Figure 2 from ATM Regulates Differentiation of Myofibroblastic Cancer-Associated Fibroblasts and Can Be Targeted to Overcome Immunotherapy Resistance
Year: 2025
Type: other
Abstract: ATM inhibition suppresses myofibroblast differentiation. A and B, Western blotting and its quantification of HFFF2 treated for 72 hours with TGFβ1 ± ATM inhibitors (13.3 μmol/L KU55933, A; 2.5 μmol/L KU60019, B). C, Western blotting quantification of SMA expression in primary fibroblasts isolated ex vivo from colon (n = 2), skin (n = 1), and oral (n = 2) tissues from healthy donors. Fibroblasts were treated as in A (see also Supplementary Fig. S2C–G). D, Collagen gel contraction assay and measurement of gel area. HFFF2 or normal primary oral fibroblasts were treated as in A. Left, representative gel images (ntr = 2). E, Representative immunofluorescence staining of SMA-positive stress fibers or collagen 1 deposition in HFFF2 treated with TGFβ1 ± ATM inhibitor as above for 3 (SMA) or 7 (collagen 1) days; relative quantification of the mean (FoV for both SMA and collagen 1 = 10). Scale bars for SMA, 100 μm and for collagen 1, 500 μm. F and G, Western blotting and quantification (F) and qRT-PCR (ntr = 3; G) of HFFF2 transfected as indicated and treated with TGFβ1 for 72 hours (ACTA2 = SMA gene). H and I, Western blotting and quantification of HFFF2 fibroblast treated for 72 hours with TGFβ1 ± ATR inhibitor (VE891; 0.5 μmol/L and 2.5 μmol/L; H) or ± DNA-PKcs inhibitor (2 μmol/L and 10 μmol/L; I) for 72 hours. J and K, qRT-PCR of HFFF2 transfected as indicated and treated with TGFβ1 for 72 hours (ntr = 3; PRKDC, DNA-PKcs). L, Western blotting and quantification of HFFF2 treated for 72 hours with TGFβ1 ± CHK2 inhibitor (1.5 μmol/L CCT241533). M, Representative immunofluorescence staining of SMA and collagen 1 in HFFF2 treated with TGFβ1 ± CHK2 inhibitor (quantified as in E). N and O, Western blotting and quantification (N) and qRT-PCR (ntr = 3; O) of HFFF2 transfected as indicated and treated with TGFβ1 for 72 hours. Heteroscedastic Student t test is used throughout the figure and is relative to TGFβ1-treated samples unless otherwise highlighted. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Authors: Massimiliano Mellone, Klaudia Piotrowska, Giulia Venturi, Lija James, Aleksandra Bzura, Maria A. Lopez, Sonya James, Chuan Wang, Matthew J. Ellis, Christopher J. Hanley, Josephine F. Buckingham, Kerry L. Cox, Gareth Hughes, Viia Valge-Archer, Emma V. King, Stephen A. Beers, Vincent Jaquet, George D.D. Jones, Natalia Savelyeva, Emre Sayan, Jason L. Parsons, Stephen Durant, Gareth J. Thomas
Language: English
Topic: TGF-β signaling in diseases
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4416587838