Journal: Cell Death and Differentiation

Article Title: The bromodomain protein BRD4 positively regulates necroptosis via modulating MLKL expression

doi: 10.1038/s41418-018-0262-9

Figure Lengend Snippet: BRD4-regulated expression of MLKL. a BRD2, BRD3, or BRD4 shRNA was transfected into 293T cells, and protein levels of BRD2, BRD3, BRD4, and MLKL were detected by western blotting after 48 h. b BRD4 shRNA or control plasmid was transfected into 293T cells, and the mRNA levels of BRD4 and MLKL were measured by q-PCR after 36 h. Columns, mean (n = 3); bars, SD. **P < 0.01; ***P < 0.001. c HT29 and HeLa cells were infected with lentiviral shRNA to BRD4. Protein levels of BRD4 and MLKL were detected by western blotting

Article Snippet: IRF1 shRNA lentiviral particles (sc-35706-v) and MLKL shRNA lentiviral particles (sc-93430-v) were purchased from Santa Cruz.

Techniques: Expressing, shRNA, Transfection, Western Blot, Control, Plasmid Preparation, Infection

Journal: Cell Death and Differentiation

Article Title: The bromodomain protein BRD4 positively regulates necroptosis via modulating MLKL expression

doi: 10.1038/s41418-018-0262-9

Figure Lengend Snippet: BRD4 regulates MLKL expression through interacting with IRF1. a Shown is the enhancement of MLKL luciferase activity by the co-expression of IRF1 or/and BRD4 in 293T cells. Columns, mean (n = 3); bars, SD. **P < 0.01; ***P < 0.001. b HT29 and HeLa cells were infected with lentiviral IRF1 shRNA or the control shRNA. The protein level of MLKL was detected by western blotting. c Co-immunoprecipitation was conducted to detect the interactions between BRD4 and IRF1 in HT29 cells treated with/without TSA for 12 h. d 293T cells were co-transfected with FLAG-BRD4 (1-470) or FLAG-BRD4 (471–1362), together with HA-IRF1 by Lipofectamine 2000. After a 48-h transfection, the cells were harvested to conduct immunoprecipitation/immunoblotting with indicated antibodies. e 293T cells were co-transfected with FLAG-BRD4 (1–470) and HA-IRF1 by Lipofectamine 2000. After a 36-h transfection, the cells were treated with/without TSA for 12 h, followed by immunoprecipitation/immunoblotting with indicated antibodies. f 293T cells were co-transfected with FLAG-BD1 or FLAG-BD2, together with HA-IRF1 by Lipofectamine 2000. After a 48-h transfection, the cells were harvested to conduct immunoprecipitation/immunoblotting with indicated antibodies. g Co-immunoprecipitation was conducted to detect the interactions among BRD4, IRF1, RNA-POL II, and P-TEFb with or without JQ-1 treatment in HT29. h ChIP-qPCR was conducted to examine whether BRD4, IRF1, RNA-POL II, and P-TEFb bind to MLKL promoter. Columns, mean (n = 3); bars, SD. ***P < 0.001. i Schematically showing the mechanism by which BRD4 regulates MLKL expression

Article Snippet: IRF1 shRNA lentiviral particles (sc-35706-v) and MLKL shRNA lentiviral particles (sc-93430-v) were purchased from Santa Cruz.

Techniques: Expressing, Luciferase, Activity Assay, Infection, shRNA, Control, Western Blot, Immunoprecipitation, Transfection, ChIP-qPCR

Journal: Cell Death and Differentiation

Article Title: The bromodomain protein BRD4 positively regulates necroptosis via modulating MLKL expression

