Fig 1: The expression of MED12 in aortic tissue. (A) Representative images of immunohistochemical staining in aortic sections from non-AD and AD patients. IgG as a negative control. (B) Quantitative analysis of MED12. (C) The mRNA expressions of MED12 in aortae of non-AD and AD patients were detected by RT-PCR, with MMP-2 as a positive control. (D) Representative images of immunohistochemical staining in aortae of AD and control mice. IgG as a negative control. (E) Quantitative analysis of Med12. (F) Representative images of immunofluorescent staining of Med12 in aortic sections from AD and control mice. Fluorescence intensities of MED12 (red) and a-SMA (green, smooth muscle cell marker) were analyzed. Right insets represent high-magnification images of each individual imaging channel and a merged image of the region (left). Pearson’s correlation coefficient (PCC) was employed to quantify colocalization. (G) Quantitative analysis of MED12 and a-SMA fluorescence intensity. Data are expressed as the mean ± SD. Statistical analyses, unpaired t-test. * p < 0.05; ** p < 0.01; *** p < 0.001.
Fig 2: Regulation of cell phenotypic conversion. MOVAS were translated with si-control or si-Med12 for 72 h. (A) Fluorescence images were taken using a 40 × objective. The length-to-width ratios of the cells were quantified using ImageJ software. (B) Expressions of col4a1 (encoding collagen IV, synthetic VSMC marker) were detected by RT-PCR. (C) Immunofluorescent staining for DAPI (blue), a-SMA (green), and SM22a (red); MERGE represents the combined image of the above three channels. Scale: 20 µm. Statistics of the mean fluorescence in each visual field (4 visual fields/sample, 3 samples/group). (D) The expressions of a-SMA and SM22a (contractile VSMC marker protein) were detected by Western blot. The experiment was repeated at least three times. Data are expressed as mean ± SD. Statistical analyses, unpaired t-test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Fig 3: Effect of Med12 on the TGFß signaling pathway. (A) MOVAS were translated with si-control or si-Med12 for 72 h, and the expressions of Tgfß1 were detected by RT-PCR. (B) MOVAS were incubated with TGFß1(4 ng/mL) for 72 h, and Western blot detected the expression of Med12 and Pcna. (C) Same experiment as in (A); the TGFßR2-phosphorylated Smad2 (p-Smad2) relative to total Smad2 and phosphorylated Smad3 (p-Smad3) relative to total Smad3, as detected by Western blot. (D) Same experiment as in (A), Western blot was used to analyze p-ERK/ERK. The experiment was repeated at least three times. Data are expressed as mean ± SD. Statistical analyses, unpaired t-test. * p < 0.05; **** p < 0.0001. ns: not significant.
Fig 4: Decreased MED12 inhibited the proliferation of MOVAS. MOVAS transfected with si-control or si-Med12 for 72 h. (A) Cell viability detected by CCK-8. (B) Expression of Med12 and Pcna detected by RT-PCR. (C) Expression of MED12 and Pcna detected by Western blot. The experiment was repeated at least three times. Data are expressed as mean ± SD. Statistical analyses, unpaired t-test. ** p < 0.01; **** p < 0.0001.
Fig 5: The decreased expression of Med12 promoted the senescence of MOVAS. MOVAS were transfected with si-control or si-Med12 for 72 h. (A) Senescent cells were stained with blue by ß-galactosidase staining kit as shown by the arrow, and the number of positive cells was quantitatively analyzed. (B) The expressions of Med12 and Cdkn1a were detected by RT-PCR. (C) The expressions of Med12 and P21 were detected by Western blot. The experiment was repeated at least three times. Data are expressed as mean ± SD. Statistical analyses, unpaired t-test. * p < 0.05; *** p < 0.001; **** p < 0.0001.
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