Fig 7. YAP activation enhances pro-inflammatory but inhibits reparative macrophage phenotype.

(A) Generation of transgenic mice expressing YAP5SA, a constitutively active form of YAP in macrophages by crossing Rosa26^YAP5SA mice with LysM^Cre mice. (B) Western blot analysis for YAP was performed using total lysates from control and LysM^Cre;Rosa26^YAP5SA BMDMs. β-actin is shown as a loading control. (C) Double immunofluorescence for β-galactosidase (red) and CD68 (green) was performed on control and LysM^Cre;Rosa26^YAP5SA BMDMs. Nuclei were visualized by DAPI staining (blue). Scale bar 50 μM. (D–F) BMDMs were isolated from control and LysM^Cre;Rosa26^YAP5SA mice and stimulated with/without LPS/IFNγ or IL4/IL13 for 24 hours. (D) qRT-PCR for pro-inflammatory marker genes, IL6, IL1β, IL12β, Nos2, TNFα, Ccl2, Rantes, and Ccr7 using RNA isolated from untreated or LPS/IFNγ-treated control and LysM^Cre;Rosa26^YAP5SA BMDMs. n = 3 in each group. (E) NO production determined by nitrite (NO₂−) levels in conditioned medium prepared from untreated or LPS/IFNγ-treated control and LysM^Cre;Rosa26^YAP5SA BMDMs. n = 3 in each group. (F) qRT-PCR for reparative marker genes, Arg1, Ym1, Cd206, and Fizz1 using RNA isolated from untreated or IL4/IL13-treated control and LysM^Cre;Rosa26^YAP5SA BMDMs. n = 3 in each group. Gene expression results were normalized to gapdh, and results are represented as fold change. (G) Masson’s trichrome staining on control and LysM^Cre;Rosa26^YAP5SA hearts at 28 days post-MI. Fibrotic area was normalized to the remaining heart for each heart section. (H) Quantification of Masson’s trichrome stained fibrotic area in control and LysM^Cre;Rosa26^YAP5SA hearts at 28 days post-MI (n = 4 per group). (I) Immunostaining showing the expression collagen type I in infarct zone on control and LysM^Cre;Rosa26^YAP5SA heart sections at 28 days post-MI. DAPI was used to stain nuclei. Scale bar 100 μM. (J) Quantification of collagen type I positive area in control and LysM^Cre;Rosa26^YAP5SA heart sections. n = 3 per group. For numerical raw data, please see S1 Data. Arg1, Arginase-I; BMDMs, bone marrow–derived macrophages; Ccl2, C–C motif chemokine ligand 2; Cd206, cluster of differentiation 206; Fizz1, found in inflammatory zone 1; IFNγ, interferon gamma; IL, interleukin; IRES, internal ribosome entry site; LPS, lipopolysaccharide; NO, nitric oxide; Nos2, nitric oxide synthase 2; NS, non-significant; qRT-PCR, real-time quantitative reverse transcription PCR; TNFα, tumor necrosis factor alpha; YAP, yes-associated protein.