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. Author manuscript; available in PMC: 2013 Apr 7.

Published in final edited form as: Mol Cancer Ther. 2009 Aug 11;8(8):2478–2489. doi: 10.1158/1535-7163.MCT-08-1132

Proliferation defect in sh-NDSP cells is reversible with overexpression of NDSP or treatment with recombinant NDSP. SK-N-AS cells were transduced with control (vector) or the NDSP shRNA-immune vector (NDSP-imm). After selection these separate cell lines were transduced with sh-Control or sh-NDSP vectors. A and C, These four cell lines, vector/sh-Control, vector/sh-NDSP, NDSP-imm/sh-Control; NDSP-imm/sh-NDSP were selected and subsequently used in the CCK-8 cell proliferation assays. These experiments were performed in triplicate and reported as the mean with standard deviations. Student's T-test was used to determine statistical significance with a p-value < 0.05 considered statistically significant. *Indicates statistical significance comparing the indicated sh-NDSP cell line (vector vs. NDSP-imm) to sh-Control. B and D, During the exponential growth phase, the cell lines were harvested, lysed and immunoblotting was performed with anti-NDSP-Ab1, anti-phospho-ERK1/2-Ab, and anti-ERK1/2-Ab. E, Recombinant NDSP-6xHis was purified by transfecting HEK293T cells with the NDSP-6xHis expression vector and collecting the supernatant. The supernatant was then incubated with nickel affinity gel and the NDSP-6xHis protein was immobilized. The protein was then eluted and immunoblotting (IB) with anti-NDSP-Ab1 was performed with the various phases of the purification process. F, SK-N-AS sh-Control and sh-NDSP cell lines were plated in 96-well plates at 1 × 10³ cells/well. Recombinant NDSP-6xHis was added to the media of the sh-NDSP cells in varying concentrations and the CCK-8 assay was used to quantify cellular proliferation relative to Day 1 absorbance measured at 450nm. These experiments were performed in triplicate and reported as the mean with standard deviations. Student's T-test was used to determine statistical significance with a p-value < 0.05 considered statistically significant. *Indicates statistical significance comparing sh-Control ( ) to sh-NDSP: 0ug ( ), **Indicates statistical significance comparing sh-NDSP: 20ug ( ) to sh-NDSP: 0ug ( ).

Inline graphic — Proliferation defect in sh-NDSP cells is reversible with overexpression of NDSP or treatment with recombinant NDSP. SK-N-AS cells were transduced with control (vector) or the NDSP shRNA-immune vector (NDSP-imm). After selection these separate cell lines were transduced with sh-Control or sh-NDSP vectors. A and C, These four cell lines, vector/sh-Control, vector/sh-NDSP, NDSP-imm/sh-Control; NDSP-imm/sh-NDSP were selected and subsequently used in the CCK-8 cell proliferation assays. These experiments were performed in triplicate and reported as the mean with standard deviations. Student's T-test was used to determine statistical significance with a p-value < 0.05 considered statistically significant. *Indicates statistical significance comparing the indicated sh-NDSP cell line (vector vs. NDSP-imm) to sh-Control. B and D, During the exponential growth phase, the cell lines were harvested, lysed and immunoblotting was performed with anti-NDSP-Ab1, anti-phospho-ERK1/2-Ab, and anti-ERK1/2-Ab. E, Recombinant NDSP-6xHis was purified by transfecting HEK293T cells with the NDSP-6xHis expression vector and collecting the supernatant. The supernatant was then incubated with nickel affinity gel and the NDSP-6xHis protein was immobilized. The protein was then eluted and immunoblotting (IB) with anti-NDSP-Ab1 was performed with the various phases of the purification process. F, SK-N-AS sh-Control and sh-NDSP cell lines were plated in 96-well plates at 1 × 10³ cells/well. Recombinant NDSP-6xHis was added to the media of the sh-NDSP cells in varying concentrations and the CCK-8 assay was used to quantify cellular proliferation relative to Day 1 absorbance measured at 450nm. These experiments were performed in triplicate and reported as the mean with standard deviations. Student's T-test was used to determine statistical significance with a p-value < 0.05 considered statistically significant. *Indicates statistical significance comparing sh-Control ( ) to sh-NDSP: 0ug ( ), **Indicates statistical significance comparing sh-NDSP: 20ug ( ) to sh-NDSP: 0ug ( ).