Tif1γ

Formalin-fixed, paraffin-embedded 3-mm thick sections were deparaffinized and rehydrated, and then the sections were, respectively, incubated with primary antibodies against p53 (Abcam, UK) (1:100), HRAS (Abcam, UK) (1:100), NSG1 (Abcam, UK) (1:200), and TIF1γ (cell signaling technology, USA) (1:1000) for 18 h at 4 °C to carry out the standard IHC staining as described previously 10 (link). The outcome of IHC staining for 30 pairs of CC tissues and matched paracancerous tissues, randomly selected from 157 cases with CC at early stage, was manually evaluated and scored by two independent certified pathologists. The intensity of staining was graded as: 0 = undetectable, 1+ = weak staining, 2+ = moderate staining and 3+ = strong staining.

Formalin-fixed, paraffin-embedded 3-mm thick sections were deparaffinized and rehydrated. These preparations were stained at room temperature. Staining was performed using an SP immunohistochemistry kit (Fuzhou Maixin Biotech, China), following the manufacturer's recommendations. The sections were, respectively, incubated with primary antibodies against TIF1γ (Cell Signaling Technology ,USA) (1:1000) for 18 h at 4 ℃, followed by incubation with the biotinylated secondary antibody for 10 min at room temperature, and horseradish peroxidase-conjugated streptavidin for 10 min at room temperature. The immnunoreactivities were visualized brown with diaminobenzidine (DAB kit; Lab Vision) and counterstained with Mayer's hematoxylin. The primary antibody was replaced with non-immune sera for negative controls, keeping all other steps in the process the same. Specimens were conducted under identical conditions. The outcome of IHC staining for 60 pairs of LC tissues and matched paracancerous tissues, randomly selected from 148 cases of adenocarcinoma, 64 cases of squamous cell carcinomas and 36 cases of small cell lung cancer, was manually evaluated and scored by two independent certified pathologists. The intensity of staining was graded as: 0=undetectable, 1+=weak staining, 2+=moderate straining, and 3+=strong staining.

Immunoprecipitation (IP) and Western blotting (WB) assays were conducted as previously described (19 (link)). The specific antibodies used were as follows: KAT2B (#3378, 1:1000, Cell Signaling Technology), H3K9ac (#9649, 1:1000, Cell Signaling Technology), H3K14ac (#26828, 1:1000, Cell Signaling Technology), H3K9ac (#9649, 1:1000, Cell Signaling Technology), Histone H3 (#4499, 1:1000, Cell Signaling Technology), TIF1α (ab38264, 1:1000, Abcam), TIF1β (#4124, 1:1000, Cell Signaling Technology), TIF1γ (#13387, 1:1000, Cell Signaling Technology), NCOA1 (#20301, 1:1000, Cell Signaling Technology), NCOA2 (#96687, 1:1000, Cell Signaling Technology), NCOA3 (#2126, 1:1000, Cell Signaling Technology), RBM3 (ab211356, 1:1000, Abcam), YAP (#14074, 1:1000, Cell Signaling Technology), Flag M2 antibody (#14793, 1:1000, Cell Signaling Technology), HA antibody (#2367, 1:1000, Cell Signaling Technology), and β-actin (#4970, 1:1000, Cell Signaling Technology).