Figure 1 from ATM Regulates Differentiation of Myofibroblastic Cancer-Associated Fibroblasts and Can Be Targeted to Overcome Immunotherapy Resistance

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ATM activation in myofibroblasts. A and B, Western blotting of HFFF2 treated with TGFβ1 over time. C and D, Representative immunofluorescence staining of pATM- (C) and pH2AX-positive (D) foci, and quantification of HFFF2 treated over time with TGFβ1 or NCS (positive control); nuclei are outlined by dotted white lines based on DAPI nuclear counterstaining (not shown). Scale bar, 10 μm. Foci counts are expressed as percentage of total cell number per FoV (FoV = ntr ≥ 8); SD and Kruskal–Wallis test are shown. E, Alkaline comet assay of HFFF2 treated with TGFβ1 for 24 hours or irradiated with 2 Gy (as positive control; ntr = 50; homoscedastic). Student t test refers to the control. F, Analysis of viability, proliferation, and cell death monitored by MTT, thymidine incorporation, and PI staining, respectively. HFFF2 were treated for 3 days with TGFβ1 or with H2O2 or cisplatin as positive controls (ntr = 2–3). G, Senescence assay of HFFF2 treated with TGFβ1 over time. The percentage of SA-β-Gal–positive cells is expressed as fold induction compared with untreated control; FoV = ntr = 10. H, Western blotting of NSCLC and HNSCC CAF. I, Volcano plots with FDR q (significance) and NES (correlation) of GSEA performed on the indicated data sets from LCMD tumor versus normal stroma. The dotted line drawn at 1.3 of −log10 FDR q axis indicates FDR q = 0.05. J–N, Representative image of HNSCC MxIHC; brightfield image of cytokeratin staining. Scale bar, 5 mm (J); scale bar, pseudocoloered images, 100 μm (K); scale bar, 20 μm (L and M). Single-stained and merged pseudocolored images with the cell regions used for the quantification are highlighted in red or green for pATM or SMA positivity, respectively (subtracted CD31 staining is shown in brown); quantification of pATM positivity in SMA+ cells (negative for CD31 and CK) in 10 HNSCC and 10 NSCLC cases (N) with significance calculated comparing pATM+/SMA+ vs. pATM−/SMA+ CAFs. Paired Student t test is used in the figure and refers to control unless otherwise stated. ns, nonsignificant; *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001.

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