Direct regulation of gene expression by nuclear PKC-θ. (A) Representative western blot of HA-tagged PKC-θ protein (HAPKC-θ) levels in non-stimulated (NS) and PMA and Ca2+ ionophore (P/I)-activated Jurkat T cells transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. (B) Representative confocal microscopy images showing subcellular localization of PKC-θ in Jurkat T cells transfected with WT or cytoplasm-restricted PKC-θ (NLS) plasmids. (C) Quantification of microscopy to show the ratio of nuclear to cytoplasmic-located HA-tagged PKC-θ in Jurkat T cells transfected with WT or cytoplasm-restricted PKC-θ (NLS) plasmids (mean±s.e.m., n=3 repeats). ***P<0.0001 (Mann–Whitney test). (D) Gene expression of transcriptional-memory-responsive genes in Jurkat T cells transfected with vector only (VO), wild-type (WT) and cytoplasm-restricted PKC-θ mutant plasmids (NLS) (mean±s.e.m., n=3) with the percentage inhibition calculated between the WT and NLS during primary (1°) activation for TNF and secondary (2°) activation for IL2, IL8, IL3, IFNG, CSF2, CCL4L1 and TNFSF9. NS, non-stimulated cells. *P≤0.05, **P≤0.01 and ***P≤0.001 (two-way ANOVA).