Fig 2: ORAI1, STIM1 and STIM2 expression in human eMSCs. (A) RT-PCR analysis of ORAI, STIM1 and STIM2 expression in eMSCs. M—size marker. Line 1—primers specific for ORAI1, STIM1 and STIM2 amplified the PCR products of the expected sizes (78, 124 and 247 bp, correspondingly). Line 2—RT-PCR negative control in which reverse transcriptase was omitted. Shown are cropped gels with enhanced contrast. Original gel is shown in Supplementary Figure S5. The distribution of ORAI1 (B), STIM1 (C) and STIM2 (D). Immunoreactivity in eMSCs was examined with confocal microscopy. No staining of the cells was observed after incubation of the cells with only fluorescent secondary antibodies (Supplementary Figure S2). Scale bar is 50 µm.

Fig 3: Effects of S66 on VSMC migration, proliferation and viability. (A, B) Mean data for S66 effects on cell migration (A) and cell count (B) as percentages of their own vehicle controls (ctrls). (B) Cell count was measured in the presence of serum and various concentrations of S66 at 1, 2, 3 and 4 day time points (1–4 days) (symbols with error bars and connecting lines). The dashed horizontal lines are comparative mean data for cell counts in serum-free conditions as percentages of their serum controls. (C) Mean numbers of cells that failed to exclude trypan blue (i.e. non-viable cells) as a percentage of the total number of cells for the vehicle control (white bar) and S66 (black bar) groups at 3 and 4 days. (D) Mean data for number of cells migrating in Boyden chambers (‘migrated’) and with trypan blue exclusion in the presence of serum over 2 days (‘count at 2 days’). Data obtained after transfection with Orai1 siRNA (Orai1 si.1) or Orai1 negative siRNA (Orai1 si.-ve) were normalized to their own scrambled siRNA controls (siRNA ctrls), even though only one control column is shown. (A, D) Raw mean migrated cell numbers in the controls (ctrls) were 28.9 and 32.5 (for the 0.1 and 5 S66 groups) and 30.4 and 31.8 (for the Orai1 si.1 and Orai1 si.-ve groups). (A–D) Data are from experiments performed at least twice with cells from at least four patients.

Fig 4: ORAI1 knockout (KO) leads to the downregulation of ORAI2 and ORAI3 messenger RNA (mRNA) expression. Dotted boxes represent the mean expression level of ORAI2 and ORAI3 mRNA measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) from four independent experiments. The levels of ORAI2 and ORAI3 mRNA in wild-type (WT) HEK-293 cells were arbitrarily set at 1. In ORAI1 KO cells, ORAI2 (low-density dots, left box) and ORAI3 (high-density dots, right box) are downregulated (ORAI2 relative mean expression level 0.42 with a 95% confidence interval [CI] of 0.19–0.87; ORAI3 relative mean expression level 0.20 with a 95% CI of 0.07–0.60 compared with WT. The significance of the results was assessed by unpaired t-test (* p = 0.02 for ORAI2 and * p = 0.01 for ORAI3).

Fig 5: Experimental design of the strategy used to create total knockout of ORAI1 protein (ORAI1 KO). (A) Structure of ORAI1 messenger RNA (mRNA) including the noncoding region (light grey), the coding sequence (dark grey), the original start codon (ATG, full-length ORAI1 origin), and the alternative start codon (ATG, ORAI1β isoform). (B) Structure of ORAI1 protein including the transmembrane domain (white boxes) and the stromal interaction molecule (STIM) interaction domain (hatched boxes). (C) Guide RNA (gRNA) strategy used to generate complete ORAI1 KO. The green dotted lines represent the localization of the two gRNAs used. Exact coordinates of gRNA double-strand break (DSB) induction sites (relative to the original ORAI1 ATG) are indicated next to the name of the corresponding gRNA (with nt standing for nucleotide and aa standing for amino acid). The light green insert displays the genomic (g), coding sequence (c), and protein (p) coordinates of the induced deletion. The dark green insert presents the sequences surrounding the deletion on genomic DNA level (g) for both alleles (underlined signs indicate the position of the original ORAI1 start codon).