Journal: Nature Communications

Article Title: A monoclonal antibody against KCNK9 K + channel extracellular domain inhibits tumour growth and metastasis

doi: 10.1038/ncomms10339

Figure Lengend Snippet: ( a ) Schematic representation of Tl + -based fluorescence assay principle (FluxOR Potassium Ion Channel Assay, Invitrogen) and readout. Compound 765 and 966 were chemical modulators used as control compounds. ( b ) Flow cytometry analysis of tetracycline-inducible hKCNK9 stable cell line stained with Y1 and Y4 in the presence of tetracycline induction, designated as +K9 or in the absence of tetracycline induction, designated as −K9. Parental HEK293 cells were used as negative control. ( c ) Time-dependent inhibition of Tl+ conductance by Y4. Tetracycline-induced cells were treated with Y4, Y1 or PBS before Tl + assay and compared with cells without treatment. Difference in Tl + conductance at time point 80 was used for comparison. Mean±s.d., n=2 4 (the experiment was replicated four times), * P< 0.0001, one-way analysis of variance (ANOVA). ( d ) Representative Tl + traces from tetracycline-induced KCNK9-expressing cells (+K9) and non-induced cells (−K9) plotted as fluorescence change over time. Mean±s.d., n=24 (the experiment was replicated four times), * P< 0.01, two-tailed Student's t -test. ( e ) Concentration-dependent inhibition of Tl + conductance by Y4. Mean±s.d., n= 6 (the experiment was replicated twice), * P< 0.01, two-tailed Student's t -test. ( f ) Internalization of hKCNK9 from cell surface induced by Y4. COS-7 cells transiently expressing hKCNK9 and mcherry or red fluorescent protein (RFP)-tagged EEA1 were incubated with Alexa488-conjugated Y4. Cells transiently expressing hKCNK3 and RFP-tagged EEA1 were control. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Co-localization (arrows) between Alexa488-conjugated Y4 and RFP-tagged EEA1 was analysed using Imaris software and representative scatter plots were shown. Statistical analysis was based on Pearson's correlation coefficient calculated from 20 fields, mean±s.d. * r >0.5. Scale bar, 20 μm.

Article Snippet: Antibodies were from the following sources: mouse anti-human growth hormone antibody (catalogue: ab9821, Abcam, 1:1,000), rabbit anti-KCNK9 antibody (catalogue: APC-044, Alomone, 1:800), goat anti-KCNK9 antibody (catalogue: sc-11317, Santa Cruz, 1:1,000), mouse anti-β-actin antibody (catalogue: ab8227, Abcam, 1:5,000), rabbit anti-cleaved caspase-3 (catalogue: 9664, Cell Signaling, 1:1,000), rabbit anti-Ki67 antibodies (catalogue: ab15580, Abcam, 1:1,000) and APC-conjugated goat anti-mouse IgG antibody (catalogue: 405308, Biolegend, 1:100).

Techniques: Fluorescence, Flow Cytometry, Stable Transfection, Staining, Negative Control, Inhibition, Expressing, Two Tailed Test, Concentration Assay, Incubation, Software

Journal: Nature Communications

Article Title: A monoclonal antibody against KCNK9 K + channel extracellular domain inhibits tumour growth and metastasis

doi: 10.1038/ncomms10339

Figure Lengend Snippet: ( a ) Kaplan–Meier curves for survival of patients with squamous cell lung cancer and breast cancer in relation to KCNK9 gene expression level. Data sets were obtained from commonly used published studies . Patients' gene expression data and clinical data were manually matched. Patients with both KCNK9 expression and survival data were segregated into two groups according to KCNK9 expression level—KCNK9 high (twofold higher than mean) and KCNK9 low (twofold lower than mean). Survival was analysed using the Prism Kaplan–Meier package and log-rank Mantel–Cox statistical test. We performed the same analysis to examine KCNK3 expression versus survival. n= 53 (lung), n= 289 (breast), * P< 0.05, log-rank Mantel–Cox. ( b ) Y4's ability to detect endogenous KCNK9 expressed in cancer cell lines verified by flow cytometry and western blotting analyses. ( c ) Treatment with Y4 for 24–72 h reduces cell viability and increases cell death of KCNK9-expressing cancer cell lines. BEN cells were sorted according to hKCNK9 expression level (K9 + or K9 − ). Mean±s.d., n= 6, * P< 0.01 at 0.1% heat-inactivated (HI) serum, ** P< 0.01 at 0.1% and 10% HI serum, two-tailed Student's t -test.

Article Snippet: Antibodies were from the following sources: mouse anti-human growth hormone antibody (catalogue: ab9821, Abcam, 1:1,000), rabbit anti-KCNK9 antibody (catalogue: APC-044, Alomone, 1:800), goat anti-KCNK9 antibody (catalogue: sc-11317, Santa Cruz, 1:1,000), mouse anti-β-actin antibody (catalogue: ab8227, Abcam, 1:5,000), rabbit anti-cleaved caspase-3 (catalogue: 9664, Cell Signaling, 1:1,000), rabbit anti-Ki67 antibodies (catalogue: ab15580, Abcam, 1:1,000) and APC-conjugated goat anti-mouse IgG antibody (catalogue: 405308, Biolegend, 1:100).

Techniques: Expressing, Flow Cytometry, Western Blot, Two Tailed Test