Bapta am

To evaluate cell proliferation upon different treatments, 1 × 10⁵ cells/well were seeded (t = 0 h) in six well plates and treated with either DMSO, BAPTA-AM (10 μM, Sigma-Aldrich), U-73343 (2.5 µM, Sigma Aldrich), U-73122 (2.5 µM, Sigma-Aldrich), Etomoxir (20 μM, Sigma-Aldrich). 24 h or 48 h later, cells were harvested and counted, normalizing their number to t = 0 h.
For cell migration, MDA-MB-231 CTRL and MDA-MB-231 MCU-KO cells were seeded at low confluency (30%) in 6-well plates. 24 h later they were treated either with DMSO, BAPTA-AM (10 μM, Sigma-Aldrich), U-73343 (2.5 µM, Sigma Aldrich), U-73122 (2.5 µM, Sigma-Aldrich), Etomoxir (20 μM, Sigma-Aldrich) or Oleic acid (500 μM, Sigma Aldrich) in 2% FCS medium. At the same time a linear scratch was obtained on cell monolayers through a vertically held P200 tip. Images were taken 24 h later. “TScratch” software (https://cse-lab.ethz.ch/software/) was used for automated image analysis.

Cells on a glass dish were washed three times with D-Hanks' solution and incubated with 5 μM Fluo 3-AM (Dojindo Laboratories, Japan) for 30 min at 37℃, followed by three washes and additional incubation in normal Hanks' solution for 15 min. To eliminate the effects of other Ca²⁺ channels or intracellular Ca²⁺ stores release, 1 mM extracellular calcium chelator EGTA, 10 μM intracellular calcium chelator BAPTA-AM (Cells were first treated with 10 uM BAPTA-AM in DMEM/high glucose medium without FBS for 30 min, then the subsequent experiments were performed), 5 μM L-type calcium channel blocker verapamil (Sigma, USA) and 5 μM selective Na⁺/H⁺ exchanger inhibitor 5-(N, N-Hexamethylene) amiloride (HMA, Alomone Labs, Israel) were added to the extracellular fluid.Using a laser scanning confocal microscope, Fluo 3-AM was excited at 488 nm, and emission was measured at 510 nm.

SKOV3-Con-sh, SKOV3-TRPM7-sh, OVCAR3-Con-sh and OVCAR3-TRPM7-sh cells (7 × 10⁴/well) were cultured in the upper chamber of 24-well transwell plates (8 μm, Corning, USA) in the presence or absence of 70 ng/ml EGF or 20 μM BAPTA (Sigma). The bottom chambers were filled with complete medium. After culture for 24 h, the cells on the surface of the upper chamber were removed with a cotton swab and the migrated on the bottom surface of the upper chamber were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. The migrated cells were counted under a microscope in a blinded manner. Similarly, these cells (1.4 × 10⁵/well) were tested for their invasion using Matrigel (BD Biosciences) coated upper chambers. In addition, SKOV3 and OVCAR3 cells were tested for their migration and invasion in the presence or absence of 30 μM MK886 and/or 70 ng/ml EGF, 30 μM MK886 and/or 20 μM BAPTA-AM, 20 μM BAPTA-AM and/or 10–15 μM LY294002 (Sigma), or 20 μM BAPTA-AM and/or 70 ng/ml IGF (Peprotech, New Jersey, USA).

For inhibition assays, the intra Ca²⁺ chelator BAPTA-AM (Sigma, St. Louis, MO, United States) and phosphoinositide specific phospholipase C inhibitor U-73122 (Sigma) were used to evaluate the effect of Ca²⁺ on sporozoite egress. In another experiment, cytochalasin D (CytoD; Merck, Darmstadt, Germany) was used to block parasitic motility. Infected PCKs were pre-treated with 10 μM BAPTA-AM, U-73122, or CytoD prepared in dimethyl sulfoxide (Sigma) for 30 min. Then, the cell cultures were incubated with 40 mM SNP for 30 min, and the number of egressed sporozoites was evaluated as described above.

All mouse cytokines were purchased from PeproTech (London, UK). The following antibodies and agonists were obtained from the specified suppliers: PAC-1 monoclonal antibody (mAb), anti-mouse P-selectin mAb (RB40.34), anti-paxillin mAb (clone 349), and anti-Hic-5 mAb (BD Biosciences, San Jose, CA); horseradish peroxidase-conjugated anti-green fluorescent protein (GFP) polyclonal antibody (Acris Antibodies, Himmelreich, Germany); phycoerythrin (PE)-Cy7-conjugated anti-mouse IgM (eBioscience, San Diego, CA); anti-talin mAb (clone 8D4); anti-phosphotyrosine mAb (clone 4G10), and BAPTA-AM (Millipore, Billerica MA); human fibrinogen and epinephrine (Sigma-Aldrich, St. Louis, MO); anti-vinculin mAb (V284) (Chemicon, Billerica, MA); anti-mouse GPVI mAb (Six.E10), anti-mouse GPIbα mAb (Xia.G5), and anti-mouse integrin αIIbβ3 mAb (Leo.D2 and clone JON/A) (Emfret Analytics, Eibelstadt, Germany); anti-α-actin mAb (D6F6), anti-FAK polyclonal antibody, and anti-Src mAb (32G6) (Cell Signaling Technology, Danvers, MA); anti-Rap1b polyclonal antibody and anti-protein kinase Cα mAb (M4) (Upstate Cell Signaling Solutions, Lake Placid, NY); allophycocyanin (APC)-conjugated anti-rat IgG polyclonal antibody (R& D Systems, Minneapolis, MN); convulxin (ALEXIS Biochemicals, Plymouth Meeting, PA); AYPGKF (Invitrogen, Carlsbad, CA); ADP (MC medical, Tokyo, Japan); U46619 (Cayman Chemical, Ann Arbor, MI).

CaMKIIα activity was measured by a commercial kinase activity kit (Promega, Madison, WI, USA). Briefly, 1 ng of CaMKIIα activity was assessed by the kinase ATP consumption kit (ADP-Glo Promega) and inferred from a consumption curve of 25 μM ATP/ADP at different ratios. The kinase was previously treated to induce its oxidation with 10 μM H₂O₂, free Ca²⁺ was chelated in some conditions using 2 mM BAPTA-AM (Millipore, Darmstadt, Germany) and the CaMKIIα inhibitor Kn-93 (MedChemExpress, Monmouth Junction, NJ, USA) was used at 1 μM. Then, the kit’s protocol was followed.