Fig 2: TGFßR and GLI1/2 inhibitors downregulate aSMA, GLI1, and tumor matrix in OFs(A) IF for aSMA and F-actin in L-OFs treated with TGFß3 ± TGFßRInh (SB431542) or ± GLI1/2 (GANT61) inhibitors.(B-K) OFs with high activation (H-OFs) were treated with TGFßRInh or GLI1/2Inh or combination; (B) gene expression was measured by qRT-PCR for ACTA2 and FAP (N = 4 donors). (C) Cells were analyzed via flow cytometry for percentage of cells aSMA+ and/or FAP+. (D) Activated OFs were visualized via IF for aSMA and FAP ± inhibitor combination. (E) GLI1/2/3 mRNA expression analyzed via qRT-PCR for OFs and (F) GLI1 visualized via IF in activated OFs or with combination. (G) mRNA expression of matrix molecules were analyzed via qRT-PCR for treated or untreated OFs (n = 4 donors). (H) IF visualized FN1, VCAN, COL11A1, COMP and COL1A1, and aSMA in activated OFs with or without combination. (I) IHC of control and combination-treated OF gels for aSMA, FAP, COL11A1, and FN1.(J–K) OF-only gels or AOCS1 co-cultures were grown for 7 days with or without inhibitors and (J) gel modulus was measured (mean of 3 gels per donor, N = 2) and (K) fixed sections stained for H&E.Scale bars (A, D, F, and H) are 50 µm and (I and K) are 500. Red lines represent control (no TGFßRINH) expression.