Cell culture
Each HEK293 Flp-In T-REx cell line (Invitrogen, #78007) was cultured in DMEM supplemented with 10% fetal bovine serum (SIGMA-Aldrich, #F7524), 5.84 mg/mL l-glutamine (Nacalai Tesque, #16919-42), 100 U/mL penicillin, and 100 µg/mL streptomycin (SIGMA-Aldrich, #P4333). Cells were grown in 5% CO2 at 37°C.
Cell line generation
For the generation of HEK293 Flp-In T-REx Jaw1 KO cells and ITPRs SE cells using the CRISPR/Cas9 system, HEK293 Flp-In T-REx cells were transfected with pSpCas9-2A-Puro Jaw1 or ITPRs sgRNA using the ScreenFect™A plus (FIJIFILM Wako Pure Chemical, 299-77103). After incubation for 48 h, the cells were subjected to selection by treatment with 2 µg/mL puromycin (Sigma-Aldrich, #P8833). The cells were then single-colonized by the limiting dilution cloning method in a 96-well plate and expanded. For the verification of the mutated sequence in each cell line by Sanger sequencing, genomic DNA was purified from each cell line using QIAamp DNA Micro Kit (QIAGEN, #51304), and the amplified PCR products containing the target region were submitted to the DNA sequence contract service (Genewiz). For the HEK293 Flp-In T-REx Jaw1 IE and Δcoil cell generation, HEK293 Flp-In T-REx Jaw1 KO #17 cells were transfected with pcDNA5 FRT/TO Hu SJaw1 or pcDNA5 FRT/TO Hu SJaw1 Δcoil using ScreenFect™A plus. After incubation for 48 h, the cells were selected by treatment with 100 µg/mL Hygromycin B (Nacalai Tesque, #07296-66) and expanded.
Anti-Jaw1 N-ter antibody production
Rabbit polyclonal anti-human Jaw1 antibody was raised against truncated Jaw1 encoding amino acids 1–440 (Operon Biotech). The sequence of human Jaw1 was referred from NCBI (NP_00191055). The serum was subjected to affinity chromatography using N-Hydroxysuccunumidyl-Sepharose 4 Fast Flow (Sigma-Aldrich, #H8280) coupled with the N-terminal region of Jaw1 encoding amino acids1–144, resulting in purified anti-Jaw1 N-ter antibody.
Plasmids
For the production of pcDNA5 FRT/TO Hu SJaw1, pcDNA3.1(+) Hu SJaw1 (previously described [35]) was digested with BamHI and XhoI, and the fragment was subcloned into a pcDNA5 FRT/TO vector digested with the same enzymes using a commercial DNA ligation kit (TaKaRa, #6023). For the production of pcDNA5 FRT/TO Hu SJaw1 Δcoil, pcDNA3.1 (+) Hu LJaw1 (previously described [35]) was first performed by inverse PCR with primer set 1 resulting in pcDNA3.1 (+) Hu LJaw1 Δcoil. pcDNA3.1 (+) Hu LJaw1 Δcoil was then digested with BamHI and XhoI, and the fragment was subcloned into a pcDNA5 FRT/TO vector (digested with the same enzymes) using the aforementioned DNA ligation kit, resulting in pcDNA5 FRT/TO Hu LJaw1 Δcoil. Furthermore, pcDNA5 FRT/TO Hu LJaw1 Δcoil was digested with HpaI and XhoI, and the fragment was subcloned into pcDNA5 FRT/TO Hu SJaw1 digested with the same enzymes to swap the N-terminal region of Hu LJaw1 to that of Hu SJaw1.
For the production of pSpCas9-2A-Puro Jaw1 or ITPRs sgRNA, the DNA cassettes containing the sgRNA sequence targeting Jaw1 and the ITPRs were inserted into a pSpCas9(BB)-2A-Puro (PX459) vector (Addgene, #48139) digested with BbsI using the aforementioned DNA ligation kit. Each DNA cassette was produced using primer sets 2–5. The sgRNA targeting ITPRs were designed according to a previous report [18].
