Fig. 7: ANKRD1 regulates CAF activation through AP-1 interaction. | Nature Communications

Fig. 7: ANKRD1 regulates CAF activation through AP-1 interaction.

From: ANKRD1 is a mesenchymal-specific driver of cancer-associated fibroblast activation bridging androgen receptor loss to AP-1 activation

Fig. 7

a Immunoprecipitation assays (IP) with anti-V5 (ANKRD1) or nonimmune (IgG) antibodies from HEK293 cells lysates infected with ANKRD1OE vector followed by immunoblotting for ANKRD1, JUN, and FRA2. IgG: nonspecific signal of IgG heavy chains. The experiment was performed twice. PLA with anti-ANKRD1 or JUN antibodies in HDFs cells infected with ANKRD1OE or CTRL vectors. For ANKRD1-JUN interaction, n(biological replicates) = 2, for ANKRD1-FRA2 interactions, n(biological replicates) = 2 (b) or CAFs matched with HDFs n(strains) = 2; (c). Fluorescence puncta from the juxtaposition of anti-ANKRD1 and JUN antibodies (red), DAPI (blue). Left: representative images. Right: number of puncta per cell, n(cells)>100 per condition, mean ± SD, unpaired two-tailed t-test. Scale bar: 20 µm. PLA with anti-JUN or FRA2 antibodies in HDFs cells infected with an ANKRD1OE or CTRL vector (d) or shCTRL or shANKRD1#1 vector-injected CAFs. Fluorescence puncta from the juxtaposition of anti-FRA2 and JUN antibodies (red), DAPI (blue). Left: representative images Right: number of puncta per cell, n(strains) = 3, n(cells) >100 per condition, mean ± SD, unpaired two-tailed t-test. Scale bar: 20 µm. For d n(biological replicates) = 3, e n(independent experiments) = 2 f ChIPmentation analysis with anti-JUN antibody and non-immune IgGs of three ANKRD1OE or CTRL-vector-infected HDF strains (coloured dots). qPCR amplification of the indicated regions of the ACTA2 and HAS2 genes, expressed as relative enrichment folds over non-immune IgG in ANKRD1 overexpressing versus control HDFs. n(strains) = 3, mean ± SD. g ChIPmentation analysis with anti-V5 (ANKRD1) antibody versus non-immune IgGs of three T-5224 or DMSO-treated (48 h) ANKRD1OE–infected HDF strains (coloured dots). Results of qPCR amplification of the indicated regions for the ACTA2 and HAS2 genes are expressed as enrichment folds over non-immune IgGs in T-5224-treated versus DMSO controls. n(strains) = 3, mean ± SD, unpaired t-test with FDR multiple comparison’s correction. h ChIPmentation analysis with anti-ANKRD1 antibody versus non-immune IgGs of two T-5224 or DMSO-treated (48 h) CAF strains. qPCR amplification of indicated regions for the ACTA2 and HAS2 genes, expressed as enrichment folds over non-immune IgGs in T-5224-treated versus DMSO controls. n(strains) = 2, mean ± SD. i RT-qPCR analysis of indicated genes in T-5224- or DMSO-treated (48 h) HDFs infected with ANKRD1OE or CTRL vectors, expressed relative to CTRL after housekeeping gene normalization. n(strains) = 3, mean ± SD. Two-way ANOVA test.

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