mcl-1 overexpression plasmid #sc315538  (OriGene)

Journal: The American Journal of Pathology

Article Title: miR-193b Regulates Mcl-1 in Melanoma

doi: 10.1016/j.ajpath.2011.07.010

Figure Lengend Snippet: Expression of miR-193b and Mcl-1 in melanoma tissue samples. A: miR-193b expression was measured by Agilent miRNA microarray. The black line indicates the mean value for each group. B: Mcl-1 staining was scored in metastatic melanomas, primary melanomas, and benign nevi. Data shown are mean ± SEM. C: Pearson correlation analysis was performed to determine the relationship between miR-193b expression and Mcl-1 expression. The results show an inverse correlation (r = −0.7 and P < 0.001). *P < 0.0001 was calculated using the independent samples t-test.

Article Snippet: A sequence-validated Mcl-1 expression plasmid (SC315538) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Expressing, Microarray, Staining

Journal: The American Journal of Pathology

Article Title: miR-193b Regulates Mcl-1 in Melanoma

doi: 10.1016/j.ajpath.2011.07.010

Figure Lengend Snippet: miR-193b down-regulates Mcl-1 and sensitizes melanoma cells to ABT-737. A: Western blot analysis of Mcl-1 and cleaved PARP expression in Malme-3M, MeWo, SK-MEL-2, and SK-MEL-28 cells transfected with miR-193b or negative control. Cells were transfected with 5 nmol/L miRNA precursor (miR-193b or negative control) and were harvested 72 hours after transfection. B: Western blot analysis of cleaved PARP and Mcl-1 in Malme-3M cells transfected with miR-193b or negative control, as indicated, incubated for 56 hours, and treated with 10 mmol/L ABT-737, as indicated. Cells were harvested 72 hours after transfection. C: Western blot analysis of MeWo and SK-MEL-28 cells, as indicated. The procedure was performed as described in B. D: One microgram of Mcl-1 plasmid (pMcl-1) was cotransfected with 5 nmol/L miRNA precursors, as indicated, and treated with 10 nmol/L ABT-737 as described in B. Gamma tubulin was used as the loading control. Representative data from one of three independent experiments are shown.

Article Snippet: A sequence-validated Mcl-1 expression plasmid (SC315538) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Western Blot, Expressing, Transfection, Negative Control, Incubation, Plasmid Preparation

Journal: The American Journal of Pathology

Article Title: miR-193b Regulates Mcl-1 in Melanoma

doi: 10.1016/j.ajpath.2011.07.010

Figure Lengend Snippet: miR-193b directly targets the 3′ UTR of Mcl-1. A: miR-193b and its predicted seed binding site in the 3′ UTR of Mcl-1 (top). The underlined 8-nucleotide sequence indicates the miR-193b seed region. Relative luciferase activity is shown for reporter constructs containing 3′ UTR or 3′ UTR M (with deleted seed pairing region) (bottom) in cells transfected with miR-193b or negative control. B: As in A, except reporter constructs carried the indicated fragment of the Mcl-1 3′ UTR (top). Four MREs predicted by RNA22 in the 3′ UTR of Mcl-1 (middle). C: As in A, except reporter constructs carried UTR1 M1 or UTR1 M2 (top). UTR1 M1 contains two point mutations, as indicated in bold italic. UTR1 M2 contains no seed binding region. Renilla luciferase activity was measured 24 hours after transfection using the Dual-Glo luciferase assay system (Promega). Data were normalized to firefly luciferase. Data shown are the mean ± SEM of three replicates and are representative of three independent experiments. *P < 0 0.05 and **P < 0.001 were calculated using the paired samples t-test.

Article Snippet: A sequence-validated Mcl-1 expression plasmid (SC315538) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Binding Assay, Sequencing, Luciferase, Activity Assay, Construct, Transfection, Negative Control

Journal: The American Journal of Pathology

Article Title: miR-193b Regulates Mcl-1 in Melanoma

doi: 10.1016/j.ajpath.2011.07.010

Figure Lengend Snippet: Expression of miR-193b and Mcl-1 in melanoma tissue samples. A: miR-193b expression was measured by Agilent miRNA microarray. The black line indicates the mean value for each group. B: Mcl-1 staining was scored in metastatic melanomas, primary melanomas, and benign nevi. Data shown are mean ± SEM. C: Pearson correlation analysis was performed to determine the relationship between miR-193b expression and Mcl-1 expression. The results show an inverse correlation (r = −0.7 and P < 0.001). *P < 0.0001 was calculated using the independent samples t-test.

Article Snippet: A sequence-validated Mcl-1 expression plasmid (SC315538) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Expressing, Microarray, Staining

Journal: The American Journal of Pathology

Article Title: miR-193b Regulates Mcl-1 in Melanoma

doi: 10.1016/j.ajpath.2011.07.010

Figure Lengend Snippet: miR-193b down-regulates Mcl-1 and sensitizes melanoma cells to ABT-737. A: Western blot analysis of Mcl-1 and cleaved PARP expression in Malme-3M, MeWo, SK-MEL-2, and SK-MEL-28 cells transfected with miR-193b or negative control. Cells were transfected with 5 nmol/L miRNA precursor (miR-193b or negative control) and were harvested 72 hours after transfection. B: Western blot analysis of cleaved PARP and Mcl-1 in Malme-3M cells transfected with miR-193b or negative control, as indicated, incubated for 56 hours, and treated with 10 mmol/L ABT-737, as indicated. Cells were harvested 72 hours after transfection. C: Western blot analysis of MeWo and SK-MEL-28 cells, as indicated. The procedure was performed as described in B. D: One microgram of Mcl-1 plasmid (pMcl-1) was cotransfected with 5 nmol/L miRNA precursors, as indicated, and treated with 10 nmol/L ABT-737 as described in B. Gamma tubulin was used as the loading control. Representative data from one of three independent experiments are shown.

Article Snippet: A sequence-validated Mcl-1 expression plasmid (SC315538) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Western Blot, Expressing, Transfection, Negative Control, Incubation, Plasmid Preparation

Journal: The American Journal of Pathology

Article Title: miR-193b Regulates Mcl-1 in Melanoma

doi: 10.1016/j.ajpath.2011.07.010

Figure Lengend Snippet: miR-193b directly targets the 3′ UTR of Mcl-1. A: miR-193b and its predicted seed binding site in the 3′ UTR of Mcl-1 (top). The underlined 8-nucleotide sequence indicates the miR-193b seed region. Relative luciferase activity is shown for reporter constructs containing 3′ UTR or 3′ UTR M (with deleted seed pairing region) (bottom) in cells transfected with miR-193b or negative control. B: As in A, except reporter constructs carried the indicated fragment of the Mcl-1 3′ UTR (top). Four MREs predicted by RNA22 in the 3′ UTR of Mcl-1 (middle). C: As in A, except reporter constructs carried UTR1 M1 or UTR1 M2 (top). UTR1 M1 contains two point mutations, as indicated in bold italic. UTR1 M2 contains no seed binding region. Renilla luciferase activity was measured 24 hours after transfection using the Dual-Glo luciferase assay system (Promega). Data were normalized to firefly luciferase. Data shown are the mean ± SEM of three replicates and are representative of three independent experiments. *P < 0 0.05 and **P < 0.001 were calculated using the paired samples t-test.

Article Snippet: A sequence-validated Mcl-1 expression plasmid (SC315538) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Binding Assay, Sequencing, Luciferase, Activity Assay, Construct, Transfection, Negative Control