Fig 1: Impaired Akt signaling in NCS1 KO and NCS1 OE cells. (A) Western blot analysis of MDA‐MB231 WT and NCS1 KO cells showing decreased basal p‐Akt in NCS1 KO compared to WT. Depicted is a representative blot (left). **P < 0.01, determined by unpaired Student's t‐test (right). (B) Western blot analysis of WT and NCS1 OE MDA‐MB231 cells showing increased p‐Akt in NCS1 OE compared to WT. Depicted is a representative blot (left). **P < 0.01, determined by unpaired Student's t‐test (right). (C–E) Akt activation following intracellular Ca2+ buffering with BAPTA‐AM. Depicted are western blot analyses for total Akt (Akt) and p‐Akt in NCS1 KO (C), WT (D), and NCS1 OE (E) MDA‐MB231 cells after treatment with DMSO (CTRL) or 1 µm BAPTA‐AM for 30 min to buffer intracellular free Ca2+. (C) Buffering of intracellular Ca2+ significantly increased Akt phosphorylation in NCS1 KO cells. N = 4 independent experiments. **P < 0.01, determined by unpaired Student's t‐test. (D, E) Buffering of intracellular Ca2+ caused only a slight and not significant increase of Akt phosphorylation in WT (D) and OE (E) cells. N = 6 and n = 4 independent experiments, respectively. Determined not significant (ns) by Mann–Whitney U‐test. Depicted are representative blots. All data are shown as mean ± SD.
Fig 2: NCS1 KO reduces cell survival and motility. (A, B) Colony formation assays of WT and NCS1 KO MDA‐MB231 cells. Cells were seeded at a density of (A) 100 cells per well or (B) 500 cells per well. After 10 days in culture, the culture area covered by colonies was analyzed with ImageJ, demonstrating that NCS1 KO cells had a diminished capacity to grow colonies and survive compared to WT cells (the area covered with colonies is shown as % total area). Depicted are box plots of n = 4 independent experiments. **P < 0.01 (A) and *P < 0.05 (B), determined by unpaired Student's t‐test. (C) Scratch assay demonstrating the wound healing capacity of MDA‐MB231 WT and NCS1 KO cells. Depicted is the mean difference between the wound width at 0 and 24 h, that is, the wound closure in 24 h in cm following wound induction. NCS1 KO cells moved significantly less compared to WT cells. The wound width was measured with ImageJ, and the wound closure is expressed in cm. Depicted are box plots of n = 4 independent experiments. *P < 0.05, by Mann–Whitney U‐test. (D) Proliferation assay demonstrating no difference in cell proliferation between NCS1 KO and WT cells. Cell proliferation was assessed with CellTiter‐Glo Luminescence Cell Viability Assay (Promega) at different timepoints (0–4 days) after plating 1000 cells per well on a 96‐well plate. Depicted is the relative Luminescence compared to timepoint 0. Each timepoint shows the mean luminescence ± SD (n = 10).
Fig 3: NCS1 as stress response protein: proposed mechanism. Environmental stressors such as TNF‐α or oxidative stress, which activate the transcription factor NFκB, lead to transcriptional up‐regulation of NCS1. NCS1 functions as cytosolic Ca2+ buffer, increases InsP3‐dependent ER Ca2+ release, and activates the Akt pathway. Increased NCS1 expression therefore causes disrupted Ca2+ signaling and increased Akt activity and consequently promotes cell survival and motility.
Fig 4: NCS1 is up‐regulated in response to NFκB‐activating stressors. (A) NCS1 promoter region predicted by TRANSFAC (Matys et al., 2006) with the TSS and binding site for the NFκB subunit RelA‐p65 located 88 kilobases downstream of the TSS. (B) Western blot analysis of SHSY5Y whole‐cell lysate after treatment with DMSO (CTRL) or 10 ng·mL−1 TNF‐α for 24 h. Depicted is a representative blot. (C) Quantification of NCS1 protein levels normalized to GAPDH shows that TNF‐α treatment significantly increased NCS1 protein levels. Depicted is the mean ± SD of n = 3 independent experiments. *P < 0.05, determined by unpaired Student's t‐test. (D) Quantification of IκBα protein levels normalized to GAPDH shows that TNF‐α treatment significantly decreased IκBα protein. Depicted is the mean ± SD of n = 4 independent experiments. *P < 0.05, determined by unpaired Student's t‐test. (E) Representative western blot of cytosolic and nuclear fractions from SHSY5Y cells after 24 h of treatment with 10 ng·mL−1 TNF‐α showing nuclear translocation of transcription factor NFκB after TNF‐α treatment. (F, G) Quantitative real‐time PCR of SHSY5Y cells treated with NFκB‐activating stimuli or DMSO control (CTRL). (F) NCS1 mRNA increase after treatment with 10 ng·mL−1 TNF‐α for 24 h. Depicted are box plots of n = 4 independent experiments. **P < 0.01, determined by unpaired Student's t‐test. (G) NCS1 mRNA increase after 20 h treatment with 1 µm tBHP. Depicted are box plots of n = 4 biological replicates. *P < 0.05, determined by unpaired Student's t‐test.
Fig 5: NCS1 and NFκB‐regulated genes are differentially expressed in human breast cancer tissue compared to normal breast tissue. (A) Expression heat map of differentially expressed (P < 0.05) NFκB‐regulated genes in normal (n = 5) versus cancerous breast tissue (n = 8). NFκB‐related genes show distinct expression patterns between groups, indicating altered NFκB‐signaling in human breast cancer. A color legend is pictured with a scale from −2 to + 2‐fold change. (B) Gene expression values of NCS1 mRNA were plotted in normal (n = 5) versus cancerous breast tissue (n = 8) and revealed increased NCS1 gene expression in human breast cancer compared to normal breast tissue. **P < 0.01, determined by Student's t‐test. RNA microarray analysis was performed as described previously (Harvell et al., 2013). Data were obtained from the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE31192).
Supplier Page from Abcam for Anti-NCS1 antibody