Fig. 2: CREM upregulation is mediated through the PKA–pCREB axis.
From: CREM is a regulatory checkpoint of CAR and IL-15 signalling in NK cells
a, Schematic of experiments for b–d. Created in BioRender. Rafei, H. (2025) https://BioRender.com/cczoc8f. b, pCREB expression measured by phospho-flow in NT, CAR70, CAR70.3ζ.Y6F and CD27(ECD) NK cells that were treated with (+) or without (–) CD70 antigen for stimulation, H89 (PKA inhibitor) or EGTA (calcium chelator) (n = 5 donors). c, FACS plots of pCREB in CAR70 NK cells under the indicated conditions. Forskolin (FSK) was used as the positive control. d, CREM expression by qPCR in the indicated NK cell conditions treated with or without CD70 antigen, H89 or EGTA for 24 h (n = 3 donors). e, Schematic of the experiments in f–j. Created in BioRender. Rafei, H. (2025) https://BioRender.com/j6djzbh. f,g, FACS plots (f) and quantification (g) of pCREB in NT NK cells that were unstimulated or stimulated for 30 min with increasing concentrations of IL-15 (n = 14 donors). h, Whole-cell lysates from NT NK cells that were treated as in f and g were analysed by western blotting for pCREB and total CREB. β-actin served as the loading control. A representative blot is shown. i, Densitometry analysis quantifying the relative band intensity of pCREB normalized to total CREB (n = 5 donors). j, CREM expression by qPCR in NT NK cells that were stimulated or not for 24 h with increasing concentrations of IL-15 in the presence or absence of H89 (n = 3 donors). k, ChIP–qPCR for the enrichment of CREB in the promoter region of CREM and a region of no enrichment (negative control) in the indicated conditions (n = 3 donors). l, ChIP–qPCR for the enrichment of pCREB in the promoter region of CREM in NT NK cells incubated for 6 h in the absence or presence of increasing concentrations of IL-15 (n = 3 donors). Statistical comparisons were performed using two-way ANOVA with Bonferroni correction (b,d), one-way ANOVA with Tukey’s correction (g,i) or two-sided multiple t-tests with Holm–Šídák correction (j–l). Data are presented as the mean ± s.e.m.
