Bapta am

Calcium concentrations were determined from the ratio of fura-2 fluorescence intensity at 340-nm excitation and 380-nm excitation. The 340-nm fluorescence of fura-2 increases and the 380-nm fluorescence decreases with increasing [Ca²⁺]i. For [Ca²⁺]i measurements HGECs and podocytes (20,000 cells/well) were plated on black 96-well plates with a clear bottom in complete medium. After 1 day the cells were serum-starved for 2-h. In the last 45 min of calcium-free serum-starvation, FURA-2AM (5 μM; Invitrogen) was added to the cells, then rinsed with Hanks Balanced Salt Solution (HBSS, Gibco BRL). FURA-2AM-loaded cells were sequentially excited at 340 and 380 nm by spectrophotometer microplate reader (Synergy MX; BioTek, Winooski, VT). Cinacalcet-induced [Ca²⁺]i were quantified by measurement of area under curve (AUC) and peak amplitude for the rise in relative [Ca²⁺]i. To evaluate cinacalcet-induced [Ca²⁺]i effects on CaMKKβ and LKB1, we used [Ca²⁺]i chelator BAPTA-AM (25 μM; Abcam).

All treatments were performed on washed isolated RBCs, except amitriptyline (AMI) which was applied on whole blood at 5 μM for 60 min at 37°C to inhibit the aSMase. To induce acute ATP depletion, RBCs were preincubated for 2 h in glucose-free DMEM (Life Technologies). ATP repletion was performed after RBC washing and resuspension in 4.5 g/l glucose-containing DMEM. To modulate the intracellular calcium content, RBCs were preincubated with 20 μM BAPTA-AM (Abcam): (i) for 15 min at 37°C, followed by a 60 min-reincubation at 37°C in the presence of Fluo-4 AM (see below), to measure its effect on the intracellular calcium content; or (ii) for 60 min at 37°C to determine EV abundance and PS exposure. To deplete membrane cholesterol, RBCs were preincubated with 0.9 mM methyl-β-cyclodextrin (mβCD; Sigma-Aldrich) for 30 min at 37°C. Cholesterol repletion was achieved with 7.5 and 15 μg/ml water-soluble cholesterol (Sigma-Aldrich) for 60 min at 37°C.

Third-instar larvae were dissected in modified HL3 saline and stained either with or without 0.03% Triton in PBS as described^{68 (link)}. The following primary antibodies were used: mouse anti-GluRIIΑ (8B4D2; 1:50; Developmental Studies Hybridoma Bank (DSHB)); rabbit anti-GluRIIIB^{70 (link)} (1:1000); rabbit anti-GluRIIC^{71 (link)} (1:2000); guinea pig anti-GluRIID^{70 (link)} (1:1000); rabbit anti-parvalbumin (Pa1-933; 1:1000; Thermo Fisher); mouse anti-DLG (4F3; 1:100; DSHB). The following primary antibodies were generated in this study, where the following peptides were injected into animals by Cocalico Biologicals (Stevens, PA, U.S.A): affinity purified rabbit anti-pCaMKII using the peptide C-VHRQET(p)VDCLKK (1:2000); guinea pig anti-CaMKII using the peptide C-VHRQET(p)VDCLKK (1:1000); guinea pig anti-GluRIIΑ^tail using the peptide C-SGSRRSSKEKSRSKTVS (1:2000). Alexa Fluor-647 conjugated goat anti-HRP (1:200; Jackson ImmunoResearch) and Donkey anti-mouse, -guinea pig, and -rabbit conjugated Alexa Fluor 488, Cy3, and DyLight 405 secondary antibodies (Jackson ImmunoResearch) were used at 1:400. For the BAPTA-AM treatment, dissected larvae were incubated in HL-3 saline with 0.1 or 0.3 mM BAPTA-AM (#120503; Abcam) for 30 min before fixation and then stained as described above. For the control conditions, dissected larvae were incubated in HL-3 saline in 0 of 1.8 mM Ca²⁺.

cis-5,8,11,14,17-Eicosapentaenoic acid (EPA), metformin, compound C (AMPK inhibitor), insulin, STO-609 (CaMKK inhibitor), SB203580 (p38 MAPK inhibitor), fluo-3, AM and a monoclonal anti-β-actin antibody were purchased from Sigma Chemical Company (St. Louis, MO, USA). BAPTA-AM (intracellular calcium chelator), monoclonal antibody against ACC and polyclonal antibodies against GLUT4 were purchased from Abcam (Cambridge, MA, USA). Monoclonal antibodies against phosphorylated AMPKα, phosphorylated p38 MAPK, p38 MAPK, phosphorylated AS160, AS160, and the polyclonal antibody against AMPKα were obtained from Cell Signaling Technology (Danvers, MA, USA). The polyclonal antibody against phosphorylated ACC was provided by Merck (Rahway, NJ, USA). Fetal bovine serum (FBS) and penicillin-streptomycin were acquired from Thermo Fisher Scientific (Foster City, CA, USA). The monoclonal antibody against c-Myc was acquired from Santa Cruz Biotechnology (Dallas, TX, USA).

Neutrophils were seeded on glass cover slips coated with 0.001% poly‐L‐lysine (Sigma‐Aldrich) with a concentration of 1 × 10⁵ cells per well if not stated otherwise. PMNs were stimulated with 4 μM ionomycin (free acid, Sigma‐Aldrich), 100 nM phorbol 12‐myristate 13‐acetate (PMA, Sigma‐Aldrich), 15 μM Ece1 peptides including candidalysin (if not otherwise stated) or infected with C. albicans yeast (MOI 2) for a defined time period, following fixed using 2% paraformaldehyde (PFA) and stored at 4°C. In the infection experiments, the fungus was added to 1 × 10⁴ PMNs 1 h after the cells were seeded.
For the pathway studies, neutrophils were incubated for 30 min before stimulation with 10 μM BB‐Cl‐amidine (PADi, Cayman Chemicals), 15 μM diphenyleneiodium (DPI, Sigma‐Aldrich), 15 μM 4‐hydroxy‐TEMPO (TEMPOL, Sigma‐Aldrich), 10/20 μM BAPTA‐AM (Abcam), 15 μM SYK inhibitors R406 (InvivoGen) and 12.5 μM piceatannol (InvivoGen), 15 μM PI3K blocker wortmannin (InvivoGen), 2.5 μM AKT inhibitor XI (InvivoGen) or NLRP3 blockage using compound 1 μM MCC950 (InvivoGen).