Journal: Nature Communications

Article Title: METTL3 promotes tumour development by decreasing APC expression mediated by APC mRNA N 6 -methyladenosine-dependent YTHDF binding

doi: 10.1038/s41467-021-23501-5

Figure Lengend Snippet: a RIP analyses of KYSE180 cells were performed with an anti-YTHDF1 or anti-YTHDF2, or anti-YTHDF3 antibody followed by qPCR analyses with primers against APC mRNA. Data represent the means ± SD of triplicate samples. *** p = 0.0006 (left), ** p = 0.0092 (right) based on two-tailed Student’s t -test. b KYSE180 cells were transfected with or without a vector expressing METTL3 shRNA. RIP analyses were performed with an anti-YTHDF2 antibody followed by qPCR analyses with primers against APC mRNA. Data represent the means ± SD of triplicate samples. *** p = 1.85E − 05 (left), ** p = 0.0023 (middle) and 0.0056 (right) based on two-tailed Student’s t -test. c KYSE450 cells were transfected with or without a vector expressing YTHDF2 shRNA. The relative mRNA expression levels of APC were measured using quantitative PCR. Data represent the means ± SD of triplicate samples. *** p = 0.0006, 0.0009, 5.58E − 05 and 0.0007 (left to right) based on two-tailed Student’s t -test. d KYSE450 cells were transfected with or without YTHDF2 shRNA. Immunoblotting analyses were performed with the indicated antibodies for three times with similar results. e KYSE180 cells were transfected with or without a YTHDF2 siRNA or combination of YTHDF1–3 siRNAs. The relative mRNA expression levels of APC were measured using quantitative PCR. Data represent the means ± SD of triplicate samples. *** p = 9.33E − 04 (left) and 2.99E − 05 (right), * p = 0.031 based on two-tailed Student’s t -test. f KYSE180 cells were transfected with or without a YTHDF2 siRNA or combination of YTHDF1–3 siRNAs. Immunoblotting analyses were performed with the indicated antibodies for three times with similar results. g KYSE180 cells expressing luciferase reporter genes fused with or without the wild-type (WT) or mutated m6A nucleotides from APC genes were transfected with or without a vector expressing YTHDF2 shRNA. The relative luciferase activity after normalization to the shControl group is shown. Data represent the means ± SD of triplicate samples. *** p = 3.94E − 06 based on two-tailed Student’s t -test. ns, not significant. Source data are provided as a Source Data file.

Article Snippet: Rabbit antibodies recognizing YTHDF1 (17479-1-AP) (for RIP, 1 : 50 dilution and immunoblotting, 1 : 1000 dilution) and YTHDF2 (24744-1-AP) (for RIP, 1 : 50 dilution and immunoblotting, 1 : 1000 dilution) and YTHDF3 (25537-1-AP) (for RIP, 1 : 50 dilution and immunoblotting, 1 : 1000 dilution) were purchased from Proteintech (IL, USA).

Techniques: Two Tailed Test, Transfection, Plasmid Preparation, Expressing, shRNA, Real-time Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

doi: 10.1038/labinvest.2017.115

Figure Lengend Snippet: Figure 5 Wogonin fails to further reduce cisplatin-induced cell injury and inflammatory response in HK2 cells where RIPK1 is blocked. (a) Western blot analysis and quantitative data of RIPK1/RIPK3/MLKL signaling in Nec-1-treated HK2 cells. (b) Western blot analysis and quantitative data of KIM-1. Results indicated that when RIPK1 was blocked, wogonin failed to further suppress the protein levels of KIM-1. (c) Real-time PCR showed that when RIPK1 was blocked, wogonin failed to further decrease IL-6 in mRNA level. Independent experiments were performed throughout the in vitro studies in triplicate or quadruplicate. *Po0.05, **Po0.01, ***Po0.001 compared with the control. ##Po0.01, ###Po0.001 compared with cisplatin-treated group. $$Po0.01, $$

Article Snippet: Then, western blot analysis was performed as described previously.16,17 After blocking nonspecific binding with 5% BSA (room temperature, 1 h), membranes were consequently incubated with the primary antibody against KIM-1, RIPK1, RIPK3, p-MLKL, cleaved-caspase-3, cleaved-caspase-8 and β-actin overnight at 4 °C, followed by treated with IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA, USA).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, In Vitro, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

doi: 10.1038/labinvest.2017.115

Figure Lengend Snippet: Figure 6 Wogonin fails to further reduce cisplatin-induced cell injury and inflammatory response in HK2 cells where RIPK3 is disrupted. (a) Western blot analysis and quantitative data of RIPK3 in HK2 cells. Results showed that RIPK3 was downregulated by transfection of RIPK3 shRNA plasmid. (b) Western blot analysis and quantitative data of RIPK1/RIPK3/MLKL signaling in RIPK3 knockdown HK2 cells. (c) Western blot analysis and quantitative data of KIM-1. Results indicated that when RIPK3 was knockdown, wogonin failed to further decrease the protein levels of KIM-1. (d) Real-time PCR identified that when RIPK3 was disrupted, wogonin failed to further decrease mRNA levels of TNF-α and IL-6. Independent experiments were performed throughout the in vitro studies in triplicate or quadruplicate. **Po0.01,***Po0.001 compared with the control. #Po0.05, ##Po0.01, ###Po0.001 compared with cisplatin-treated group. $$Po0.01,$$$Po0.001 compared with RIPK3 EV group. Cis, cisplatin; Wog, wogonin; EV, empty vector; KD, knockdown.

