Fig 2: Srf is required for proliferation and myofibroblast differentiation(A–C) Schematic of the genetic labeling strategy for Pdgfra-Srfflox/+ control mice (A) and Pdgfra-Srfflox/flox knockout mice (B) and the timeline (C).(D) Stain for SRF in day 7 wound tissue. Scale bars 100 μm.(E–H) Histology and histomorphometry at the wound center on day 7 (n = 6–7 of each genotype). H&E staining: granulation tissue has not filled the knockout wound bed, leaving a gap (asterisk) with downward migrating epithelial tongues (arrows). Dotted line indicates the wound bed area. (E). Wound bed cross-sectional area (F). Contraction (G). Epithelialization (H). Scale bars, 200 μm.(I and J) Mice have a Col1a1-GFP reporter to identify fibroblasts. EdU/GFP co-labeling of proliferating cells on day 4. Arrowheads indicate EdU+GFP+ cells (I). Quantification of EdU in GFP+ cells (n = 4 mice per genotype) (J). Scale bars, 50 μm.(K and L) Tomato and αSMA at the wound center on day 7. Asterisk indicates tissue gap in the wound center. Dotted lines indicate the wound bed area. Scale bars, 200 μm.(M) Quantification of αSMA+ area on day 7 (n = 4–5 mice per genotype).(N and O) αSMA/Tomato co-labeling on day 7 (N). Quantification of αSMA in Tomato+ cells (n = 3 mice per genotype) (O). Scale bar, 50 μm. Data plotted as mean ± SEM. Each point represents one mouse.See also Figure S7.

Fig 3: Mrtfa/Mrtfb are required for myofibroblast differentiation but not proliferation(A–C) Schematic of the genetic labeling strategy for Pdgfra-Mrtfa+/−Mrtfbflox/flox control mice (A) and Pdgfra-Mrtfa−/−Mrtfbflox/flox knockout mice (B) and the timeline (C).(D) Stain for MRTFB in day 7 wound tissue. Scale bars, 100 μm.(E–H) Histology and histomorphometry at the wound center on day 7 (n = 5–6 of each genotype). H&E staining: the knockout wound bed is filled with granulation tissue, but the wound is uncontracted with widely separated epithelial tongues (arrows). Dotted line indicates the wound bed area.(E). Wound bed cross-sectional area (F). Contraction (G). Epithelialization (H). Scale bars, 200 μm.(I and J) EdU/Tomato co-labeling of proliferating cells on day 4. Arrowheads indicate EdU+Tomato+ cells (I). Quantification of EdU in Tomato+ cells (n = 3 mice per genotype) (J). Scale bars, 50 μm.(K and L) Tomato and αSMA at the wound center on day 7. Dotted lines indicate the wound bed area. Scale bars, 200 μm.(M) Quantification of αSMA+ area on day 7 (n = 4 mice per genotype).(N and O) αSMA/Tomato co-labeling on day 7 (N). Quantification of αSMA in Tomato+ cells (n = 3 mice per genotype) (O). Scale bar, 50 μm. Data plotted as mean ± SEM. Each point represents one mouse.

Fig 4: PDGFRαK opposes myofibroblast differentiation and is opposed by TGF-β.(A–F) Primary dermal fibroblasts (DFs) assayed at passage 2–3.(A) Collagen lattice contraction, expressed as a percentage of the initial diameter, with representative images at 24 h after lattice detachment (n = 3 biological replicates per genotype). Data are plotted as mean ± SD. Each point represents one biological replicate.(B) DF stained for F-actin and αSMA. Scale bar, 50 μm.(C) DF serum starved and treated with 10 ng/mL TGF-β1, then processed for western blot. TGF-β1 downregulates PDGFRα while upregulating αSMA with SMAD3 phosphorylation. Effects of TGF-β1 are delayed in PdgfraK/+ DFs.(D) qPCR for Pdgfra or Acta2 in DFs treated with TGF-β1 as above (n = 2 biological replicates).(E) Western blot for αSMA, SRF, MRTFA, and MRTFB in serum-starved DFs.(F) Western blot with nuclear (n) and cytoplasmic (c) fractions after serum starvation followed by serum for 30 min(G) Summary of the proposed PDGFRα-TGF-β-myofibroblast relationship.

Fig 5: PDGFRα and RAC1 regulate SRF nuclear localization.(A–H) Wild-type 3T3 cells and 3T3 cells with Cas9-mediated Pdgfra knockout (aR-KO).(A) Cells serum starved and treated with 50 ng/mL PDGF-AA or PDGF-BB for 30 min, then processed for western blot.(B) Cells stained for F-actin and αSMA. Scale bar, 50 μm. Total SRF levels are similar between genotypes.(D) Collagen lattice contraction, expressed as a pecentage of the initial diameter, with representative images at 24 h after lattice detachment (n = 3 biological replicates per genotype). Data are plotted as mean ± SD. Each point represents one biological replicate.(E) Western blot with nuclear (n) and cytoplasmic (c) fractions after serum for 30 min. SRF and MRTFB are predominantly cytoplasmic in aR-KO cells.(F) aR-KO cells with a doxycycline (dox)-inducible constitutively active (CA−) MRTFA were serum starved and treated with dox for 48 h. Western blot of nuclear (n) and cytoplasmic (c) fractions shows that CA-MRTFA restores SRF nuclear localization and αSMA expression.(G) aR-KO cells fail to activate Rac1 when serum starved and treated with 10% FBS for 10 min(H) siRNA knockdown of RAC1 reduces SRF nuclear localization.(I) Summary of the proposed PDGFRα-RAC1-SRF relationship.See also Figure S6.