Extended Data Fig. 6: Calculation of KAT8 and IRF1 endogenous protein concentrations and the staining of KAT8 or IRF1 in patient samples.
a, Representative 3D reconstruction of confocal image of 143B cells stained with Hoechst 33342. The nuclear volumes were calculated by the ellipsoid volume formula indicated on top of the plot. A total of 30 randomly selected cells were measured and analyzed. The statistical data were plotted on the right. Error bar indicates the mean ± SD. b, SFB-KAT8 and IRF1-SFB were expressed in HEK293T cells and purified by streptavidin beads. Coomassie blue staining were applied to validate the protein purity and were quantified by Quantity One. Protein concentrations were calculated by the standard curve generated from Bovine Serum Albumin (BSA) protein. c,d, Quantification of endogenous KAT8 (c) and IRF1 (d). Quantified SFB-KAT8 and IRF1-SFB were used to generate standard curves to estimate the endogenous KAT8 and IRF1 protein numbers from 100, 000 143B cells with or without IFN-γ 100 U/mL for 12 h. Western blot signals were quantified by Quantity One. Protein concentrations were estimated by the protein numbers per cell divided by the mean nuclear volume. e, pTeton-mEGFP-KAT8 and IRF1-mCherry were integrated into the genome of 143B cells by transposase (see Method for details). A concentration gradient of doxycycline from 0.125 to 2 μg/mL were used to induce mEGFP-KAT8 and IRF1-mCherry expressions for 12 hours in the presence of IFN-γ 100 U/mL, then cells were fixed and visualized by confocal microscope (on the left side), proteins were harvested and detected by western blot (on the right side). f-h, Indicated patient tissue samples were stained with anti-PD-L1 antibodies and either anti-KAT8 or anti-IRF1 antibodies as indicated. Representative images are shown (f). The fluorescence intensities of KAT8, IRF1 and PD-L1 of each cell in the representative images were measured using Fiji. Data of KAT8 and PD-L1 (g), IRF1 and PD-L1 (h) are plotted. Spearman’s correlation coefficients (ρ) and the two-sided p values are shown. The fitted linear regression lines are also depicted in grey. The experiments in b-e were repeated three times with similar results. The experiments in f were repeated twice with similar results.
