Fig 1: TET inhibits CAMKIIδ activity and B cell lymphoma proliferation in vitro and in vivo. A) Molecular structure of Tetrandrine (TET). B) CETSA assay to determine the physical interaction between TET and CAMKIIδ. Cells were incubated with either DMSO or 10 µM TET for 1 h and cell lysates were incubated with gradient temperature from 45 °C to 52.5 °C. CAMKIIδ protein levels were detected by western blot assay. C) In vitro kinase assay of CAMKIIδ on FOXO3A in present with TET. HEK293 cells were transfected with 3xFLAG‐FOXO3A plasmid and FOXO3A protein was pulled down by FLAG‐beads. In vitro kinase assay was performed with purified CAMKIIδ protein on FLAG‐tagged FOXO3A, incubated with calcium, calmodulin, ATP, and indicated concentrations of TET. Total phosphorylated serine and threonine levels were detected by western blotting. D) Representative western blot of SU‐DHL‐6 CAMK2D WT and KO cells treated with indicated dosages of TET for 24 h. Tumor volume growth curve E) and tumor gross pictures F) of NSG mice treated with either PBS or TET. G) Tumor weight at the endpoint of the 32nd day. **p < 0.01 versus PBS group. H) Representative western blot analysis of FOXO3A and CAMKIIδ protein levels in tumor tissues from PBS and TET treated groups. GAPDH is used as the loading control. I) Representative images of H&E and IHC staining of Ki67 and FOXO3A with sections of tumors from PBS and TET‐treated groups. The bar represents 50µm. J) IHC scores for CAMKIIδ and FOXO3A. K) Correlation of overall CAMKIIδ and nuclear FOXO3A levels. The P‐value was evaluated by Pearson Correlation assay. T‐test was used for analysis unless otherwise indicated. All the results shown here are representative of three independent experiments.
Fig 2: CAMKIIδ phosphorylates and destabilizes FOXO3A. A) Identification of FOXO3A S7 and S12 phosphorylation by CAMKIIδ from proteomic analysis. The mass shift of phosphorylation is 79.9663. Determination of S7: The theoretical molecular weight of SPA's three amino acid residues is 255.12. The calculation (b9−b6 = 1135.61−800.4249 = 355.185) matches the theoretical shift for SPA plus phosphorylation, calculated as (255.12+79.9663 = 355.086). Thus, it can be determined that the serine residue in SPA underwent phosphorylation modification. Determination of S12: The theoretical molecular weight of the peptide plus the charge is 2909.4. The actual mass shift across the entire peptide was calculated as (m/z × charge = 1023.118 × 3 = 3069.354), suggesting there are two phosphorylation sites on this peptide (2909.4+79.9663 × 2 = 3069.33). Furthermore, the y2−y1 fragment analysis indicates that S26 did not undergo phosphorylation modification. Since S7 was confirmed to be phosphorylated, it can be inferred that S12 is another phosphorylation site. B) HEK293 cells transfected with 3xFLAG‐FOXO3A, and CAMK2D‐HA or CAMK2G‐HA plasmids were co‐immunoprecipitated with HA‐beads; protein levels of 3xFLAG‐FOXO3A were analyzed by western blot. C) (left) In vitro kinase assay of CAMKIIδ on FOXO3A wild‐type and site mutants. HEK293 cells were transfected with 3xFLAG‐FOXO3A wild‐type, 3xFLAG‐FOXO3A S7A, S12A, and S7/12A plasmids. Proteins were then pulled down by FLAG beads, followed by an in vitro kinase assay supplied with Ca2+, calmodulin, and ATP. Total phosphorylated serine and threonine levels were detected by western blots. (right) In vitro kinase assay of CAMKIIδ and CAMKIIγ on FOXO3A protein. D) Radioisotope‐based in vitro kinase assay was performed on purified FOXO3A protein using 4 nM CAMKIIγ and 0.1 nM CAMKIIδ. E) Mutu CAMK2D wild‐type and knockout cells were treated with cycloheximide (CHX) for the indicated times. FOXO3A protein levels were then analyzed by western blot with Hsp90 as the loading control (left panel). Protein levels were measured with densitometric intensity. FOXO3A levels were quantified relative to Hsp90 levels and graphed as the percentage of remaining FOXO3A protein after treatment (right panel). F) Wild‐type BCL cells were treated with MG132, chloroquine, or DMSO control for indicated times, and FOXO3A expression was determined by western blot. GAPDH was used as a loading control. G) Venn diagram showing identified potential ubiquitylation regulator for FOXO3A by overlapping the MS results and online prediction. H) HEK293 cells transfected with vector control, WT or S7&12 mutant 3xFLAG‐FOXO3A were co‐immunoprecipitated with FLAG‐beads; protein levels of USP7 were analyzed by western blot. I) HEK293 cells overexpressing FLAG‐FOXO3A were transfected with HA‐ubiquitin and treated with 0.5µM USP7 inhibitor for 24 h. FOXO3A immunoprecipitated with FLAG beads, and HA‐ubiquitin was examined by western blot. All the results shown here, except panel A and G, are representative of three independent experiments. T‐test was used for analysis, unless otherwise indicated.
