Fig. 2: CaMK4 modulates Bcl6 gene expression through CREMα.
a Wild-type (Wt, red) or Camk4−/− T cells (blue) CD62L+ CD4+ T cells were differentiated in vitro in Tfh cells for 3 days (n = 4 mice). Gating strategy for differentiation Tfh (left panel) and cumulative data (right panel; n = 4 mice). b Mean fluorescence intensity (MFI) of Bcl6 and PD1 in in vitro differentiated Tfh (n = 4 mice). c Representative western blot of CaMK4, Bcl6, and ß-actin protein expression in in vitro differentiated Tfh (left panel) and densitometry (right panel; each point represents an independent experiment). d Design of the luciferase reporter vector including the full Bcl6 gene promoter (control) or the Bcl6 gene promoter with deletion of the CRE-binding site (Mut). e Relative luciferase activity in in vitro differentiated Tfh transfected with the control and the mutated luciferase promoter (n = 5 mice, conducted in two independent experiments). f Chromatin immunoprecipitation (CHIP) was conducted on the DNA of murine iTfh cell using a CREMα or control antibody. The qPCR targeted the Bcl6 gene promoter region, and relative binding was calculated by comparing the ΔCt between CREMα or control antibody with the input. Three technical replicates are shown for each mouse (n = 3 mice per group). Unpaired two-tailed Student’s t-test (a, c) one-way ANOVA (b) or one-way paired ANOVA (e) with Holm–Sidak’s correction. Bars indicate mean ± s.e.m.
