Fig. 1: ANKRD1 is a mesenchymal-specific cancer-associated fibroblast (CAF) marker.

a Differentially expressed transcription factors and co-factors (TFs) in published data sets. (1) TFs upregulated (UP) in cancer-associated fibroblasts (CAFs, red, GSE122372), and present in Animal Transcription Factor database (http://bioinfo.life.hust.edu.cn/AnimalTFDB/). (2) TFs downregulated (DOWN) in CAFs treated with BET inhibitor JQ1 (blue, GSE81406). (3) TFs upregulated (UP) in human dermal fibroblasts (HDFs) following AR silencing (green, GSE107321). For all datasets: logFC > 2, p < 0.05 two-way ANOVA test. b RT-qPCR analysis of ANKRD1 expression in skin-SCC patient-derived CAFs and matched normal fibroblasts (NFs) (#8 to #18), amplification cycle normalized to RPLP0 (ΔΔCT). n(strains) = 11, two-tailed unpaired t-test. c ANKRD1 and GAPDH Immunoblot analysis (WB). Top: one experiment including 2 patient’s CAFs and matched HDFs n(strains) = 4. Bottom: second independent experiment with melanoma-derived CAFs and unmatched HDFs n(strains) = 6. d Representative immunofluorescence (IF) images of ANKRD1 (red), DAPI (blue) in CAFs and matched NFs. n(strains) = 2, n(fields) = 11 for NF17 and NF18, 14 for CAF17, 10 for CAF18, mean ± SD, two-tailed unpaired t-test. Scale bar: 20 µm. e RT-qPCR analysis of ANKRD1 mRNA in Laser capture microdissection (LCM) samples from patients’ stroma underlying and flanking in situ skin SCC lesions. Amplification cycles were normalized to ACTB (ΔΔCT). n(strains) = 6, two-tailed unpaired t-test. f ANKRD1 (green), Pan-KRT (red) and VIMENTIN (blue) IF in skin SCC and matched normal skin (NS). n(fields) = 7 for normal, and 8 for SCC, mean ± SD, two-tailed paired t-test with Mann-Whitney correction. Scale bar: 50 µm. The experiment was repeated in other 2 SCC tissues as reported in Supplementary Fig. 1e. g, h ANKRD1 expression in CAFs versus NFs from breast (GSE29270, n = 63), lung (GSE22862, n = 30), colon (GSE46824, n = 34), normal fibroblasts (NF), n = 9, primary tumor CAF (P-CAF), n = 14, metastatic CAF (M-CAF), n = 11. ANKRD1 expression in fibroblasts from hypertrophic scar and keloids (E-MTAB-2509, n = 27), normal fibroblasts = 9, hypertrophic fibroblasts = 9, keloid fibroblasts = 9, and from idiopathic pulmonary fibrosis (IPF) (GSE40839, n = 21), control fibroblasts = 10 and IPF = 11. Moderated t-statistic for two-class comparison. Moderated F-statistic for three-group comparison with Benjamini-Hochberg correction. Minimum value, first quartile (lower box bound), median (centre), third quartile (top box bound), maximum value. Whisker length is a maximum of 1.5 times the interquartile range. Points represent values outside intervals.