Fig. 3: BMP4 regulation of cardiac fibroblasts.

Cardiac fibroblasts were isolated from hearts of 8-week-old Ccl19-Cre R26R-EYFP mice. a, Representative dot plots showing the gating strategy for FACS of PDPN⁺CD31⁻, Bmp4-deficient (EYFP⁺) and Bmp4-proficient (EYFP⁻) cardic fibroblasts. b, Purity of Bmp4-deficient (EYFP⁺) and Bmp4-proficient (EYFP⁻) cardiac fibroblasts after 10 d of culture. Representative dot plots from three independent isolations. c, Production of the indicated BMPs by Bmp4^−/− (EYFP⁺) or Bmp4^+/+ (EYFP⁻) fibroblasts. d,e, Production of the cytokines IL-6 (d) and TNF (e) by Bmp4^−/− (EYFP⁺) or Bmp4^+/+ (EYFP⁻) fibroblasts after 24 h of culture (n = 5; pooled data from independent wells from two independent experiments). f,g, Expression of ICAM1 by Bmp4^−/− (EYFP⁺) or Bmp4^+/+ (EYFP⁻) fibroblasts after exposure to medium or BMP4 (10 ng ml⁻¹) (f) or IL-1β (1 ng ml⁻¹) (g) as assessed by flow cytometry with MFI of ICAM1 in f and MFI fold change relative to ICAM1 expression by untreated Bmp4^+/+ fibroblasts in g. Dots represent values from individual wells; pooled data of two independent experiments (n = 5). All box plots as in Fig. 1. Statistical analysis was performed using Student’s t-test (c–e) or one-way ANOVA with Dunnett’s multiple comparison test (f,g) with *P < 0.05, **P < 0.01 and ***P < 0.001. FACS, fluorescence-activated cell sorting.

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