Western blotting
Each cell line was seeded onto six-well plates (TrueLine, #TR5000) and incubated overnight. The cells were then treated for an additional 24 h with each Dox concentration to induce Jaw1 expression. After that, the cells were harvested by gentle pipetting, washed once with PBS, and lysed with RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing a protease inhibitor cocktail (Nacalai Tesque, #04080-24). The samples were sonicated for 10 min on ice and centrifuged at 12,000 × g for 30 min. The supernatants were added to SDS-PAGE sample buffer and heated at 95℃ for 5 min. The samples were loaded onto a poly acrylamide gel, electrophoresed, and transferred onto polyvinylidene difluoride membranes (GE Healthcare, #10600029). The membranes were blocked with tris-buffered saline containing 0.1% Tween20 (TBS-T) and 3% skim milk (FIJIFILM Wako Pure Chemical, #190-12865) for 1 h at room temperature and incubated overnight at 4℃ with the following primary antibodies: rabbit anti-Jaw1 N-ter (1:1000, produced in our lab), rabbit anti-ITPR1 (1:1000, ABclonal, #A7905), mouse anti-ITPR2 (1:1000, Santa Cruz Biotechnology, #sc-398434), mouse anti-ITPR3 (1:1000, BD Bioscience, #610312), mouse anti-α-Tubulin (1:1000, FUJIFILM WAKO Pure Chemical, #013-25033), or mouse anti-GAPDH (1:1000, FUJIFILM WAKO Pure Chemical, #016-25523). The membranes were washed with TBS-T three times and incubated for 3 h with the following secondary antibodies: anti-Mouse IgG, HRP-Linked Whole Ab Sheep (1:5000, GE Healthcare, #NA931) or anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (1:5000, GE Healthcare, NA934). The membranes were washed with TBS-T three times and reacted with Immuno-star Zeta (FIJIFILM Wako Pure Chemical, #297-72403) or SuperSignal™ West Atto Ultimate Sensitivity Substrate (Thermo Fisher Scientific, #A38554). The bands were detected using an LAS4000 imaging system.
Immunostaining
Each cell line was seeded onto 96-well plates (TrueLine, TR5003) and incubated overnight. The cells were treated with each Dox concentration to induce Jaw1 expression. After incubation for 24 h, the cells were fixed with 4% PFA in PBS buffer for 15 min. The cells were then washed with PBS once and permeabilized with 0.2% Triton-X (FIJIFILM Wako Pure Chemical, #162-24755) in PBS for 10 min. Next, the cells were washed with PBS three times and blocked with 2% bovine serum albumin in PBS for 1 h. The cells were then incubated with the following primary antibodies: rabbit anti-Jaw1 N-ter (1:200, produced in our lab) and rat anti-Jaw1 coil (1:200, produced in our lab [16]). After the incubation for 1 h, the cells were washed with PBS three times and incubated for 1 h with Hoechst 33342 along with the following secondary antibodies: goat anti-rat IgG conjugated with Alexa Fluor 488 (1:500, Thermo Fisher Scientific, #A-11006) and goat anti-rabbit IgG conjugated with Alexa Fluor 568 (1:500, Thermo Fisher Scientific, #A-11011). The cells were then washed with PBS three times and observed using fluorescence microscopy (Leica, AF6000-DMI6B).
Co-immunoprecipitation
Each cell line was cultured on a six-well plate and incubated overnight. The cells were treated with 200 ng/mL Dox to induce Jaw1 expression. After incubation for 24 h, the cells were collected by gentle pipetting and centrifuged at 500 × g for 10 min at 4°C. The pellets were lysed in a lysis buffer (50 mM Tris–HCl, pH 7.6, 150 mM NaCl) containing 0.5% NP-40 and 1 µL of a protease inhibitor cocktail (Nacalai Tesque, 04080-24). The lysates were sonicated for 10 min on ice and centrifuged at 12,000 × g for 30 min at 4°C. The lysates were diluted with lysis buffer into 0.1% NP-40, then incubated with anti-Jaw1 antibody-conjugated beads for 1 h at 4℃. The conjugated beads were prepared by mixing the anti-Jaw1 N-ter antibody and Protein A Mag Sepharose (Cytiva, #28944006) for 1 h at 4℃. The beads reacted with the cell lysate were then collected by a magnetic stand and washed with lysis buffer three times. For elution, the beads were mixed with SDS-PAGE buffer and heated at 95°C for 5 min. On the magnetic stand, the supernatants were collected and subjected to western blotting.
