Plasmids and peptides

The pcDNA4/TO-NCL (4TO-NCL) and GFP-Orai1 plasmids were described previously [36, 37]. NCL (residues 284–710, residues 630–710) were cloned into the pGEX-6P-1 vector to generate GST-NCL fragments. Flag-NCL fragments (residues 1–283, residues 284–629, residues 284–710, residues 630–710) were PCR amplified from 4TO-NCL and cloned into the pcDNA4/TO vector. GFP-Orai1 R91W mutant plasmid was created by using the overlap extension PCR site-directed mutagenesis technique. Biotin-labeled Orai1 peptides and cell-penetrating (trans-activator of transcription) TAT-Orai1 peptides were purchased from GL Biochem (Shanghai, China).

Cell culture and transfection

Human breast cancer MCF-7 and MDA-MB-231 cell lines were obtained from the National Collection of Authenticated Cell Cultures. All the cell lines were authenticated by short tandem repeat (STR) profiling and tested for mycoplasma contamination. MCF-7 cells were cultured in Dulbecco’s modified Eagle medium (DMEM), and MDA-MB-231 cells were cultured in L15 medium supplemented with 10% fetal bovine serum (Hyclone). Cells were transfected using Lipo8000 (Beyotime, China). For stable expression of NCL in breast cancer cells, the NCL gene was cloned into the lenti-CMV vector to package lentiviruses. Breast cancer cells were infected with lentiviruses expression control or NCL-Flag and treated with puromycin. The selected cells were used to assay proliferation and migration of cancer cells.

Protein expression

E. coli strain BL21 cells containing pGEX-6P-1-NCL were cultured at 37 °C in LB medium to an OD 600 of 0.8 then induced with 0.3 mM isopropyl β-D-thiogalactoside at 25 °C for 16 h. Cells were harvested and lysed by sonication. The GST-NCL proteins were purified by Glutathione Sepharose 4FF (GE healthcare).

Biotin-based pull-down assay

Biotinylated Orai1-NT or CT peptides were immobilized on streptavidin beads, then mixed with the MCF-7 cell lysate expression NCL-Flag fragments or purified GST-NCL proteins. Beads were washed three times in PBS containing 0.1% Triton X- 100. Proteins were eluted from the beads with 2xSDS sample buffer, then resolved by SDS-PAGE.

Western blotting

The whole cell lysates were extracted using RIPA buffer, and the protein concentration was determined using the BCA protein assay. Samples were resolved by SDS-PAGE and analyzed by standard Western blotting. The following antibodies were used and purchased from Proteintech Group. Rabbit polyclonal antibodies against NCL (10556-1-AP), Flag (80010-1-RR), PCNA (10205-2-AP), STIM1 (11565-1-AP), PKC (21991-1-AP), AKT (10176-2-AP); Mouse monoclonal antibody against Orai1 (66223-1-Ig), GAPDH (60004-1-Ig). Antibody against Phospho-AKT (CST, 4060) and Phospho-PKC (CST, 9375) were purchased from Cell Signaling Technology.

Co-immunoprecipitation

The Co-IP assay was performed in MCF-7 cells transfected with NCL-Flag vector using protein A/G magnetic beads (Thermo scientific). After cells were lysed in IP buffer, Orai1 was immuno-precipitated from cell lysate using Flag antibody, and mouse IgG antibodies were used as a negative control. Bound proteins and cell lysate were resolved by SDS-PAGE, followed by western blotting using anti-Flag and anti-Orai1 antibodies.

Immunofluorescence

MCF-7 cells were plated on confocal glass-bottomed dishes and co-transfected with GFP-Orai1 and NCL-Flag fragments plasmids with or without thapsigargin (TG) treatment. Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 3% BSA. Cells were stained with Alexa-Fluor-594-conjugated Flag antibody (Proteintech, CL594–66008). Nuclei were counterstained with DAPI for 10 min. Images were taken from confocal microscope.

