Figure 5

CBP/p300-deficient cells revealed a diminished NKG2D-L upregulation in response to HDACi and DNA damage. (a–g) HEK-293 CBP/p300 double-knockout (dKO) cells were generated using the CRISPR/Cas9 nuclease (dKOnuc) or CRISPR/Cas9 nickase (dKOnic) approach. (a, left panel) Western blot of CBP/p300 dKO or WT HEK-293 cells using an anti-CBP antibody (C-terminal, clone A22) detecting both CBP and p300. GAPDH was used as loading control. (a, right panel) Real-time PCR for CBP and p300 mRNA expression levels relative to GAPDH. (b) Basal expression levels of MICA/B measured by flow cytometry. (c) Cells were treated with 100 nM LBH589 for indicated time periods and MICA/B expression was analyzed by FACS. (d, e) Flow cytometric analysis of MICA/B (left panel) and ULBP2 (right panel) of CBP/p300 dKO or WT HEK-293 treated with 100 nM LBH589 or 10 μM Ara-C for 16 h. (f) Intracellular FACS for acetylated lysine after 1 h of treatment with 100 nM LBH589. (g) FACS-based NK cell killing assay. CBP/p300 dKO or WT HEK-293 cells were treated with 100 nM LBH589 for 16 h, washed and incubated with primary human NK cells in indicated ratios for 3 h. (g, left panel) One representative experiment and (g, right panel) mean and s.e. of four independent experiments (ratio 5:1).