Pa5 99584

Cells were lysed with RIPA lysis buffer (Solarbio, R0020) containing PMSF (Solarbio, P0100), phosphatase inhibitor cocktail I (MCE, HY-K0021) and phosphatase inhibitor cocktail II (MCE, HYK0022). Total protein levels in cell lysates were determined by the BCA kit (Thermo Fisher Scientific). Lysates containing 10-20 μg of protein were subjected to SDS-PAGE separation and transferred to the PVDF membrane (Millipore) for determination. The antibody used for A3A (PA5-99584) was from Thermo Fisher. Vinculin (ab219649), STAT1 (ab234400), γH2AX (phospho S139, ab26350) antibodies were from Abcam, while antibodies against cGAS (#79978), STING (#13647), phospho-STING (p-STING) (Ser366; #50907), IRF3 (#11904), phospho-IRF3 (p-IRF3) (Ser396; #29047), TBK1 (#3504), phospho-TBK1 (p-TBK1) (Ser172; #5483), and phospho-STAT1 (p-STAT1) (Tyr701; #7649) were from Cell Signaling Technology. Antibodies against for PD-L1 (66248-1-Ig) was from Proteintech. All the antibodies were diluted in Universal Antibody Diluent (NCM Biotech, WB500D). The membrane was incubated overnight at 4°C with primary antibody and 2 hours with secondary antibody. The signal was detected with a SuperSignalTM West Pico/Femto Chemiluminescent Substrate kit (Thermo Fisher, 34580) through the Amersham Imager 600.

The sections were deparaffinized, and rehydrated as described for the IHC procedure. Opal multiplex staining was performed according to the Opal 5-Color Manual IHC Kit (PANOVUE). The sections were incubated by antibodies against A3A (1:300, PA5-99584, Thermo Fisher), CD8 (1:800, ab93278, Abcam), Granzyme B (1:300, #17215, CST), PD-L1 (1:400, ab205921, Abcam), and FOSL1 (1:50, sc-28310, Santa Cruz) at 4°C overnight. Opal 520 corresponding to the A3A antibody, Opal 570 to Granzyme B (or PD-L1) antibodies and Opal 650 to CD8 (or FOSL1) antibodies were used to generate different immunofluorescent signals. Slides were counterstained with DAPI for nuclei visualization, and subsequently coverslipped using a VectaShield Hardset mounting media. The slides were imaged using Vectra Polaris Automated Quantitative Pathology Imaging System (Perkin Elmer). We used inForm software (Perkin Elmer) to unmix and remove autofluorescence and analyze the multispectral images.

Formalin-fixed paraffin-embedded (FFPE) tissue sections were deparaffinized with Histo-Clear II twice and rehydrated with gradient ethanol, followed by inactivating of endogenous peroxidase and retrieval antigen. For IHC staining, the sections were stained by antibodies against A3A (1:300, PA5-99584, Thermo Fisher), and FOSL1 (1:50, sc-28310, Santa Cruz) at 4°C overnight and then detected with the ABC Kit (Pierce). The labeling score of intensity was estimated as negative (0), weak (1), moderate (2) and strong (3). The extent of staining, defined as the percentage of positive stained cells, was scored as 1 (≤ 10%), 2 (11%-50%), 3 (51%-80%) and 4 (> 80%). The total immune reactive score was obtained by multiplying the staining score of intensity and that of extent, ranking from 0 to 12.