Journal: Cancers

Article Title: Loss of PARP7 Increases Type I Interferon Signaling in EO771 Breast Cancer Cells and Prevents Mammary Tumor Growth by Increasing Antitumor Immunity.

doi: 10.3390/cancers15143689

Figure Lengend Snippet: Figure 4. Nuclear factor κB (NF-κB) is involved in the increased type I IFN signaling observed in EO771 Parp7KO cells. (A) Inhibition of TANK-binding kinase 1 (TBK1) with MRT67307 significantly decreases Ifnb1 mRNA levels in EO771 Parp7KO cells but not in WT cells. Inhibition of both TBK1 and the IκB kinases (IKKs) with BX795 blocks Ifnb1 mRNA expression in all cases. Cells were treated with either 1 µM of MRT67307 or 100 nM of BX795 for 24 h, followed by 10 µg/mL of DMXAA for 2 h. (B) Treatment with DMXAA and/or RBN-2397 increases protein levels of both p50 and RelA in EO771 WT cells while levels are higher in Parp7KO cells in both untreated and treated cells. Cells were treated with 10 µg/mL of DMXAA (+/−100 nM of RBN-2397) for 24 h and proteins were visualized by Western blotting. (C) Levels of expressed NF-κB subunits are higher in response to PARP7 inhibition, and levels of RelA are significantly higher in the Parp7KO cells. Protein levels of p50 and RelA in untreated samples were quantified by normalizing to the loading control. (D) Expression levels of Rela mRNA are not affected by DMXAA or PARP7. (E) Knockdown of either TBK1, RelA, or both, in EO771 WT and Parp7KO cells. After 48 h of transfection with 25 nM of siRNA targeting Tbk1, Rela, or both, protein levels were visualized by Western blotting. (F) Knockdown of TBK1 and RelA results in significantly decreased Ifnb1 mRNA levels in Parp7KO cells but not in WT cells. After 48 h of transfection with 25 nM of siRNA for Tbk1, Rela, or both, cells were treated with 10 µg/mL of DMXAA for 2 h. (G) p50 and RelA interact with WT and catalytically inactive PARP7, and RelA is MARylated by WT PARP7. Cos-1 cells were transfected with FLAG-tagged p50 or RelA together with either GFP-tagged WT PARP7 or the catalytically inactive H532A mutant. The FLAG-tagged p50 or RelA was immunoprecipitated, and the interaction and modification was examined by Western blotting. * Denotes statistical significance (p < 0.05) compared to DMSO; # denotes statistical difference due to the loss of PARP7; “a” denotes statistical difference compared to no inhibitor (A) or to the nontargeting siRNA (F). Original western blots have been presented in File S1.

Article Snippet: The pSpCas9(BB)-2A-Puro (PX459) (plasmid #62988), p50 cFlag pcDNA3 (plasmid #20018), and RelA cFlag pcDNA3 (plasmid #20012) were obtained from Addgene (Watertown, MA, USA).

Techniques: Inhibition, Binding Assay, Expressing, Western Blot, Control, Knockdown, Transfection, Mutagenesis, Immunoprecipitation

Journal: Oncogenesis

Article Title: A novel role of MNT as a negative regulator of REL and the NF-κB pathway

