U73122

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.

Mz treatment for confocal observations except for the fatty acid add-back experiment was performed on seedlings grown for 5 days on half MS plates containing 10, 50, or 100 nM Mz (Cayman Chemical). Mz was added to the medium from a 100 mM stock in dimethylsulfoxide by using an intermediate diluted stock at 100 μM (extemporarily prepared). The treatments for the fatty acid add-back and SVs/TGN immuno-purification experiments are described hereafter. FB1 treatment for the confocal observations was performed by transferring the seedlings grown on drug-free half MS plates into the liquid half MS medium containing 2.5 μM FB1 (Sigma–Aldrich) 20 h before observation. For the sphingolipid analysis, plants were grown on half MS agar plates containing 0.5 μM FB1 for 5 days. FB1 stock solution was prepared in dimethylsulfoxide at 0.5 mM. β-Estradiol treatment for amiRNA induction was performed by growing the seedlings on half MS agar plates containing 5 μM β-Estradiol (Sigma–Aldrich) for 5 days. PI-PLC-inhibitor treatment (U73122 and its inactive analog U73343, Sigma–Aldrich) was performed on seedlings grown on drug-free half MS plates for 5 days and transferred in liquid half MS medium containing 1 or 5 µM of either U73122 or U73343 for 90 min. The drug concentrations are also indicated in the figure legends.

Antibodies: monoclonal anti-HCV core antibody (ThermoFisher), anti-influenza-M2 and anti-Caspase-1 (Santa Cruz Technologies), anti-Flag (Sigma), anti-actin (Millipore), anti-IL-1β (Cell Signaling, detects only cleaved IL-1β protein), goat-anti-mouse or goat-anti-rabbit secondary antibodies (ThermoFisher). HCV E2 antibody (Austral Biologicals). Other reagents: Recombinant human TNF and m-CSF were purchased from Peprotech and recombinant HCV core protein (rHCV-core) (Meridian Life Science, Inc). The recombinant core protein is produced in Pichia pastoris and was used at 10-30ug/ml. Nigericin, ATP, D609, u-73343, u-73122, DMSO, and PMA (Sigma). Indo-I-AM calcium flux detection kit was purchased from Calbiochem and LPS from Addipogen. For polyU/UC RNA transfection and/or DNA transfection, Mirus Trans-IT mRNA transfection (for RNA transfection) and Mirus low-toxicity (LT1) (for DNA transfection) were used. The polyU/UC RNA was made as previously described [52 (link)]. The p7 inhibitor (JK3/32), which is the active analog, and (R-21), which is the inactive analog, were dissolved in DMSO and used at 1-10uM with final DMSO content of 0.25%. For all kits used, the manufacturer’s protocol was followed and if a modification is made, it is noted.

Twenty-one-day-old seedlings of the wild type and ZmNrt2.1-expressing plants grown on MS medium were removed from the medium, gently rinsed with water and incubated with gentle shaking for 1 h in water, 5 mM LaCl₃, 10 μM U73122 or 10 μM U73343, followed by incubation for 1 h in either 5 mM KNO₃ or 5 mM KCl, according to Riveras et al. (2015 (link)). Roots and shoots were collected, used immediately for RNA extraction or flash frozen in liquid N₂ and stored at – 80 °C. LaCl₃, U73122 and U73343 were purchased from Sigma-Aldrich (USA). LaCl₃ was dissolved in water. U73122 and U73343 were dissolved in a minimal amount of dimethyl sulfoxide and diluted in water to the final concentration. All the solutions were adjusted to pH 6.5.

The smooth muscle cells were freshly dispersed for approximately 40 minutes at 37 °C in digestion solution containing the following (mg/mL): 2.0 type II collagenase, 2.0 BSA, and 2.0 trypsin inhibitor (all from Sigma-Aldrich, St. Louis, MO). After washing in Hank’s (with calcium) or D-Hank’s (without calcium) Balanced Salt Solution, the cells were filtered away from the tissue fragments with a 200-mesh cell strainer. Next, the cells were seeded onto a glass coverslip at the bottom of a recording chamber filled with Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin-streptomycin at 37 °C, 95% O₂ and 5% CO₂. Eight hours later, the cells were washed with PBS solution and loaded with Fluo-4 AM (5 μM, Calcium Ion Probes) for 30 minutes at 37 °C. Ca²⁺ imaging experiments were captured at an emission wavelength of 488 nm. NTs (1 μM), the NTSR antagonist SR142948A (1 μM), the non-selective cation channels blocker FLA (100 μM) and the PLC signal pathway inhibitor U73122 (1 μM) (Sigma-Aldrich, St. Louis, MO) were used to examine the effects on real-time [Ca²⁺]i with laser confocal microscopy. The effects are presented as the relative fluorescence intensities RFI = F1/F0, where F1 is the real-time fluorescence intensity, and F0 is the baseline fluorescence intensity before administration.

We have analyzed the involvement of phospholipases C in the regulation of AGPs excretion to the culture medium. 100 μM of the aminosteroid U73122 (Sigma-Aldrich), a specific phospholipase C inhibitor, was applied to the control and salt-adapted cells during 24 h. The inactive analog U73343 (Sigma-Aldrich) was also used as a negative control. Inhibitors were dissolved in 500 μM of dimethylsulfoxide (DMSO). Similarly, control and salt-adapted cells were also treated with the same amount of DMSO solvent as a control. Immuno-dot blot assay of the culture medium was developed as described above. At least three independent experiments, each with five replicates, were conducted for each treatment.