Fig 1.
Differential expression and genomic alteration of SOCE components in HNSCC.
(A) Boxplot representation of mRNA expression levels of ORAI1, ORAI2, ORAI3, STIM1, and STIM2 across normal solid tissue (red), primary tumors (blue), and metastatic tissues (orange). Data were obtained from the UCSC Xena Browser using the TCGA HNSCC cohort (n = 566). Expression is shown as log₂(normalized count + 1). One-way ANOVA revealed significant differences across groups: ORAI1 (p < 0.001), ORAI2 (p < 0.001), ORAI3 (p < 0.01), STIM1 (p < 0.001), and STIM2 (p < 0.001). (B) Dot plot of ORAI and STIM isoform expression across HNSCC anatomical subsites in the TCGA cohort (n = 604), t-test (ORAI1: p > 0.05; ORAI2: p < 0.01; ORAI3: p < 0.01; STIM1: p < 0.05; STIM2: p > 0.05). (C) OncoPrint visualization of somatic alterations in ORAI/STIM genes in the TCGA PanCancer Atlas HNSCC cohort (n = 523). (D) OncoPrint visualization and heatmap of the same cohort showing mRNA alterations relative to normal samples.
Table 1.
Molecular characterization of HNSCC cell lines.
Fig 2.
Variable molecular profiles of SOCE components and EGFR in HNSCC cell lines.
(A, B) Copy number alterations (A) and mRNA expression levels (B) of ORAI1, ORAI2, ORAI3, STIM1, and STIM2 in FaDu and Detroit-562 HNSCC cell lines, retrieved from the Cancer Cell Line Encyclopedia (CCLE) using the UCSC Xena browser. Heatmaps illustrate relative gene copy number (A) and transcript abundance (B). (C) Quantitative RT-PCR validation of ORAI and STIM isoform expression in FaDu and Detroit-562 cells (n = 3). Expression was normalized to GAPDH, and relative levels were calculated using the 2 (-ΔCt) method. Data represent mean ± SEM. (D) EGFR mRNA expression (RPKM) in FaDu and Detroit-562 cells, retrieved from CCLE/Xena. Values represent summary statistics; error bars are not shown.
Fig 3.
Pharmacological inhibition of SOCE with 2-APB induces cytotoxic effects in HNSCC cells.
(A, B) Dose–response curves of FaDu (A) and Detroit-562 (B) cell lines treated with increasing concentrations of 2-APB for 48 hours. Cell viability was assessed using the MTT assay. Viability was normalized to vehicle-treated control, and IC₅₀ values were calculated using non-linear regression (log[2-APB] vs. % viability) in GraphPad Prism 8.0. Data represent the mean ± SD of at least three independent experiments performed in sextuplicate. (C) Representative bright-field images of FaDu and Detroit-562 cells after 24-hour treatment with 2-APB at corresponding IC₅₀ concentrations. Representative images are shown to illustrate qualitative morphological changes (cell shrinkage, rounding, detachment) associated with cytotoxicity. Quantitative assessment of viability at 48 hours is provided in panels A-B. Images were captured at 10X magnification; scale bar = 500 µm.
Fig 4.
Pharmacological inhibition of SOCE with 2-APB suppresses migration of HNSCC cells.
(A, B) The effect of 2-APB on cell migration was assessed using a wound healing assay in FaDu (A) and Detroit-562 (B) cells. Cells were treated with 200 µM (FaDu) or 100 µM (Detroit-562) 2-APB for 12 hours. Representative bright-field images were captured at 0 and 12 hours using a light microscope at 5 × magnification. Scale bar = 200 µm. Migration rates (%) were calculated using ImageJ software with the formula: Migration (%) = [(Distance at 0 h – Distance at 12 h) / Distance at 0 h] × 100. Data represent the mean ± SEM of at least three independent experiments performed in triplicate. Statistical significance was assessed using Student’s t-test. ***p < 0.001.
Fig 5.
2-APB impairs clonogenic potential of HNSCC cells under long- and short-term exposure conditions.
(A) FaDu and Detroit-562 cells were exposed to continuous 2-APB treatment (200 µM and 100 µM, respectively) for 3–4 weeks to assess long-term clonogenic survival. Representative images show the absence of colony formation in treated cells compared to robust colony growth in untreated controls. (B) Cells were treated with 2-APB for 72 hours, after which the drug was removed and replaced with drug-free medium. Following a 3–4 weeks recovery period, colonies were fixed and stained. Colony area (%) was quantified using ImageJ, and results represent the mean ± SEM of at least three independent experiments. Statistical significance was assessed using a Student’s t-test. **p < 0.01.