Fig. 8: ANKRD1 targeting reproduces the effects of AP1 inhibition on CAF activation. | Nature Communications

Fig. 8: ANKRD1 targeting reproduces the effects of AP1 inhibition on CAF activation.

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

Fig. 8

a RT-qPCR analysis of indicated genes in multiple CAF strains transfected with 100 nM of ANKRD1-FANA or Scrambled-FANA for 72 h. Values are expressed relative to Scrambled-FANA; after house-keeping gene normalization. n(biological replicates) = 6, mean ± SD, two-tailed unpaired t-test. b Immunofluorescence images of ANKRD1 (green), αSMA (red), or DAPI (blue) in CAF#7 transfected with 100 nM of ANKRD1-FANA or Scrambled-FANA for 72 h (left) and quantification (right). n(independent experiment) = 3, mean ± SD, n(fields/condition>10), n(cells/field)>100, two-tailed unpaired t-test. Scale bar: 100 µm. c PLA with anti-JUN and FRA2 antibodies in CAF#7 transfected with 100 nM of ANKRD1-FANA or Scrambled-FANA for 72 h. Fluorescence puncta from the juxtaposition of anti-FRA2 and JUN antibodies (red), DAPI (blue), number of puncta per cell shown. n(independent experiment) = 2, n(cells) >100 per condition, mean ± SD, two-tailed unpaired t-test. Scale bar: 20 µm. d ChIPmentation analysis with anti-JUN antibody of three CAF strains (CAF#1, 2, 7) with an additional independent repeat of CAF7 (CAF7 rep), transfected with 100 nM of ANKRD1-ASO or Scrambled-ASO for 72 h. Results of qPCR amplification of indicated regions of the ACTA2 and HAS2 genes are expressed as enrichment folds over non-immune IgG in ANKRD1-ASO treated CAFs versus scrambled ASO. Results per individual CAF strains are shown as coloured dots. n(biological replicates) = 4, mean ± SD, multiple unpaired t-test. e IF for Pan-KRT (red) and VIM (green) to identify FaDu cells and CAFs, respectively. FaDu cells were co-cultured for 5 days with CAF#7 transfected ANKRD1-FANA or Scrambled-FANA (100 nM, 72 h). n(biological replicates) = 4, n(fields/condition>10), n(cells/field)>100. Mean ± SD, unpaired two-tailed t-test. Scale bar: 100 µm. f Images of H&E-stained back lesions formed by FaDu cells co-injected with CAF#2 transfected with ANKRD1-FANA or Scrambled-FANA (100 nM, 72 h) intradermally in contralateral mouse back skin, following cell embedding in Matrigel. n(mice) = 4, mean ± SD, unpaired two-tailed t-test. Scale bar: 500 µm. g FaDu cell density and proliferation, quantified as Pan-KRT positive cells per field. n(mice) = 4; 4–5 fields/tumor were analyzed. n(fields) = 25 for Scrambled and n(fields) = 27 for ANKRD1-ASO. Mean ± SD, unpaired two-tailed t-test. Scale bar: 100 µm.

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