Kn 93

Murine CD62L⁺CD4⁺ T cells were isolated using magnetic cell sorting from mouse splenocytes according to the manufacturer’s instruction (Miltenyi Biotec, Charlestown, MA, USA). In total, 3 × 10⁵ cells were cultured in a 48-well plate coated with goat anti-hamster crosslinking antibody in RPMI 1640 medium complemented with penicillin/streptomycin, 10% fetal bovine serum, and ß-mercaptoethanol. To differentiate the cells into T_fh, the medium was supplemented with anti-CD3 (0.25 µg/ml, 145-2C11; BioLegend, San Diego, CA), anti-CD28 (0.5 µg/ml, 37.51; BioXcell, Lebanon, NH), interleukin-21 (50 ng/mL, R&D systems, Minneapolis, MN, USA) interleukin-6 (100 ng/mL, R&D systems), anti-TGFß (0.5 µg/mL, clone 1D11; R&D systems) anti-IFN-gamma (10 µg/ml; BioXcell, Lebanon, NH, USA), and anti-IL-4 (10 µg/ml; BioXcell). The cells were retrieved for analysis on day 3 of the culture.
For the coculture experiment using human T_fh and B cells, 50,000 freshly sorted autologous T_fh and B cells were cocultured at a 1:1 ratio in a 96-well plate with staphylococcal enterotoxin B (SEB, 1 ng/mL, Fisher Scientific) for 7 days at +37 °C. The CaMK4 inhibitor KN93 (Selleckchem, Houston, TX, USA) or its vehicle (dimethysulfoxide, DSMO) was used at the concentration of 10 µM.

Anti-CaMKIIδ (Item No. sc-100362) was purchased from Santa Cruz (USA), anti-CaMKII (phospho T287) (Item No. ab182647), anti-β Tubulin (Item No. ab6046), anti-cardiac troponin T (Item No. ab209813) were purchased from Abcam (Cambridge, UK), Tropomyosin-1/3 (Item No. D17B8) were purchased from Cell Signaling Technology (Beverly, MA, USA), Anti-MYOM2 (Item No. A20526) was purchased from ABclonal (Wuhan, China). KN-93 and KN-92 were obtained from Selleck (Houston, TX, United States).

H-89 and KN-93 (1 μmol/L; Selleck), specific inhibitors for PKA and CaMKII, respectively, were used to investigate PKA/Ser¹⁶-PLB, and CaMKII/Thr¹⁷-PLB signaling pathways mediated the effect of OPD. Cells were treated with the abovementioned medium containing H-89 and KN-93 for 6 h, finally cultured in a medium containing ISO (1 μmol/L) and the highest concentration of OPD (1 μmol/L).

Staurosporine (ThermoFisher, Waltham, MA, United States) was made as a 2.15 mM stock solution in DMSO and used at a final concentration of 100 nM. KN93 (Selleckchem, Houston, TX, United States) was made up as a 3.35 mM stock solution in MQ water and used at a final concentration of 1 μM. KN92 (Biovision, Milpitas, CA, United States) was made up as a 3.6 mM stock in DMSO and used at a final concentration of 1 μM. Myristoylated-AIP (mAIP; Tocris, Bristal, United Kingdom), a membrane permeant analog of the CaMKII inhibitor AIP (Autocamtide-2-related inhibitory peptide), was made as a 293 μM stock solution in MQ water and used at a final bath concentration of 100 nM. In the isolated cell experiments, drugs were added to the external bath solution. For drugs made up in DMSO, addition of DMSO to the bath solution at the same concentration as in the test condition had no effect on pSTX3B or pCaMKII levels (not shown). For electrophysiological measurement of exocytosis, a 1 mM stock solution of AIP (Tocris, Bristal, United Kingdom) was made in MQ water and added to the internal pipette solution at a final concentration of 25 μM (Sakaba and Neher, 2001 (link); Tatone et al., 2002 (link); Gao et al., 2006 (link); Mockett et al., 2011 (link)). All other chemicals were purchased from Sigma.

For B-cell differentiation, isolated naive B cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and 1% penicillin/streptomycin (Life Technologies). Cells were stimulated with antihuman IgM (5 μg/ml, Sigma, #10759) and antihuman CD40 (1 μg/ml, Bioxcell, #BE0189) antibodies in the presence of interleukin (IL)-2 (20 ng/ml, PeproTech), IL-4 (25 ng/ml, Sino Biological) and IL-21 (50 ng/ml, Sino Biological) for 8 days.
For signaling study, B cells were stimulated with antihuman IgM (5 μg/ml) and antihuman CD40 (1 μg/ml) antibodies for 72 h. CRAC channel inhibitor YM-58483 (50 nM, MedChemExpress, USA) or CaMK2 inhibitor KN-93 (5 μM, Selleck) was included in some of the experiments. Cells were maintained in a humidified atmosphere at 37°C with 5% CO₂. Cells were collected for Western blot or FACS analysis.