Alexa fluor 488

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.

A liver sample was fixed in 4% Paraformaldehyde at 4 °C for one day, cryoprotected in 30% sucrose, frozen and sectioned at 20 µm. Protein detection was performed as described^{34 (link)}. Briefly, the sections were washed in and permeabilized for 20 min with PBS-0.4% (v/v) Triton X-100. Nonspecific antibody binding sites were blocked by incubation with a blocking solution (PBS, 10% goat serum, 0.1% Triton X-100) for one hour. The liver sections were then incubated overnight (4 °C) with Rabbit anti-SIRT1 antibody diluted 1:100 in blocking solution. For fluorescence detection was used, Alexa Fluor® 488 coupled to an anti-rabbit secondary antibody (GeneTex, Inc., San Antonio, CA, USA) diluted 1:200 in a blocking solution. The nucleus was stained by adding 1 µg/ml of 4,6-diamidino-2-phenylindole (DAPI) in PBS. Finally, the liver sections were mounted on a Fluoromount G mounting medium (EMS, Hatfield, PA, USA). The images were taken with a TCS SP2 confocal laser microscopy system (Leica Microsystems, Wetzlar, Germany) equipped with an inverted DMIRE2 Leica microscope. Confocal fluorescence images were analyzed using LCS Lite software from Leica.