(A) Proximity ligation assays (PLAs) with antibodies against AR and CSL of 2 HDF strains with or without CSL gene silencing as specificity control. Red fluorescence puncta resulting from the juxtaposition of anti-AR and anti-CSL antibodies were visualized by confocal microscopy with concomitant DAPI nuclear staining. Shown are representative images and quantification of number of puncta per cell. Scale bar: 10 μm. Values of PLA puncta for each individual cell are indicated with mean ± SD. n(cell measurements per condition) = 35; ***P < 0.005, 2-tailed unpaired t test. (B) PLAs with antibodies against AR and CSL in 3 HDF strains in the absence or presence of the AR agonist DHT (10 nM, 24 hours). Scale bar: 10 μm. Values of PLA puncta for each individual cell are indicated with mean ± SD. n(HDF strains) = 3, n(CAF strains) = 3; ***P < 0.005, 2-tailed unpaired t test. Additional PLAs carried out in HDFs versus CAFs are shown in Supplemental Figure 10. (C) Reciprocal coimmunoprecipitation assays of CSL and AR association. HDF total cell extracts were immunoprecipitated with anti-AR or anti-CSL rabbit polyclonal antibodies or nonimmune IgG (mock). Immunoblotting was carried out with anti-AR or anti-CSL mouse monoclonal antibodies. (D) Total cell extracts of 2 HDF strains and WI38 lung fibroblasts were processed for RNA immunoprecipitation with anti-CSL antibodies versus nonimmune IgGs. Immunoprecipitated RNAs were analyzed by RT-qPCR with specific primers for HOTAIR lncRNA, SRA lncRNA, H19 lncRNA, and GAPDH mRNA. Results are expressed as fold enrichment in immunoprecipitates with anti-CSL antibodies versus nonimmune IgG (dashed line), and values for each HDF strain are indicated as dots with mean ± SD. n(fibroblast strains) = 3; ***P < 0.005, 2-tailed unpaired t test.