RT-qPCR
Each cell line was cultured on a six-well plate and incubated overnight. The cells were treated with 200 ng/mL Dox to induce Jaw1 expression. After incubation for 24 h, total RNA was isolated using RNAiso Plus (TaKaRa, 9108). Total RNA was then reverse transcribed using PrimeScript RT Master Mix (TaKaRa, RR036A), and subsequent PCR was performed using TB Green Premix EX Taq II (Tli RNaseH Plus, TaKaRa, RR820A) on Thermal Cycler Dice Real Time System II MRX (TP960, TaKaRa). For jaw1 and gapdh gene detection, primer sets 6 and 7 were used, respectively.
Calcium assay
Each cell line was seeded onto 96-well black wall plates (Greiner Bio-One, #655090) and incubated overnight. The cells were treated with each Dox concentration to induce Jaw1 expression.
For the calcium assay with extracellular Ca2+ condition, the cells were incubated for 24 h, washed with PBS once, and incubated with recording buffer (20 mM HEPES, pH 7.4, 115 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 13.8 mM D-Glucose, and 1.25 mM probenecid) containing 2 µM Fluo-4 AM (dojindo, #F311) or 2 µM Rhod-2 AM (dojindo, #R-002) for 30 min. The cells were then washed with PBS once and incubated with HBSS containing 1.25 mM probenecid for 30 min. For the well scanning calcium assay using a plate reader, Varioskan LUX (Thermo Fisher Scientific) was used. The fluorescence values were recorded every second, and ATP solution was added at 10 s. The data was measured from three independent wells. For the calcium imaging, we used a fluorescent microscope (Leica, AF6000-DMI6B) equipped with a 20X fluor objective. The fluorescent images were captured every 2 s, and ATP solution was added at 10 s. The data was measured from four independent wells. For the calcium assay under Ca2+ free conditions, the cells were incubated with Ca2+ free recording buffer containing Fluo-4 AM for 30 min, washed with PBS once and incubated with Ca2+ free recording buffer. For the well scanning calcium assay for SOCE activity quantification, a plate reader was used and 2 µM thapsigargin solution was added at 10 s and recording buffer with 2 mM CaCl2 at 480 s. For the calcium imaging under Ca2+ free conditions, fluorescence microscopy was used and the 100 µM ATP solution without Ca2+ was added at 10 s.
Relative Fluo-4 fluorescence intensity quantification
The fluorescence intensity at 0 s was defined as F0 and the relative fluorescent intensity at each time point was calculated as F/F0. The calculated data was then graphed in GraphPad Prism7.
For the calcium imaging, fluorescent images of the Fluo-4-loaded cells upon ATP stimulation were analyzed and quantified by Fiji. The calcium imaging was performed in the four independent wells and data from 50 cells in each image was used for the quantification. We randomly chose 50 cells from each image, and the center of each cell (8 × 8 pixel) was selected as a representative area. The fluorescent intensity in the chosen area was measured over time for 5 min.
Ca2+ influx type classification
Relative Fluo-4 intensity of each cell was plotted for 5 min using GraphPad Prism 7. The relative Fluo-4 intensity curves were then transferred to layouts and manually classified into four Ca2+ influx types as follows: (1) The first group (single type) included the cells with amplitudes only once for 5 min. (2) The second group (oscillation type) included the cells with more than two oscillations. The oscillation was defined as the relative Fluo-4 intensity decreasing below 125% with a peak that rose again during 5 min. (3) The third group (steady reduction type) included the cells that maintained the relative Fluo-4 intensity higher than 125% of the initial values for 5 min. (4) The fourth group (no response type) included the cells maintaining the amplitude less than 300% of the initial value for 5 min. To calculate the ratio, we used 50 cells for each of the four measurements (total of 200 cells).
Statistical analysis
All statistical tests were calculated using GraphPad Prism 7, and the data are represented as the mean ± S.D. The sample numbers are included in each figure legend. Unpaired, nonparametric Student’s t-test was used to compare two sample groups. To compare more than two groups, one-way ANOVA followed by Tukey–Kramer’s t-test was used. *, **, ***, and **** indicate statistically significant P-values of p < 0.05, < 0.01, < 0.001, and < 0.0001, respectively. n.s.: non-significant.