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. 2015 Dec 16;6:10084. doi: 10.1038/ncomms10084

Figure 5. MBNL1-regulated calcineurin Aβ mRNA levels underlies myofibroblast differentiation.

Figure 5

(a) RT–PCR of a calcineurin Aβ splice variant 1 (CnAβ1) from Adβgal (control), TGFβ-treated and AdMBNL1-infected fibroblasts. 18S RNA expression was used for normalization. (b,c) RT–PCR gel and quantification of the two major calcineurin Aβ-spliced isoforms, CnAβ and CnAβ1, from Adβgal- or AdMBNL1-infected fibroblasts, with Gapdh as a processing and loading control. N=3 experiments, t-test, *P<0.05 versus Adβgal. (d) RT–PCR analysis of the same two calcineurin Aβ spliced isoforms from fibroblasts of hearts subjected to sham or MI injury in Mbnl1+/+ and Mbnl1−/− mice. N=3 experiments. (e) NFAT-luciferase reporter activity in Adβgal- and AdMBNL1-infected fibroblasts. Average luciferase activity is expressed relative (Rel.) to Adβgal (control), N=3, t-test, *P<0.05 versus Adβgal. (f) Immunofluorescence for αSMA (green) stress fibres and nuclei (blue) in Ppp3r1+/+ and Ppp3r1−/− (calcineurin B1 protein) primary MEFs infected with Adβgal or AdMBNL1. (g,h) Immunofluorescent staining and quantification of αSMA (green) stress fibres and nuclei (blue) in Mbnl1+/+ or Mbnl−/− MEFs infected with Adβgal or constitutively active calcineurin (AdΔCnA). Data represent the average number of fibroblasts with αSMA+ stress fibres per total nuclei±s.e.m., N⩾375, analysis of variance (ANOVA) and Newman–Keuls pairwise analysis, *P<0.05 versus Adβgal. (i) Quantification of skin wound closure at 6 days after 6 mm biopsy injury in Mbnl1+/+ and Mbnl1−/− mice treated with either Adβgal or AdΔCnA directly applied throughout the wound. Average closure±s.e.m., N⩾14 mice per group, ANOVA and Newman–Keuls pairwise analysis, *P<0.05 versus Adβgal Mbnl1+/+; #P<0.05 versus Adβgal Mbnl1−/−. All scale bars, 50 μm.