Article Snippet: Then, western blot analysis was performed as described previously.16,17 After blocking nonspecific binding with 5% BSA (room temperature, 1 h), membranes were consequently incubated with the primary antibody against KIM-1, RIPK1, RIPK3, p-MLKL, cleaved-caspase-3, cleaved-caspase-8 and β-actin overnight at 4 °C, followed by treated with IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA, USA).

Techniques: Western Blot, Transfection, shRNA, Plasmid Preparation, Knockdown, Real-time Polymerase Chain Reaction, In Vitro, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

doi: 10.1038/labinvest.2017.115

Figure Lengend Snippet: Figure 9 Wogonin prevents RIPK1-mediated necroptosis in cisplatin nephropathy. (a) Electron microscope. Results clearly demonstrated that wogonin largely reduced nuclear structure damage and abnormal cell organelle content in injured kidney. (b) Western blot analysis of RIPK1/RIPK3/MLKL signaling. Results showed that treatment of wogonin significantly suppressed the activation of RIPK1/RIPK3/MLKL axis in cisplatin-injured kidney. Data represent the mean ± s.e.m. for 6–8 mice. ***Po0.001 compared with control. ##Po0.01, ###Po0.001 compared with model. Cis, cisplatin; Wog, wogonin.

Article Snippet: Then, western blot analysis was performed as described previously.16,17 After blocking nonspecific binding with 5% BSA (room temperature, 1 h), membranes were consequently incubated with the primary antibody against KIM-1, RIPK1, RIPK3, p-MLKL, cleaved-caspase-3, cleaved-caspase-8 and β-actin overnight at 4 °C, followed by treated with IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA, USA).

Techniques: Microscopy, Western Blot, Activation Assay, Control

Journal: Nature Communications

Article Title: METTL3 promotes tumour development by decreasing APC expression mediated by APC mRNA N 6 -methyladenosine-dependent YTHDF binding

doi: 10.1038/s41467-021-23501-5

Figure Lengend Snippet: a RIP analyses of KYSE180 cells were performed with an anti-YTHDF1 or anti-YTHDF2, or anti-YTHDF3 antibody followed by qPCR analyses with primers against APC mRNA. Data represent the means ± SD of triplicate samples. *** p = 0.0006 (left), ** p = 0.0092 (right) based on two-tailed Student’s t -test. b KYSE180 cells were transfected with or without a vector expressing METTL3 shRNA. RIP analyses were performed with an anti-YTHDF2 antibody followed by qPCR analyses with primers against APC mRNA. Data represent the means ± SD of triplicate samples. *** p = 1.85E − 05 (left), ** p = 0.0023 (middle) and 0.0056 (right) based on two-tailed Student’s t -test. c KYSE450 cells were transfected with or without a vector expressing YTHDF2 shRNA. The relative mRNA expression levels of APC were measured using quantitative PCR. Data represent the means ± SD of triplicate samples. *** p = 0.0006, 0.0009, 5.58E − 05 and 0.0007 (left to right) based on two-tailed Student’s t -test. d KYSE450 cells were transfected with or without YTHDF2 shRNA. Immunoblotting analyses were performed with the indicated antibodies for three times with similar results. e KYSE180 cells were transfected with or without a YTHDF2 siRNA or combination of YTHDF1–3 siRNAs. The relative mRNA expression levels of APC were measured using quantitative PCR. Data represent the means ± SD of triplicate samples. *** p = 9.33E − 04 (left) and 2.99E − 05 (right), * p = 0.031 based on two-tailed Student’s t -test. f KYSE180 cells were transfected with or without a YTHDF2 siRNA or combination of YTHDF1–3 siRNAs. Immunoblotting analyses were performed with the indicated antibodies for three times with similar results. g KYSE180 cells expressing luciferase reporter genes fused with or without the wild-type (WT) or mutated m6A nucleotides from APC genes were transfected with or without a vector expressing YTHDF2 shRNA. The relative luciferase activity after normalization to the shControl group is shown. Data represent the means ± SD of triplicate samples. *** p = 3.94E − 06 based on two-tailed Student’s t -test. ns, not significant. Source data are provided as a Source Data file.

Article Snippet: Rabbit antibodies recognizing YTHDF1 (17479-1-AP) (for RIP, 1 : 50 dilution and immunoblotting, 1 : 1000 dilution) and YTHDF2 (24744-1-AP) (for RIP, 1 : 50 dilution and immunoblotting, 1 : 1000 dilution) and YTHDF3 (25537-1-AP) (for RIP, 1 : 50 dilution and immunoblotting, 1 : 1000 dilution) were purchased from Proteintech (IL, USA).