Immunohistochemistry

Breast cancer patient tissues were from the first affiliated hospital of Bengbu medical college. The tissues were dehydrated and embedded in paraffin, and then tissues blocks were sectioned at 4-μm thickness for hematoxylin and eosin (H&E) or immunohistochemistry (IHC) analysis. For IHC analysis, tissues were stained with anti-NCL antibody (Proteintech, China) according to the manufacturer’s instruction. Images were randomly taken at a magnification of 400x using a light microscope and analyzed using the ImageJ software.

siRNA transfection

MCF-7 cells were seeded in a 6-well plate and transfected with 5 μg siRNA with Lipo 8000 reagent. The following siRNA were used: negative control siRNA (siNC) (5′-UUCUCCGAACGUGUCACGU-3′), small interfering RNA for NCL (siNCL) (5′-UUUCUCAAACGAAGUAAGCUUdTdT-3′) and siRNA for STIM1(siSTIM1) (5′-GGCUCUGGAUACAGUGCUCdTdT-3′). Gene silencing was detected by western blotting.

Determination of changes in intracellular calcium

Calcium imaging was performed as described previously [37]. After transfected, HEK293T cells were loaded with 2 μM Fura-2 AM for 30 min. Cells were excited at 340 nm and 380 nm, and F340/F380 ratios were calculated. For measurement of intracellular calcium using confocal microscopy, cells were plated on confocal glass-bottomed dishes. Cells were then loaded with CalbryteTM 630 AM (final concentration 10 μM) for 40 min. Cells were washed three times in calcium-free buffer. Calcium level (red fluorescence, Ex/Em = 610/640 nm) and GFP signal (green fluorescence, Ex/Em = 488/510 nm) were analyzed with a confocal microscope. The initial fluorescence was recorded for 30 s, then 2 mM CaCl2 and 1 μM TG were added, and cellular fluorescence intensity was recorded every 10 s for 5 min.

Cell proliferation assay

MTT (3- [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and EdU (5-ethynyl-2′-deoxyuridine) assay were done on MCF-7 or MDA-MB-231cells with stable expression of NCL as described previously [36]. Cells were plated into 96-well plate or 24-well plate for 24 h, then treated with 2-Aminoethoxydiphenyl borate (2-APB) or peptide for 24 h. The cell viability was assessed using MTT assay kit and Edu staining proliferation kit (KeyGEN, China) based on the manufacturer’s instructions.

Colony formation assay

200 MCF-7 cells with stable expression of NCL were plated into 12-well plates. After being cultured for 7 days, cell clones were treated with 2-APB for 7days. Then all clones were fixed with 4% paraformaldehyde, followed by staining with crystal violet for 5 min. Finally, clones were washed with PBS and photographed.

Wound healing assay

The transfected cells were plated into a 6-well plate and allowed to grow to a monolayer. The monolayer was then scratched and washed with PBS to remove the detached cells. Cells were maintained in media with or without 2-APB (50 μM) for 24 h. The wounded areas were imaged, and the change in the scratch gap was measured.

Xenograft animal model

BALB/c nude mice (female, 6 weeks old) were purchased from Wei-tong Lihua experimental animal technical company (Beijing, China). The mice were housed in a specific pathogen-free environment. To establish a breast cancer xenograft model, MCF-7 breast cancer cells (2 × 106) with stable expression of NC or NCL were injected into the nude mice. When tumor volumes reached about 100 mm3, NCL group mice were randomly divided into the untreated group, the 2-APB-treated group (20 mg/kg, three times weekly, ip.) and the Orai1 peptide-treated group (50 mg/kg, once daily, ip.), (n = 5 mice per group). Mice were monitored up to 14 days after initiation of treatment. Tumor size was measured using digital calipers, and volumes were calculated according to the formula: V (mm3) = (width2 × length)/2. Tumor weights were measured using an electronic scale after the mice were sacrificed.

Statistical analysis

Statistical significance was evaluated with data from at least three independent experiments. Statistical analysis was performed using the unpaired t test or one-way ANOVA (SPSS 20.0, Chicago). Data are presented as the mean ± SD. For all statistical tests, P < 0.05 was considered statistically significant.