smooth muscle cells (VSMC) accelerates osteodifferentiation and augments BMP (bone morphogenetic protein) signaling. A, Schematic illustration of the experiment. The pooled VSMCs from 2 to 3 wild type (WT) mice constituted 1 biological replicate. Cultured VSMCs were treated with negative control (NC) siRNA or Txnip siRNA (si-Txnip) and were subjected to osteodifferentiation by osteogenic cocktail (β-glycerophosphate, L-ascorbic acid, and H2O2) incubation. B, qRT-PCR results showing the changes in gene expressions of Ly6a and Lum (upregulated in the modulated VSMC cluster), and the Myh11 (contractile gene; VSMC marker) along the culture passages. n=4. C, Western blot confirming knockdown of TXNIP by si-Txnip. TXNIP expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for quantification. n=4. D, Alizarin Red staining results of osteodifferentiation (end point). The staining was quantified by cetylpyridinium chloride extraction. n=5. E, qRT-PCR results showing the expressions of Sp7, Bglap, Ibsp (which are the osteochondrogenic marker genes), and Myh11 (which is a contractile gene; VSMC marker) in the NC and si-Txnip treated VSMCs with or without osteodifferentiation. n=4. F, Feature plots showing Bmp2 and Bmp4 expression in WT and Txnip knockout mice (Txnip KO) single-cell RNA-sequencing (scRNA-seq) data. G, qRT-PCR results showing the expressions of Bmp2 and Bmp4 in the NC and si-Txnip treated VSMCs with or without osteodifferentiation. n=4. H, qRT-PCR results showing the mRNA expressions of inhibitor of DNA binding (Id) proteins, which are transcribed by BMP as a crucial target of BMP signaling. n=4. E, G, and H, Hmbs was used as a housekeeping gene for qRT-PCR. The applied statistical tests and results are summarized in Table S11. The error bars denote standard deviation. The exact P values are specified. EC indicates endothelial cell; Fibro, fibroblast-like; Mac, macrophage; Modul, modulated; OD, osteodifferentiation; Osteochon, osteochondrogenic; and P, passage.