Fig 2: Discovery of STIM1β as a STIM1 alternative splicing variant that is upregulated in certain types of cancer. Data were shown as mean ± sem. a) Schematic of exon boundaries and alternative splicing of a previously‐unrecognized STIM1 variant (STIM1β). The primer pairs used to amplify the splice variant STIM1β or conventional STIM1 are shown as arrows. cEF/hEF, canonical or hidden EF‐hand; SAM, sterile alpha‐motif; TM, transmembrane domain; CC1, coiled coil region 1; CAD/SOAR, the STIM ORAI‐activating region; ID, inhibitory domain; PAD, pro‐activation domain; P/S, proline/serine‐rich region; PB, polybasic tail. b) The expression abundance of exon 10, the extra exon found in STIM1β (red), and exon 11 revealed by RNA‐seq. Boxplots denote expression distribution of each exon and each dot denotes RNA‐seq signals from one dataset. c,d) Comparison of the mRNA levels of STIM1 and STIM1β splice variants in selected cells by RT‐PCR. c) Agarose gel electrophoresis of PCR amplification of the region between exon 10 and exon 11. d) The relative expression ratio of STIM1β over STIM1 in the indicated cells. e) Immunostaining of STIM1β (green) in selected cell lines using a home‐made antibody specifically against STIM1β. Red, SEC61β as an ER marker. Scale bar, 10 µm. f) Detection of STIM1β protein in U87 cells with mass spectrometry. The polypeptide region containing PAD (underlined) was selected to perform MS/MS analysis to characterize its sequence identity. Assigned bn/yn fragments were listed with the corresponding peptide sequences. An anti‐STIM1β antibody was used to enrich STIM1β from cell lysates. g) IHC staining of STIM1β in normal, normal adjacent tissues of tumor (NAT), and cancerous tissues from patients with brain tumors. h) Quantification of STIM1β expression in patient samples shown in panel (g) (Normal, n = 10; NAT, n = 4; GBM, n = 80). Also see Figure S3, Supporting Information.

Fig 3: Silencing of Stim1, Orai1 and Orai3 in mCRC cellsShort interfering RNA (siRNA) and scrambled siRNA (Ctr) were transfected in mCRC cells as described in Materials and methods. Western blot and densitometry demonstrate a significant reduced Stim1 (A), Orai1 (B) and Orai3 (C) protein expression in silenced cells compared to controls (*, p<0.05; Student’s t test). Blots representative of three were shown. Lanes were loaded with 30 μg of proteins, probed with affinity purified antibodies and processed as described in Materials and Methods. The same blots were stripped and re-probed with anti-beta-2-microglobulin (B2M) antibody. Bands of the expected molecular weights were shown. Bands were acquired with the Image Master VDS (Amersham Biosciences Europe, Italy). Densitometric analysis of the bands was performed by Total Lab V 1.11 computer program (Amersham Biosciences Europe, Italy) and the results were normalized to the corresponding B2M.

Fig 4: Expression of Stim1-2, Orai1 and Orai3 proteins in patients-derived colorectal cancer cellsBlots representative of four (each from a distinct patient) were shown. Lanes were loaded with 30 μg of proteins, probed with affinity purified antibodies and processed as described in Materials and Methods. The same blots were stripped and re-probed with anti-beta-2-microglobulin (B2M) polyclonal antibody, as housekeeping. Major bands of the expected molecular weights were observed: Stim1 (A), Stim2 (B), Orai1 (C) and Orai3 (D). Bands were acquired, densitometric analysis of the bands was performed by Total Lab V 1.11 computer program (Amersham Biosciences Europe, Italy) and the results were normalized to the corresponding B2M.

Fig 5: The expression of Stim1-2, Orai1-3, TRPC1 and TRPC4 transcripts in breast cancer-derived endothelial colony forming cellsqRT-PCR showing increased expression of Stim1 (A) mRNA in BC-ECFCs compared to N-ECFCs. Conversely, Stim2 (B), Orai1 (C), Orai2 (D), Orai3 (E), TRPC1 (F), were not differently expressed in BC-ECFCs. Also, the expression of TRPC4 mRNA was enhanced in BC-ECFCs (G). Bars represent mean±SE of at least 4 different experiments each from different RNA extracts. The asterisk indicates p<0.05 vs. N-ECFCs (ANOVA followed by Newman-Keuls’ Q test). The PCR products were of the expected size (not shown): Orai1, 257 bp; Orai2, 334 bp; Orai3, 159 bp; Stim1, 347 bp; Stim2, 186 bp; TRPC1, 307 bp and TRPC4, 300 bp.