Fig 2: Effect of silencing of myocardin-related transcription factor (MRTF) on single-cell outgrowth of breast cancer cells in MoT assay. (A) Representative immunoblots of MRTF-A and MRTF-B from the extracts of MDA-231 andT47D cells transfected with the indicated siRNAs (tubulin blot––loading control). (B) Representative images of outgrowth of MDA-231 and T47D cells treated with the indicated siRNAs. (C) A bar graph summarizing the relative outgrowth of MRTF-silenced culture compared to control siRNA-transfected culture (summarized from three independent experiments). (D–F) Representative phase-contrast images (panel D; scale bar—200 µm) and fluorescence images of phalloidin-stained cells (panel E; scale bar 10 µm) showing morphological changes and defect in FLP formation (arrows) caused by MRTF depletion in MDA-231 cells in 3D culture. The insets show magnified images of the regions of interest outlined by squares. The bar graph in panel F summarizes the average number of FLP per cell of control versus MRTF-depleted cells. Values are presented as mean ± SD summarized from two independent experiments with at least 20 cells analyzed from each group (**p < 0.01)

Fig 3: PDGFRa 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 aSMA. 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 aSMA 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 PDGFRa-RAC1-SRF relationship.See also Figure S6.

Fig 4: 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 5: PDGFRaK opposes myofibroblast differentiation and is opposed by TGF-ß.(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 aSMA. Scale bar, 50 µm.(C) DF serum starved and treated with 10 ng/mL TGF-ß1, then processed for western blot. TGF-ß1 downregulates PDGFRa while upregulating aSMA with SMAD3 phosphorylation. Effects of TGF-ß1 are delayed in PdgfraK/+ DFs.(D) qPCR for Pdgfra or Acta2 in DFs treated with TGF-ß1 as above (n = 2 biological replicates).(E) Western blot for aSMA, 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 PDGFRa-TGF-ß-myofibroblast relationship.