Journal: Frontiers in Immunology

Article Title: PDLIM7 Synergizes With PDLIM2 and p62/Sqstm1 to Inhibit Inflammatory Signaling by Promoting Degradation of the p65 Subunit of NF-κB

doi: 10.3389/fimmu.2020.01559

Figure Lengend Snippet: PDLIM7 promotes p65 degradation in a LIM3 domain-dependent manner. (A) PDLIM7 interacts with p65 but not with p50 subunit of NF- κ B. 293T cells were transfected with a FLAG-tagged p65 or p50 expression plasmid along with or without PDLIM7. Whole cell extracts were immunoprecipitated with anti-c-Myc and immunoblotted with anti-FLAG. Western blots are representative of four independent experiments. (B) Effect of PDLIM7 on cytoplasmic, soluble and insoluble nuclear p65 in NIH3T3 cells transfected with plasmids encoding FLAG-tagged p65 and increasing amounts of c-Myc-tagged PDLIM7. Cells were subjected to fractionation and analyzed with the indicated antibody. Western blots are representative of three independent experiments. (C) Western blot analysis for soluble and insoluble nuclear p65 in NIH3T3 cells transfected with plasmids encoding p65 without or with PDLIM7, then left untreated or treated for 4 h with MG132 (10 μM) and analyzed as in (B) . (D) NIH3T3 cells were transfected with FLAG-p65 along with or without c-Myc-PDLIM7, left untreated or treated for 4 h with MG132 (10 μM) or bafilomycin (100 μM). Cells were subjected to fractionation and analyzed with anti-FLAG antibody. Western blots are representative of three independent experiments. (E) Ubiquitination assay for p65 in 293T cells cotransfected with plasmids encoding His-tagged ubiquitin (His-Ub) and p65, together without or with wild-type or ΔLIM3 PDLIM7 and analyzed as in . Western blots are representative of three independent experiments. (F) Effect of ΔLIM3 PDLIM7 on cytoplasmic, soluble and insoluble nuclear p65 in NIH3T3 cells transfected with plasmids encoding p65, together without or with wild-type or ΔLIM3 PDLIM7 and analyzed as in (B) . Western blots are representative of five independent experiments. (G) Luciferase activity in MEFs transfected with an ELAM-1-luc and pGL4.32-Null renilla construct with or without plasmids encoding p65 in the absence or presence of expression plasmids encoding wild-type or ΔLIM3 PDLIM7. Data are representative of three independent experiments. Shown are the mean values ± SD. ** P < 0.01.

Article Snippet: FLAG-tagged p50 expression plasmid (p50 cFLAG pcDNA3) was purchased from Addgene (#20018).

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Fractionation, Ubiquitin Assay, Luciferase, Activity Assay, Construct

Journal: Oncotarget

Article Title: A c-Jun N-terminal kinase inhibitor, JNK-IN-8, sensitizes triple negative breast cancer cells to lapatinib

doi: 10.18632/oncotarget.20581

Figure Lengend Snippet: (A) Cells were treated with 5μM JNK-IN-8 (JIN) and/or 3μM lapatinib (Lap) for MDA-MB-231 cells, and 4μM JIN and/or 7μM Lap for MDA-MB-436 cells for various time points. Cells were also transfected with NFκB luciferase reporter plasmids. Line graph points represent normalized luciferase values from 3 technical replicates. (B) MDA-MB-231 cells were treated with either vehicle (DMSO), 5μM JIN and/or 3μM Lap for 48 hours in full media. Whole cell lysates were probed for phospho-p65(ser536), p65, p50, IKKβ, and IKKα. (C) MDA-MB-231 cells were treated with 5μM JIN and/or 3μM Lap for various time points and transfected with a luciferase reporter plasmid under the control of the NQO1 promoter containing Nrf2/ antioxidant response elements (ARE). Line graph points represent normalized luciferase values from 3 technical replicates. (D) Relative levels of glutathione were measured in MDA-MB-231 and MDA-MB-436 cells after 72 hours of treatment in DMSO, JIN, Lap, or JNK-IN-8 and lapatinib (J+L). (E) Levels of NADP + and NADPH were measured in MDA-MB-231 and MDA-MB-436 cells after 72 hours of treatment in DMSO, JIN, Lap, or J+L. (F) MDA-MB-231 cells were non-transfected, transfected with empty vector (EV), or expression plasmids for p65 (p65 OE) and Nrf2 (Nrf2 OE). Twenty four hours after transfection cells were treated with either vehicle (DMSO), 5μM JIN and/or 3μM Lap for 72 hours in full media. Cell viability was assayed using MTS.

Article Snippet: The next day cells were transfected with 200ng (96 well) or 2μg (6 well) of empty vector (pcDNA.3), p65 (RelA cFlag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20012)), or Nrf2 (pCDNA3-Myc3-Nrf2 was a gift from Yue Xiong (Addgene plasmid # 21555)).

Techniques: Transfection, Luciferase, Plasmid Preparation, Control, Expressing

Journal: Oncotarget

Article Title: A c-Jun N-terminal kinase inhibitor, JNK-IN-8, sensitizes triple negative breast cancer cells to lapatinib

doi: 10.18632/oncotarget.20581

Figure Lengend Snippet: Lapatinib and JNK-IN-8 jointly inhibit the transcriptional activities of AP-1, NFκB, and Nrf2. This disrupts the cell's natural antioxidant response resulting in accumulation of ROS leading to apoptosis.

Article Snippet: The next day cells were transfected with 200ng (96 well) or 2μg (6 well) of empty vector (pcDNA.3), p65 (RelA cFlag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20012)), or Nrf2 (pCDNA3-Myc3-Nrf2 was a gift from Yue Xiong (Addgene plasmid # 21555)).

Techniques: