SM22 alpha Antibody - #AF9266

Figure 5 Characterization of OAF cells during early IVD formation. A) UMAP visualization of the OAF cluster during human early IVD formation at the Noto, Trans, and NPL stages. B) Representative violin plots showing the expressions of PAX1, PAX9, MKX, COL5A1, SOX9, PDGFRL, TAGLN, FMOD, and BGN during human early IVD formation. C) Representative IHC images of TAGLN (upper) and FMOD (lower) in human axial skeleton sections at the indicated developmental stages. Scale bar, 100 µm. D) UMAP visualization of the OAF cluster during mouse early IVD formation at the Noto, Trans, and NPL stages. E) Representative violin plots showing the expressions of Pax1, Mkx, Col5a1, Sox9, Pdgfrl, and Fmod during mouse early IVD formation. F) Representative IF images of Fmod (left) and Tagln (right) in mouse axial skeleton sections at E14.5. The dotted line indicates the presumptive OAF. The asterisks indicate the presumptive IAF. Triangles indicate NP. VB, vertebral body. Scale bar, 200 µm. G) Dot plot showing the mean expression of selected mechanosensitive ion channel genes in the human OAF along with early IVD formation. Dot size indicates the percentage of cells with detected expression. H) Representative images of lineage tracing in Piezo2‐Cre;Ai9 mouse IVDs. Scale bar, 200 µm.

Fig. 2. HLJDD inhibited VSMC-derived foam cell formation. Primary VSMCs were identified by (a) bright field examination, α-SMA expression, and Phalloidin staining. Scale bar: 200 and 100 μm. (b, n = 6) VSMC viability was evaluated in different doses of HLJDD using the cell counting kit-8. The effect of HLJDD on foam cell formation was tested in (c) ORO, (d) Nile Red, (e) Annexin V/PI staining, and (f) ROS generation. n = 3, 3, 5, and 5, respectively. (g) Phalloidin and Giemsa staining on VSMCs and A7r5 cell line, respectively. Scale bar: 100 μm. Foam cell formation was also evaluated via (h) Western blot for (i, n = 4) α-SMA, (j, n = 5) SM22-α, (k, n = 4) TNF-α, (l, n = 3) IL6, (m, n = 8) OPN, (n, n = 7) CD36, and (o, n = 5) Caspase 3. Data were presented as mean ± standard deviation in one-way ANOVA with Dunnett post hoc tests. ns: not significant

Fig. 1. Effect of HOXA5 overexpression on PDGF-BB-induced VSMC phenotypic changes. A) Immunofluorescence staining against α-SMA was used to identify VSMCs. B, C) VSMCs were starved for 24 h and subsequently incubated with PDGF-BB (10, 20 and 30 ng/ml) for 24 h. The mRNA and protein levels of HOXA5 were determined by qRT-PCR and Western blot. D, E) HOXA5 mRNA and protein levels were examined in VSMCs infected with NC or HOXA5 overexpression lentivirus using qRT-PCR and Western blot. VSMCs were infected with NC or HOXA5 overexpression lentivirus for 48 h and then cultured with 10 ng/ml PDGF-BB for 24 h (F–K). F, G) Relative expression of HOXA5 mRNA and protein was determined by qRT-PCR and Western blot. H) Western blot analysis of the protein levels of calponin, α-SMA, SM22α, vimentin, PCNA and thrombospondin. I, J) Immunofluorescence staining was utilized to assess levels of calponin and vimentin in VSMCs. The nucleus was stained with DAPI. K) VSMC proliferation was detected using the MTT assay. Scale bar, 50 μm &, p < 0.05.

Fig. 3 Cap inhibits Pi-induced calcification of VSMCs by activating the TRPV1 receptor. A) RT-qPCR showing the mRNA levels of TRPV1; B) Western blot depicting the protein expression levels of TRPV1; C) Alizarin Red staining assay; D) Calcium content in VSMCs assessed using a calcium colorimetric assay kit. E) RT-qPCR illustrating the expression of osteogenesis-specific and phenotypic markers in VSMCs calcification. F) Western blot revealing the protein expression levels of osteogenesis-specific and phenotypic markers in VSMCs calcification. VSMCs cultured in the complete and pro-calcifying medium were defined as the control and Pi group, respectively; Pi + Cap: VSMCs cultured in the pro-calcifying medium with 20 μM Cap; Pi + Cap + CPZ: VSMCs cultured in the pro-calcifying medium with 20 μM Cap and 10 μM CPZ; Data are mean ± SD (n = 3); #: p < 0.05, ##: p < 0.01, ###: p < 0.001 vs Control; *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs Pi; ※: p < 0.05, ※※: p < 0.01, ※※※: p < 0.001 vs Pi + Cap; ns: p > 0.05 vs Control; p > 0.05 vs Pi; p > 0.05 vs Pi + Cap. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Figure 6 Overexpression of SPINT2 prevents PDGF-BB-induced SMC phenotypic switching. SMCs were incubated with 20 ng/ml PDGF-BB for 24 h following 24 h of adenoviral infection. (A) The expression levels of the synthetic proteins (vimentin and collagen I) were detected by western blotting. (B) The expression levels of the contractile proteins (α-SMA and SM22α) were detected by western blotting. (C) The expression levels of vimentin were assessed by immunofluorescence analysis. The nuclei were stained with DAPI. Scale bar, 100 µm. ***P

Fig. 2. |MiR-155-5p overexpression inhibited calcification in rVSMCs. Cells were transfected with LV-rno-miR-155-5p for 48 h, followed by cultured in CM. The expression level of miR-155-5p was detected by real-time PCR at day 3, 7, 14 and 21 (A), the calcification degree was detected by alizarin red staining at day 7 and 21(B), ALP activity was assessed by ALP activity kit (C), the levels of ALP, TGF-β1, pSmad-2, Smad-2, pSmad-3, Smad-3, and SM22a were detected by western blot (D).

Fig. 6. Immunohistochemistry of SM22α and OPN in the thoracic aorta among the groups.

Fig. 7. Western blot of SM22α and OPN in the thoracic aorta among groups.