doi: 10.1038/s41418-018-0262-9

Figure Lengend Snippet: BET inhibitors do not protect MLKL-independent cell death. a HT29 cells were pretreated with BET inhibitors at the indicated concentrations for 3 days. Then apoptosis was induced by TS [TNFα (10 ng/ml), Smac-mimetic (100 nM)]. In 24 h, cell viability was determined by CCK8 staining. Columns, mean (n = 3); bars, SD. ***P < 0.001. b HT29 cells were transfected with MLKL shRNA lentiviral particles or control in this experiment. In 48 h, apoptosis was induced by TS [TNFα (10 ng/ml), Smac-mimetic (100 nM)]. Cell viability was determined by CCK8 staining 24 h after TS treatment. Columns, mean (n = 3); bars, SD. ***P < 0.001. RNA interference efficiency was measured by western blotting. c HT1080 cells were pretreated with BET inhibitors for 3 days. Then ferroptosis was induced by erastin (10 μM) for 24 h. Cell viability was determined by CCK8 staining. d HT29 was treated with JQ-1 (0.3 μM) for 48 h. Then, JQ-1-treated HT29 cells were transfected with lentiviral particles of pCDH-MLKL-GFP or pCDH-RFP vector. In 48 h, TNFα/Smac mimetic/Z-VAD-FMK (TSZ) was added to induce necroptosis. Cell viability was measured by CCK8 staining. Columns, mean (n = 3); bars, SD. **P < 0.01; ***P < 0.001. e Protein levels of MLKL in HT29 cells changed following JQ-1 treatment and transfection with lentiviral particles of pCDH-MLKL-GFP or pCDH-RFP vector. Transfection efficiency was shown by GFP or RFP fluorescence. Scale bar, 200 μm

Article Snippet: IRF1 shRNA lentiviral particles (sc-35706-v) and MLKL shRNA lentiviral particles (sc-93430-v) were purchased from Santa Cruz.

Techniques: Staining, Transfection, shRNA, Control, Western Blot, Plasmid Preparation, Fluorescence

Journal: Cell Division

Article Title: Interferon gamma-induced apoptosis of head and neck squamous cell carcinoma is connected to indoleamine-2,3-dioxygenase via mitochondrial and ER stress-associated pathways

doi: 10.1186/s13008-016-0023-4

Figure Lengend Snippet: a Western blot analysis of JAK1, IRF-1, IDO and HO-1 in CLS-354 and RPMI 2650 cells following the exposure to IFNγ for 48 h. Actin was used as an internal control for loading and transfer. EMSA demonstrates the enhancement of the DNA-binding activity of the transcription factors IRF-1 ( b ) and STAT1 ( c ) following the exposure of CLS-354 and RPMI 2650 cells to IFNγ for 48 h. Data are representative of three independent experiments with similar results

Article Snippet: Briefly, the double stranded synthetic oligonucleotides that represent the specific binding sites of the corresponding transcription factors including, AP-1, ATF-2, p53, NF-κB, STAT1, IRF-1 each purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA.

Techniques: Western Blot, Binding Assay, Activity Assay

Journal: Cell Division

Article Title: Interferon gamma-induced apoptosis of head and neck squamous cell carcinoma is connected to indoleamine-2,3-dioxygenase via mitochondrial and ER stress-associated pathways

doi: 10.1186/s13008-016-0023-4

Figure Lengend Snippet: Proposed model for IFNγ-induced apoptosis of HNSCC cells. The activation of IFNγ receptor of HNSCC cells with IFNγ results in the activation of JAK-STAT pathway that subsequently enhances the DNA binding activity of the transcription factor interferon regulatory factor-1 (IRF-1) that is essential for the induction IDO expression. The increased level of IDO results in the suppression of the anti-oxidant protein heme oxygenase-1 (HO-1) that, in turn, leads to the accumulation of reactive oxygen species (ROS). The increased accumulation of ROS mediates oxidative stress-associated pathways such as apoptosis signal-regulating kinase 1(ASK1)/c-Jun-N-terminal kinase (JNK)-AP-1/p53, IKK-NF-κB, and p38-ATF-2. The formation of a transcriptional complex from the transcription factors NF-κB, AP-1 and p53 results in the transcriptional activation of the promoter of Noxa protein. The localization of Noxa protein to mitochondria results in the loss of mitochondrial membrane potential (Δ ψ m) that is characterized by the release of cytochrome c (Cyt.C) into the cytoplasm where it initiates apoptosis by formation of apoptosome complex leading to the cleavage of caspase-9, caspase-3 and PARP. Whereas the localization of Noxa to endoplasmic reticulum (ER) results in ER-stress that is associated with the activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) and inositol-requiring-1α (IRE1α) pathways as well as cleavage of caspase-4 that, in turn, mediates the cleavage of caspase-3

Article Snippet: Briefly, the double stranded synthetic oligonucleotides that represent the specific binding sites of the corresponding transcription factors including, AP-1, ATF-2, p53, NF-κB, STAT1, IRF-1 each purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA.

Techniques: Activation Assay, Binding Assay, Activity Assay, Expressing