Fig. 5: ATF4-deficient fibroblasts fail to support endothelial tube formation and secrete reduced levels of specific angiogenic cytokines.

a, Representative images of vasculature from B16F10 tumours grown in Atf4^WT/WT and Atf4^Δ/Δ mice. b, Box and whisker plot of the number of sprouts per field from a (n = 3 biologically independent samples per group). c, EC^WT/WT were treated with CM collected from LFB^WT/WT or LFB^Δ/Δ for 24 h and plated for tube formation assay and analysed 4 h after plating. Magnification, ×19. d, Box and whisker plots of the number of tubes and number of junctions per field from c. e, CM collected from LFB^WT/WT, LFB^Δ/Δ and LFB^Δ/Δ + AdmATF4 cells was used for analysis of pro-angiogenic cytokines using antibody arrays. Green boxes indicate the reference spots. Red boxes refer to the analysed proteins (VEGF, CXCL12, IGFBP-2 and IGFBP-9). f, Membranes were subjected to immunoblotting and protein levels were quantified from e. Values represent the mean ± s.e.m., unpaired two-sample t-test. g, Tumour lysates from equal volume B16F10 tumours collected from two Atf4^WT/WT and two Atf4^Δ/Δ mice were analysed for pro-angiogenic cytokines using the same antibody array as in e. h, Representative IF images from B16F10 tumours stained for VEGF (red) and CD31 (green). Magnification, ×20. Right: cropped images from ×20 original magnification. i, Box and whisker plot of the percentage VEGF⁺CD31⁺ colocalization area from h (n = 5 biologically independent samples per group). j, Representative IF images from B16F10 tumours stained for CXCL12 (red) and CD31 (green). Magnification, ×20. Right: cropped images from ×20 original magnification. k, Box and whisker plot of the percentage CXCL12⁺CD31⁺ colocalization area from j (n = 5 biologically independent samples per group). l, Proteins were detected by immunoblotting in untreated or TGF-β1-treated LFB^WT/WT or LFB^Δ/Δ (6 h). β-tubulin was used as a loading control. Numbers below blots represent relative band intensities, normalized to β-tubulin. Unpaired two-sample t-test in all box and whisker plots. Scale bars, 100 μm (a), 50 μm (h and j) and 5 μm (c).

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