Chamber slide

Cells were seeded onto chamber slides (Merck Millipore Ltd., Watford, UK) at 3 × 10⁴ cells/chamber for 48 h where after fixing with 4% paraformaldehyde (Histolab Products AB, Västra Frölunda, Sweden) in PBS for 5 min at room temperature (RT). Fixed cells were washed twice with PBS. After washing with PBS, the slides were blocked with 1% BSA for 45 min before incubated with CD24 (Invitrogen, #MA5–11833) diluted in 1% BSA/PBS overnight. Cells were washed three times with PBS, followed by incubation with Alexa Fluor 568 conjugated anti-mouse (Invitrogen #A11004) dissolved in 1% goat serum/1% BSA for 1 h at RT. After washing three times with PBS, the coverslips were stained with DAPI (Sigma-Aldrich) for 3 min. Stained cells were washed twice with PBS and once with water before cells were mounted on cover glass slides with prolong diamond anti-fade mountant media (Life Technologies, Carlsbad, CA, USA). Cells were examined under a Zeiss Axio Vert.A1 microscope (Carl Zeiss, Oberkochen, Germany) at ×5 magnification.

Metformin and phenformin were purchased from Sigma-Aldrich (St. Louis, MO, USA); human TGF-β1 and antibodies against β-actin, p-AMPKα Thr172, AMPKα, p-ACC Ser79, ACC, Slug, p-Smad3 Ser423/425, and Smad2/3 were from Cell Signaling Technology (Beverly, MA, USA); ELISA kits for human and mouse TGF-β1, antibodies against E-cadherin, and vimentin were from Abcam (Cambridge, MA, USA); monoclonal antibody against LKB1 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); growth factor-reduced Matrigel and monoclonal antibody against β-catenin were from BD Biosciences (San Jose, CA, USA); Lipofectamine 2000, 4′,6-diamidino-2-phenylindole (DAPI), fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit antibodies were from Life Technologies (Grand Island, NE, USA); Chamber slides was from EMD Millipore (Billerica, MA, USA).

MDA-MB-231, BT-474, SK-BR-3 and JIMT-1 BC cell lines were cultured in chamber slides (Merck-Millipore, Darmstadt, Germany) up to a confluence of 90%. Immunofluorescence staining was performed according to manufactures protocol of the primary anti-PD-L1 antibody (Cell Signaling). In brief, the medium was removed and the cells were washed with PBS, PBS was discarded, ice-cold 100% methanol was added and incubated for 10 min at −20 °C. Then the slides were rinsed with PBS for 5 min and incubated with blocking buffer for 60 min. The blocking buffer was aspirated afterwards and the primary anti-PD-L1 antibody (1:100) (D8T4X, Cell Signaling, Danvers, MA, USA) or the isotype control (1:833) (DA1E, IgG XP^® Isotype Control, Cell Signaling, Danvers, MA, USA) were applied in appropriate dilution for overnight incubation at 4 °C. After a 3 × 5 min washing step with PBS, the slides were incubated for 1 h at room temperature in the dark with goat anti-rabbit IgG AlexaFluor 532 secondary antibody (1:500) (Thermo Fisher Scientific, Bremen, Germany). The slides were washed again (3 × 5 min) with PBS and were cover-slipped with VECTASHIELD Antifade Mounting Medium with DAPI (VECTOR Laboratories Inc., Burlingame, CA, USA).