Fig 3: CAMKIIδ regulates lipid homeostasis in B cell lymphoma. A) Confocal microscope imaging of BODIPY 493/503 staining in cells as indicated. Blue represents DAPI staining, and green represents BODIPY staining. The bars represent 10 µm. **p < 0.01. B) Indicated cells were stained with BODIPY 493/503 and analyzed by flow cytometry. Mean fluorescent intensity (MFI) was normalized by fluorescent mode. Representative overlays between indicated cells were shown. **p < 0.01. Data are presented as mean ± SD from three technical replicates per group. C) Representative images of IHC staining for perilipin3 on EµMYC; Camk2dwt/wt and EµMYC; Camk2d ‐/‐ mice spleen sections. The bars represent 50 µm. H score was determined by ImageJ. **p < 0.01. D) Lipidomic analysis was performed to compare the lipid profile of SU‐DHL‐6 CAMK2D wild‐type with knockout cells. The bar plot showed a Log2 fold change of individual (left) triglycerides, and (right) phospholipids. PC: Phosphatidylcholine; PE: Phosphatidylethanolamine; PS: Phosphatidylserine; PI: Phosphoinositide. E) mRNA expression of genes related to lipolysis, fatty acid oxidation (FAO), and lipid droplets (LD) in indicated cells. The data is representative of three experiments (average of three values ± standard error). *p < 0.05, **p < 0.01. F) Summary of the lipid components change after CAMK2D inhibition. The results shown here, except panel D, are representative of three independent experiments. T‐test was used for analysis, unless otherwise indicated.
Fig 4: FOXO3A is a key substrate of CAMKIIδ. A) Proteomic analysis was conducted between Mutu CAMK2D wild‐type and knockout cells. The Venn diagram showed FOXO3A was one of the 18 proteins with both expression and phosphorylation changes. B) Representative western blots for FOXO3A protein levels in CAMK2D wild‐type (WT) and knockout (KO) cells. Data are presented as mean ± SD from three independent biological samples per group. **p < 0.01. C) Representative western blot and D) IHC staining of FOXO3A between EµMYC; Camk2d wt/wt and EµMYC; Camk2d‐/‐ mice. The bars represent 50µm. H score was determined by ImageJ. **p < 0.01. E) Growth curve of Mutu CAMK2D knockout cells with either FOXO3A knockdown (SH) or scramble control (SCR). Data are presented as mean ± SD from four independent biological samples per group. F) (upper) Representative images of the electron microscope of lipid droplets in the indicated cells. The bars represent 2 µm. (lower) Confocal microscope imaging of BODIPY 493/503 staining in the indicated cells. Blue represents DAPI staining, and green represents BODIPY staining. The bars represent 10 µm. G) The median fluorescent intensity (MFI) of BODIPY 493/503 in the indicated cells was analyzed by flow cytometry. MFI was normalized by fluorescent mode. Representative overlays among the indicated cells were shown. Data are presented as mean ± SD from three technical replicates. H) Diagram of FOXO3A binding on IRF4 promoter region. I) The enrichment of FOXO3A on the IRF4 promoter region was examined by ChIP. The enriched DNA sequence was detected by qPCR. **p < 0.01 in FOXO3A‐ChIP compared to IgG. All the results shown here, except panel A, are representative of three independent experiments. T‐test was used for analysis, unless otherwise indicated.
Fig 5: CAMK2D inhibition impairs mitochondria functions. A) GSEA analysis showed top hallmark pathways enriched in CAMK2D‐/‐ cells. B) Heatmap analysis of genes in OXPHOS pathways from RNA‐seq between CAMK2D WT and KO cells. C) Representative measures of oxygen consumption rate (OCR) between Mutu CAMK2D WT and KO cells upon addition of Oligomycin, FCCP, Rotenone, and AntimycinA. Quantified ATP production. **p < 0.01. Mutu CAMK2D WT and KO cells were treated with either control or BPTES (D), UK5099 (E), and etomorxir (F). Oxygen consumption rate (OCR) upon the addition of indicated inhibitors was measured by Agilent Seahorse XF Analyzers. Data in panels C‐F are presented as mean ± SD from four independent biological samples for each group. G) Representative images of the electron microscope of mitochondria in the indicated cells. The bars represent 2 µm (upper) and 1 µm (lower). The results shown in panels C‐G are representative of three independent experiments. T‐test was used for analysis, unless otherwise indicated.
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