Jasplakinolide

For in vivo jasplakinolide treatments, wild-type embryos were collected on E15.5 and incubated with 3μM jasplakinolide (Sigma-Aldrich), 30 nM Calyculin A (Cell Signaling Technology), or DMSO in serum-free DMEM (Biological Industries) at 37°C for 2 h before embedding in OCT or processed for whole-mount preparation and immunofluorescence microscopy as described above. For in vitro treatments, keratinocytes were infected with shScr;puromycin or shAnln;puromycin, selected with 3 μg/ml puromycin (Sigma) and plated on fibronectin-coated coverslips (40,000 cells in a single well of a 24-well plate). Twenty-four hours later, the medium was switched to high calcium (1.5mM Ca²⁺) and cells were treated with 100nM jasplakinolide (Sigma-Aldrich) or 2nM Calyculin A for 5 min, and then with 8μM nocodazole for 6 h. Cells were then fixed and labeled with Phalloidin-iFluor 647 (Abcam, ab176759) and pERM (Cell Signaling Technology, 1:200) overnight at 4°C. After washing, sections were incubated with a secondary antibody (1: 500 dilution) at room temperature for 1 h. Data was collected using a Nikon C2+ 60×/1.4 objective that generates optical sections of 0.49 μm at the middle of the cell.

After excystation (prior to plunge freezing), sporozoites resuspended in DMEM-10 were incubated at 37 °C with 5 µM A23187 (Sigma-Aldrich, Cat# C7522) for 4 min or with 1 µM Jasplakinolide (Sigma-Aldrich, Cat# J4580) for 40 min (or with 5 µM Jasplakinolide for 65 min).

After excystation (prior to plunge freezing), sporozoites resuspended in DMEM-10 were incubated at 37 °C with 1 µM jasplakinolide (Sigma-Aldrich, Cat# J4580) for 40 min (or with 5 µM jasplakinolide for 65 min).