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. 2025 Jan 1;15(3):1185–1204. doi: 10.7150/thno.98660

Figure 2.

Figure 2

OTUB2 accelerates the development of VC. (A) Western blot analysis of OTUB2, RUNX2, BMP2 and α-SMA expression in aortas. n = 6 per group. (B) Representative images of Von Kossa staining, Alizarin red staining, and immunohistochemical staining for RUNX2 and BMP2 in aortas from the indicated experimental cohorts. Scale bars, 200 μm. (C) Representative images of immunofluorescence staining for RUNX2 and α-SMA in aortas from the different experimental groups. Scale bars, 200 μm (left panels), 100 μm (right panels). (D) Representative images of Alizarin red staining of whole aortas from the indicated experimental groups. Scale bars, 5 mm. (E) Representative images of immunofluorescence staining for RUNX2 and α-SMA in aortas from the different experimental groups. Scale bars, 200 μm (left panels), 100 μm (right panels). (F) Western blot analysis of OTUB2, RUNX2, BMP2 and α-SMA expression in aortas. n = 6 per group. (G) Representative images of Von Kossa staining, Alizarin red staining, and immunohistochemical staining for RUNX2 and BMP2 in aortas from the indicated experimental cohorts. Scale bars, 200 μm. (H) Western blot analysis of OTUB2, RUNX2, BMP2 and α-SMA expression in aortas. n = 6 per group. (I) Representative images of Von Kossa staining, Alizarin red staining, and immunohistochemical staining for RUNX2 and BMP2 in aortas from the indicated experimental cohorts. Scale bars, 200 μm. (J) Western blot analysis of OTUB2, RUNX2, BMP2, and α-SMA expression in VSMCs overexpressing OTUB2. n = 3 per group. (K) Representative images of Alizarin red and Von Kossa staining of VSMCs after transfection of the indicated constructs and CM exposure for another 7 days. Scale bars, 5 mm (upper panels), 100 μm (lower panels). (L) Western blot analysis of OTUB2, RUNX2, BMP2, and α-SMA expression in VSMCs with OTUB2 depletion. n = 3 per group. (M) Representative images of Alizarin red and Von Kossa staining of VSMCs after the transfection of the indicated constructs and CM exposure for another 7 days. Scale bars, 5 mm (upper panels), 100 μm (lower panels). Statistical significance was assessed using one-way ANOVA followed by Dunnett's test (A, F, H, J and L). All values are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.