Jetprime system

Cav1 knockdown was performed by human and mouse Cav1 siRNA, respectively, as reported previously (32 (link)). HPAECs and HEK 293 cells were grown to 60% confluency and transfected with human Cav1 siRNA using the JetPRIME system (Polyplus-transfection) according to the manufacturer's protocol. After cells were incubated with siRNA for 3 days, they were split and plated onto 55 mm dishes. Similarly, MEF cells were treated with mouse siRNA for 3 days.

2.5 × 10⁵ cells were seeded in a 6-well plate. Experiments were initiated on the following day, and cells were analyzed 48 h later for transfections, or 6 h later for drug treatments. All DNA/RNA transfections were performed with the jetPRIME® system (Polyplus-transfection) according to the manufacturer’s instructions. For plasmid transfections, 1 µg of plasmid was transfected. For siRNA transfections, 30 nM final concentration siRNAs were transfected. For CRISPR/dCas9 experiments, 1 µg dCas9 plasmid and 1 µg of each gRNA plasmid were transfected. Drugs were solubilized in 100% dimethyl sulfoxide (DMSO) as stock solutions, aliquoted and kept at −20 °C. Stocks solutions were: 5,6-dichlorobenzimidazole 1-β-d-ribofuranoside (DRB) 10 mM; actinomycin D (ActD) 1 mM; (S)-(+)-camptothecin (CAM) 10 mM; 1-hydroxypyridine-2-thione zinc salt (zinc) 50 mM. Working solutions were prepared on the day of the experiment and pre-diluted with 100% DMSO to a 200-fold higher concentration than the desired final concentration. Finally, 10 µl of the drug solutions, or 100% DMSO (0.5% final concentration of DMSO) were added to a 6 well plate (2 ml medium), mixed and incubated for 6 h at 37 °C.

The plasmid for dual expression of Lyn-iRFP-FKBP12 and FRB-Inp54 was prepared by subcloning the genes encoding pPBH-TRE_tight-Lyn-iRFP-FKBP12 and pCMV-FRB-Inp54 into a PiggyBac vector using In-Fusion Cloning Kit. The pCMV-FRB-Inp54 expression vector was controlled by the Tet-On system for reduced basal expression (54 (link)). The resulting dual expression plasmid (1.5 μg) and the recombination helper plasmid pSPB-Transposase (0.6 μg) were transfected into 70 to 80% confluent HeLa cells plated in a 6-well plate using the JetPRIME system (Polyplus-transfection) according to the manufacturer’s protocol. Cells in a separate well were kept without transfection as a control. After 24 h transfection, the growth media were replaced with the selection media (Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 200 mg/ml hygromycin, 1% penicillin and streptomycin). The growth medium was replaced every other day until the cells in the control well were completely dead. Successful transfection and stable expression of Lyn-iRFP-FKBP12 and FRB-Inp54 were confirmed with the iRFP signal on the cell membrane by confocal microscopy. These stably transfected cells were maintained in the growth media containing 100 mg/ml hygromycin. PI(4,5)P₂ depletion in these cells was induced by 1 μM of rapamycin and confirmed by ratiometric PI(4,5)P₂ imaging.