Sterilized cover glasses

OEC-M1 and SCC-25 cells were exponentially grown on sterilized cover glasses (Paul Marienfeld, Lauda–Königshofen, Germany) and treated with 10⁻⁷ M T₄ in the presence or absence of a STAT3 inhibitor, S31-201 (Sigma, SML0330) for another 24 h. Samples were immediately fixed with 4% paraformaldehyde in PBS for 10 min. Cells were permeabilized in 0.1% Triton X-100 in PBS for 20 min. After 1 h of 1% BSA blocking, cells on the slides were incubated with an anti-PD-L1 (GeneTex, San Antonio, TX, USA, GTX104763) and P300 (GeneTex, GTX30618) antibody overnight at 4 °C. Then, cells were incubated with secondary antibody conjugated with Alexa Fluor 647 (abcam, Cambridge, UK, ab150079), Alexa Fluor 488 (GeneTex, GTXGTX213110-04) or Alexa Fluor 594 (GeneTex, GTX213111-05) and DAPI (ThermoFisher Scientific, Waltham, MASS, USA, S36938) stain for nuclei. The fluorescent signals of PD-L1 or P300 were recorded and analyzed with a TCS SP5 Confocal Spectral Microscope Imaging System (Leica Microsystems, Wetzlar, Germany). The figures shown are representative of four fields for each experimental condition.

Exponentially growing primary colorectal cancer cells and HT-29 were seeded on sterilized cover glasses (Paul Marienfeld, LaudaKönigshofen, Germany). After treatment of NDAT (0.1 μM) or LY294002 (10 μM) for 24 h, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min and then permeabilized in 0.06% Triton X-100 for 30 min. Cells were incubated with monoclonal rabbit anti-PD-L1 antibody, followed by an Alexa-647-labeled goat anti-rabbit antibody (Abcam, Cambridge, MA, USA) and mounted in EverBrite Hardset mounting medium with DAPI (Biotium, Fremont, CA). The fluorescent signals were recorded and analyzed with the TCS SP5 Confocal Spectral Microscope Imaging System (Leica Microsystems). The figures shown are representative of at least four fields for each experimental condition.