Fapα h 56

Cells were grown on a cover slip, serum deprived for 18 h and exposed to treatments for 4 h, when required. Cells were then fixed in ice-cold methanol at room temperature for 10 min, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated with 1% BSA in PBS at room temperature for 1 h. After washing with PBS, cells were incubated with primary antibodies against AHR (D5S6H) (Cell Signalling technology, Euroclone, Milan, Italy), vimentin (V9), cytokeratin 14 (LL001) and FAPα (H-56) (Santa Cruz Biotechnology, DBA, Milan, Italy) (diluted in 1% BSA/PBS) at 4 °C for 18 h. After incubation, cells were washed three times with PBS and incubated with Alexa fluor conjugated secondary antibodies (Thermofisher Scientific, Milan, Italy) for 1 h at room temperature. Finally, cells were washed three times with PBS, incubated with DAPI (4′,6-diamidino-2-phenylindole) (1:1000) for 3 min and, after washing, immunofluorescence images for the characterization of CAFs were obtained by Cytation 3 Cell Imaging Multimode reader (BioTek) and analyzed using the software Gen5. As it concerns the evaluation of AHR nuclear translocation, immunofluorescence images were obtained by Leica inverted TCS SP8 confocal scanning laser microscope, with a 63X oil immersion objective.

MDA-MB-231, MCF-7, and SkBr3 breast cancer cells were obtained from the ATCC (Manassas, USA). MDA-MB-231 and MCF-7 cells were maintained in DMEM/F12 (Life Technologies, Milan, Italy), 10% fetal bovine serum (FBS) and 1% of penicillin/streptomycin, while SkBr3 cells were maintained in RPMI-1640 (Life Technologies, Milan, Italy), 10% fetal bovine serum (FBS), and 1% of penicillin/streptomycin (Life Technologies, Milan, Italy). Cells were used less than six months after resuscitation and mycoplasma negativity was tested monthly. CAFs were extracted from invasive mammary ductal carcinomas obtained from mastectomies, while normal fibroblasts (NFs) were isolated from a non-cancerous breast tissue at least 2 cm from the outer tumor margin, as previously described [25 (link),28 (link),34 (link)]. Primary cells cultures of breast fibroblasts were characterized by immunofluorescence. Cells were incubated with anti-vimentin (V9, sc-6260), anti-cytokeratin 14 (LL001 sc-53,253) and anti-fibroblast activated protein α (FAPα) (H-56) antibodies that were obtained from Santa Cruz Biotechnology (DBA, Milan, Italy) (Supplementary Figure S1). All cells were grown in a 37 °C incubator with 5% CO₂ and switched to medium without serum and phenol red the day before treatments to be processed for immunoblot and quantitative PCR (qPCR) assays.

Cells were grown in regular media on a cover slip and then fixed in ice-cold methanol at room temperature for 10 min, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated with 1% bovine serum albumin (BSA) in PBS at room temperature for 1 h. After washing with PBS, cells were incubated with primary antibodies against vimentin (V9), cytokeratin 14 (LL001) and FAPα (H-56) (Santa Cruz Biotechnology, DBA, Milan, Italy) (diluted in 1% BSA/PBS) at 4 °C for 18 h. After incubation, cells were washed three times with PBS and incubated with Alexa fluor conjugated secondary antibodies (Thermofisher Scientific, Milan, Italy) for 1 h at room temperature. Finally, cells were washed three times with PBS, incubated with DAPI (4′,6-diamidino-2-phenylindole) (1:1000) for 3 min and, after washing, immunofluorescence images for the characterization of CAFs were obtained by Cytation 3 Cell Imaging Multimode Reader and analyzed using the software Gen5 (BioTek, Ahsi, Milan, Italy).