Cell line nucleofector kit

ORAI1 sequence-specific guide RNAs were inserted into the lentiCRISPR v2 vector (Addgene, Plasmid #52961) with the BsmBI restriction site to create a gRNA-Cas9-encoding plasmid. The complete list of primers used is included in Supplementary Table 2. HEK293 cells (ATCC, CRL-1537) were transfected with the gRNA-Cas9 plasmid using the Amaxa Nucleofector (Lonza, Nucleofector™ 2b Device) and the cell line Nucleofector Kit (Lonza, VCA-1003) according to the manufacturer’s protocol (Q-001). Forty-eight hours after transfection, cells were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin containing puromycin (2 μg/ml) (Gemini Bio Products, West Sacramento, CA) in 5% CO₂ at 37 °C. Six days after puromycin selection, cells were collected and seeded at one cell per well into 96-well plates. Disruption of the ORAI1 gene in individual colonies was detected using the Guide-it Mutation Detection Kit (Clontech Laboratories, 631443) and confirmed by sequencing, as well as western blotting and functional Ca²⁺ imaging assays. The ORAI1 knockout HEK293 cell line generated was named ORAI1-KO HEK293. The ORAI1 guide RNA used in this study was: 5′-GTTGCTCACCGCCTCGATGT-3′.

Mouse preadipocyte (3T3-L1) and macrophage (RAW264.7) cells were purchased from the American Type Culture Collection (VA, USA) and then cultured as previously described^{12 (link),44 (link)}. Briefly, 3T3-L1 cells were normally differentiated into mature adipocytes until day 8, and in indirect co-culture, 3T3-L1 cell differentiation was induced in conditioned media from RAW264.7 cells. Oil red O staining of lipid droplets was used to monitor cell differentiation as previously described^{12 (link)}.
Human UCN3 gene cloned in pCMV6 vector tagged with Myc-DDK (OriGene, Rockville, MD, USA) was used for transfection. Myc-DDK tagged pCMV6 vector without UCN3 (OriGene, Rockville, MD, USA) was used as a control. 3T3-L1 adipocytes were transfected via electroporation using the Cell Line Nucleofector Kit (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. Briefly, 2 × 10⁶ cells were electroporated with 2 μg of plasmid using Nucleofector program, A-033. About 24 h after transfection, the cells were treated with 400 µm of PA (Sigma-Aldrich, St. Louis, MA, USA) in low glucose (1 g/l) and 1% (w/v) serum medium for 24 h with and without insulin (20 nM for 10 min). PA was prepared in 10% (w/v) fatty acid free bovine serum albumin (BSA). The cells were then processed for RNA and protein analyses or plated for glucose uptake assay.

Phosphorothioate-modified antisense oligodeoxynucleotides (ASOs) were synthesized at BioSune (Shanghai, China), and transduced into Neuro-2a cells using Lipofectamine® 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol at 100 nmol/L. For transfecting primary NPCs, ASOs were introduced into tertiary cortical NPCs derived from E14.5 CD-1 mouse cortex by nucleofection (Lonza) according to the manufacturer’s protocol at 1 μmol/L for 1 × 10⁶ cells. Optimal programs and solutions of the Lonza Cell Line Nucleofector Kit for the ASO delivery were tested. Total RNAs were collected for RT-qPCR analysis two days post-transfection. Total protein was collected for immunoblotting analysis four days post-transfection. See Table S4 for ASO sequences.