lentiviral vector plvct ttr krab  (Addgene inc)

Journal: Journal of translational medicine

Article Title: Cycling hypoxia induces chemoresistance through the activation of reactive oxygen species-mediated B-cell lymphoma extra-long pathway in glioblastoma multiforme.

doi: 10.1186/s12967-015-0758-8

Figure Lengend Snippet: Fig. 2 Cycling hypoxia mediates Bcl-xL expression via HIF-1α or NF-κB activation. Bcl-xL mRNA (a) and protein (b) levels in GBM8401 glioblastoma cells collected before and after 1 h of each of the 3 cycles of hypoxia (<1 % O2) during cycling hypoxia (CyH) and after 4 h of uninterrupted hypoxia (NiH). *P < 0.05, **P < 0.01, ***P < 0.001 compared to normoxia. c Western blot analysis of HIF-1α or p65 knockdown in GBM8401 glioblastoma cells via the lentiviral-based HIF-1α or p65 shRNA. d, e Bcl-xL mRNA and protein levels in GBM8401 glioblastoma cells transfected with or without scram‑ ble (Scr.), HIF-1α, or p65 shRNA for 48 h followed by stimulation with cycling hypoxic stress for 4 h. ***P < 0.001 compared to normoxia (c). #P < 0.01, ##P < 0.001 compared to Scr. shRNA treatment. f, g Bcl-xL mRNA and protein levels in U251 and U87 cells treated with cycling hypoxic stress for 4 h in the absence or presence of HIF-1α (YC-1) and NF-κB (Bay 11-7082) inhibitors. Error bars denote the standard deviation within triplicate experi‑ ments. ***P < 0.001 compared to normoxia (Nor). ##P < 0.01, ###P < 0.001 compared to vehicle treatment

Article Snippet: The lentiviral vector pLVCTtTR-KRAB (Addgene) was used to express Bcl-xL shRNA (Sigma-Aldrich) following the manufacturer’s protocol.

Techniques: Expressing, Activation Assay, Western Blot, Knockdown, shRNA, Transfection, Standard Deviation

Journal: Journal of translational medicine

Article Title: Cycling hypoxia induces chemoresistance through the activation of reactive oxygen species-mediated B-cell lymphoma extra-long pathway in glioblastoma multiforme.

doi: 10.1186/s12967-015-0758-8

Figure Lengend Snippet: Fig. 3 Cycling hypoxia-mediated Bcl-xL expression results in chemoresistance. a Verification of Bcl-xL knockdown by Tet-regulatable lentiviral knockdown system at the mRNA (a) and protein (b) levels. The lentiviral infected cells were treated for 48 h with Dox (0.04 μg/mL) to induce Bcl-xL knockdown. Error bars denote the standard deviation among triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to no Dox treat‑ ment. Caspase-3 activity (c, d) and percentage of apoptotic cells (e, f) in U251 and U87 cells cultured in normoxia, uninterrupted (NiH) hypoxia (<1 % O2), and cycling (CyH) hypoxia (<1 % O2) with or without Bcl-xL knockdown in response to temozolomide (TMZ) treatment. Cells were pretreated for 48 h with Dox (0.04 μg/mL) to induce Bcl-xL knockdown and exposed to hypoxic stresses before TMZ treatment. Error bars denote the standard deviation among triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control without any treatment. #P < 0.05, ##P < 0.01 compared to normoxia with TMZ treatment

Article Snippet: The lentiviral vector pLVCTtTR-KRAB (Addgene) was used to express Bcl-xL shRNA (Sigma-Aldrich) following the manufacturer’s protocol.

Techniques: Expressing, Knockdown, Infection, Standard Deviation, Activity Assay, Cell Culture, Control