Figure 5. Myofibroblasts and not senescent fibroblasts mediate collagenous ECM deposition. (A) Schematic of experimental procedure for B and C. (B) Representative image of immunofluorescent staining for Fibronectin (FN) in fibroblast-derived matrices (FDM) produced by HFFF2s treated as indicated; FN (orange; pseudo-colored in Fiji) and Dapi (blue) as nuclear counterstain; scale bar represents 50µm. (C) Transwell assay examining OE33 invasion through FDM deposited by HFFF2 fibroblasts induced to transdifferentiate through treatment with TGF-β1 or γ-irradiation (IR). (D-F) Analysis of xenografts formed from 5PT cells injected s.c. into RAG1-/- mice with HFFF2 fibroblasts treated as indicated. (D) Representative images of SMA immunochemistry in 5PT xenografts co-injected with HFFF2s treated as indicated. Histogram shows SMA quantification expressed as % positive area. (E) Representative images of Masson’s trichrome staining for collagen (royal blue) with HFFF2s treated as indicated. Histogram shows quantification expressed as % positive area. (F) Representative images showing multi-photon excitation (MPE) filtered for second harmonic generation to identify collagen fibers on sections from the xenograft tumors as indicated (Scale Bar indicates 100µm). Histogram shows quantification of collagen fiber elongation. Data are presented as mean ± SEM and statistics are shown for T-test compared to controls unless otherwise indicated.