Fig 2: Arginine-induced glucagon secretion was decreased by Pkcδ knockdown in InR1G9 cells. (a) Glucagon secretion was significantly increased by 15-mM arginine and 100-mM IBMX in InR1G9 cells. (b–c) InR1G9 cells were treated with or without 15-mM arginine for 30 min; then cell lysates were examined for Pkcδ and pPkcδ (Thr505) by Western blotting. β-actin was employed as an internal control. Arginine significantly increased Pkcδ (Thr505)/Pkcδ relative ratio in InR1G9 cells. (d) InR1G9 cells were transfected with plasmid encoding MARCKS conjugated with GFP for 48 h followed by 100-mM PMA, 15-mM arginine, or 15-mM arginine plus 10-µM rottlerin administration. MARCKS localized on the cell membrane was translocated to the cytoplasm by PMA. Arginine also translocated MARCKS to the cytoplasm, which was canceled by rottlerin. Representative photomicrographs are shown. Scale bars represent 20 µm. (e) Arginine-induced glucagon secretion was significantly reduced by Prkcd #2 siRNA in InR1G9 cells. We performed triplicate and three independent experiments. * p < 0.05; t-test (c) or ANOVA (Bonferroni adjustment) (a,e). Data are expressed as means ± SEM.

Fig 3: Genetic and clinical features of a patient with autosomal recessive PKCδ deficiency. a Pedigree of the family, showing familial segregation of the PRKCD alleles. Generations are indicated by Roman numerals (I–II), and each individual is indicated by an Arabic numeral (1–2). The patient is represented by closed black symbol. b Images of the patient presenting left submandibular (top) and left lower anterior cervical (bottom) lymphadenitis. c Flow cytometry images of intracellular ROS production in neutrophils (top) and monocytes (bottom) from a healthy control (Ctrl) and the patient before (NS) and after PMA stimulation. d Electropherogram of exons 4 and 5 showing the variants (p.C95* and D126Y) found in the patient and their comparison with a healthy control, the patient’s parents and brother. e Minor allele frequency (MAF) and combined annotation-dependent depletion (CADD) score of the heterozygous PRKCD variants (red triangles) found in the patient, variants reported in other PKCδ-deficient patients (blue lozenges) or found in the homozygous state in gnomAD v2.1.1 (black circles). The dotted line represented the mutation significant cutoff (MSC) with its 99% confidence interval. f Schematic representation of the PRKCD gene/protein. Coding exons are numbered from 3 to 19. The PKCδ protein is presented with the C2-like domain (gray), C1 domain (light blue), ATP-binding domain (green), and the substrate-binding domain (orange). Mutations reported in PKCδ-deficient patients are indicated in black and the two heterozygous mutations found in the patient are indicated in red

Fig 4: Functional analysis of the patient’s variants by overexpression and investigation of primary cells. a Proportions of the various PRKCD transcripts identified by TOPO-TA cloning on monocyte-derived macrophages (MDMs) from the patient (P) and a healthy control (Ctrl). b Schematic representation of the different splice variants found in P and their predicted consequences for the PKCδ protein. The positions of affected amino acids are indicated by red triangles. c Western blot of total cell extract from HEK293T cells not transfected (NT), or transfected with the pCMV6 empty vector (EV), wild-type (WT), or mutated PRKCD pCMV6. PKCδ phosphorylation was detected with antibodies against the phosphorylated p.T505 and p.S643 residues. d RT-qPCR for PRKCD on MDMs from a healthy control (Ctrl) and the patient (P), with probes spanning the junction between exons 3 and 4 (left) and exons 17 and 18 (right). GUSB was used for normalization (technical duplicates ± SD). e Western blot of total protein lysates isolated from polymorphonuclear neutrophils (PMNs) from a healthy control (Ctrl), the patient (P), her mother (M), and father (F). The asterisk indicates nonspecific bands. f Extracellular H2O2 release, measured in the Amplex Red assay after PMA stimulation with or without IFN-γ priming, for MDMs from a local control (Ctrl), the patient (P), and her WT/WT father (F) (technical triplicates ± SD). g Western blot on whole-cell lysates from MDMs of the patient (P) and a healthy control (Ctrl) not stimulated (NS) or stimulated with PMA for 15 min. The phosphorylation of p40phox was assessed with an antibody against the phosphorylated p.T154 site

Fig 5: Regulation of prkcda expression by an intronic enhancer element(A) Expression of PKC isoforms in kidney marrow cell clusters identified in Figure 3.(B) UMAP representation of global chromatin accessibility in single zebrafish kidney marrow cells. Samples are from the same kidney marrow pools shown in Figure 3.(C) Chromatin accessibility at the prkcda locus in kidney marrow cells by cluster. Peaks of chromatin accessibility and Cicero links identifying co-accessible peaks are shown.(D) Bulk ATAC sequencing showing chromatin accessibility at the human PRKCD locus in E4-HUVEC cells. Peaks were identified using MACS2.(E) FIMO analysis of zebrafish and human intron enhancer peak sequences. The number of putative binding sites with q < 0.05 in each set is indicated.(F) Expression of an mClover reporter construct driven by the zebrafish prkcda intronic enhancer in uninjected (i) and injected (ii) animals. Arrowhead indicates the bulbus arteriosus. Bar, 25 μm.(G) Immune fluorescence in a 72 hpf zebrafish embryo expressing the prkcda intronic enhancer reporter. Cryosections were probed with anti-GFP/mClover and anti-Fli1 antibodies and fluorescent secondaries (see STAR Methods). The outline indicates pericardium (P), ventricle (V), and bulbus arteriosus (BA, cardiac outflow tract). Bar, 10 μm. See also Figure S3 and Video S1.