doi: 10.1038/s41389-020-00298-4

Figure Lengend Snippet: a Co-immunoprecipitation assay with anti-MNT and anti-MAX antibodies in URMax34 cells after Zn 2+ treatment. MAX was used as a positive control for MNT immunoprecipitations. The asterisk marks the position of the heavy IgG band. b Co-immunoprecipitation assay with anti-MNT in mouse Neuro-2a cells . c Co-immunoprecipitation assay in human LoVo cells with anti-MNT and anti-REL. Two immunoblot images of the blots with high and low intensity are shown. Note that the hypotonic buffer required for MNT–REL co-immunoprecipitation does not extract efficiently MNT protein. d Co-immunoprecipitation assay in rat glioma C6 cells with anti-MNT and anti-REL antibodies. The asterisk marks the position of the light IgG band. e rpoximity ligation assay (PLA) performed in C6 cells 48 h after transfection with WT MNT-HA and REL-flag overexpressing vectors. Antibodies anti-MNT/anti-REL, anti-MYC/anti-MAX (positive control), and anti-MYC/anti-REL (negative control). The PLA-positive signal in red and DAPI as a nuclear marker . f PLA in LoVo cells (untransfected cells) with anti-MNT/anti-REL, anti-p65/anti-REL (positive control), and anti-MNT/anti-MYC (negative control) antibodies. The PLA-positive signal is in red and DAPI staining was used as a nuclear marker.

Article Snippet: As for REL plasmids, we used mouse c-Rel cFlag pcDNA3 (gift from Stephen Smale, RRID:Addgene_20013, http://n2t.net/addgene:20013 ); human pcDNA-FLAG-REL (RRID:Addgene_27253, http://n2t.net/addgene:27253 ) and human pcDNA-FLAG-RELΔRID with a deletion of 323–422 amino acids (Addgene # 27265; http://n2t.net/addgene:27265 ; RRID: Addgene_27265) (both gifts from Thomas Gilmore).

Techniques: Co-Immunoprecipitation Assay, Positive Control, Western Blot, Immunoprecipitation, Ligation, Transfection, Negative Control, Marker, Staining

Journal: Oncogenesis

Article Title: A novel role of MNT as a negative regulator of REL and the NF-κB pathway

doi: 10.1038/s41389-020-00298-4

Figure Lengend Snippet: a Co-immunoprecipitation assays with anti-MNT and anti-p65 antibodies (the IgG as a negative control) in URMT and URMax34 after Zn 2+ treatment. The inputs are shown in the left panel. b Co-immunoprecipitation assay with MNT antibodies (MNT 50 , an antibody against the 50 first amino acids of MNT protein; MNT 582 , an antibody against the 532–582 amino acids of MNT protein), and with p65 and p105/p50 antibodies, and IgG as a negative control. Two immunoblots for REL are shown (low and high intensity). c Working hypothesis with two options: MNT bound to REL homodimers, MNT bound to REL and other yet unknown protein(s). d Schematic representation of the mouse MNT deletion constructs used for the co-IP assays, ΔbHLH and ΔCt1 MNT-HA. The dotted red lines connect the domains of MNT and REL necessary for the interaction. e C6 cell lysates 48 h after transfection with REL-flag (mouse) and ΔbHLH or ΔCt1 MNT-HA (mouse) were immunoprecipitated with anti-HA antibodies (IgG as negative control). The immunoblot of HA and REL is shown. The asterisk marks the heavy IgG band. f C6 cells were lysed following the nucleus/cytoplasm fractionation protocol, both in basal conditions or 30 min after treatment with TNFα (100 ng/mL) and immunoprecipitated with an anti-MNT antibody or the IgG (the latter as a negative control). The presence of REL, MNT, and MAX was determined in the immunoprecipitates by immunoblot. RhoGDI and SIN3B were analyzed as cytoplasm and nucleus markers, respectively. Coomassie blue was used as a protein loading control for the inputs. The asterisk marks the light IgG band.

Article Snippet: As for REL plasmids, we used mouse c-Rel cFlag pcDNA3 (gift from Stephen Smale, RRID:Addgene_20013, http://n2t.net/addgene:20013 ); human pcDNA-FLAG-REL (RRID:Addgene_27253, http://n2t.net/addgene:27253 ) and human pcDNA-FLAG-RELΔRID with a deletion of 323–422 amino acids (Addgene # 27265; http://n2t.net/addgene:27265 ; RRID: Addgene_27265) (both gifts from Thomas Gilmore).

Techniques: Immunoprecipitation, Negative Control, Co-Immunoprecipitation Assay, Western Blot, Construct, Transfection, Fractionation