Techniques: Two Tailed Test, Transfection, Plasmid Preparation, Expressing, shRNA, Real-time Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay

Journal: Nature Communications

Article Title: METTL3 promotes tumour development by decreasing APC expression mediated by APC mRNA N 6 -methyladenosine-dependent YTHDF binding

doi: 10.1038/s41467-021-23501-5

Figure Lengend Snippet: a RIP analyses of KYSE180 cells were performed with an anti-YTHDF1 or anti-YTHDF2, or anti-YTHDF3 antibody followed by qPCR analyses with primers against APC mRNA. Data represent the means ± SD of triplicate samples. *** p = 0.0006 (left), ** p = 0.0092 (right) based on two-tailed Student’s t -test. b KYSE180 cells were transfected with or without a vector expressing METTL3 shRNA. RIP analyses were performed with an anti-YTHDF2 antibody followed by qPCR analyses with primers against APC mRNA. Data represent the means ± SD of triplicate samples. *** p = 1.85E − 05 (left), ** p = 0.0023 (middle) and 0.0056 (right) based on two-tailed Student’s t -test. c KYSE450 cells were transfected with or without a vector expressing YTHDF2 shRNA. The relative mRNA expression levels of APC were measured using quantitative PCR. Data represent the means ± SD of triplicate samples. *** p = 0.0006, 0.0009, 5.58E − 05 and 0.0007 (left to right) based on two-tailed Student’s t -test. d KYSE450 cells were transfected with or without YTHDF2 shRNA. Immunoblotting analyses were performed with the indicated antibodies for three times with similar results. e KYSE180 cells were transfected with or without a YTHDF2 siRNA or combination of YTHDF1–3 siRNAs. The relative mRNA expression levels of APC were measured using quantitative PCR. Data represent the means ± SD of triplicate samples. *** p = 9.33E − 04 (left) and 2.99E − 05 (right), * p = 0.031 based on two-tailed Student’s t -test. f KYSE180 cells were transfected with or without a YTHDF2 siRNA or combination of YTHDF1–3 siRNAs. Immunoblotting analyses were performed with the indicated antibodies for three times with similar results. g KYSE180 cells expressing luciferase reporter genes fused with or without the wild-type (WT) or mutated m6A nucleotides from APC genes were transfected with or without a vector expressing YTHDF2 shRNA. The relative luciferase activity after normalization to the shControl group is shown. Data represent the means ± SD of triplicate samples. *** p = 3.94E − 06 based on two-tailed Student’s t -test. ns, not significant. Source data are provided as a Source Data file.

Article Snippet: Rabbit antibodies recognizing YTHDF1 (17479-1-AP) (for RIP, 1 : 50 dilution and immunoblotting, 1 : 1000 dilution) and YTHDF2 (24744-1-AP) (for RIP, 1 : 50 dilution and immunoblotting, 1 : 1000 dilution) and YTHDF3 (25537-1-AP) (for RIP, 1 : 50 dilution and immunoblotting, 1 : 1000 dilution) were purchased from Proteintech (IL, USA).

Techniques: Two Tailed Test, Transfection, Plasmid Preparation, Expressing, shRNA, Real-time Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay

Journal: Viruses

Article Title: Swine Influenza Virus Induces RIPK1/DRP1-Mediated Interleukin-1 Beta Production

doi: 10.3390/v10080419

Figure Lengend Snippet: The SIV-induced IL-1β production requires receptor-interacting protein kinase 1 (RIPK1) kinase activity. ( A ) PAMs were transfected with a negative control siRNA (siControl) or RIPK1 -targeting siRNA (siRIPK1) for 24 h and infected with Sk02 at an MOI of 1 for 16 h. Porcine IL-1β levels from supernatants were measured by ELISA. Expression of pro-IL-1β was analyzed by Western blotting. RIPK1 knockdown efficiency was determined by real-time PCR. ( B ) PAMs were infected with Sk02 at an MOI of 1 for 20 h in the presence of a vehicle (DMSO) or increasing concentrations (80 and 160 μM) of RIPK1 kinase inhibitor, Nec-1. Porcine IL-1β levels from supernatants were measured by ELISA. Expression of pro-IL-1β and viral NS1 was analyzed by Western blotting. ( C ) Amino acid sequences of the N-terminal ends of the kinase domains in porcine, murine, and human RIPK1 (based on the GenBank accession numbers XM_005665536, NM_009068, and NM_003804, respectively) were aligned using Clustal Omega. Amino acid positions are based on the human RIPK1 and asterisks indicate conserved residues among the three species. K41/K42 in porcine RIPK1, K45/K46 in murine RIPK1, and K45 in human RIPK1 are underscored. ( D ) For the NLRP3 inflammasome reconstitution assay, HEK293T cells were co-transfected with plasmids expressing porcine NLRP3 inflammasome components (NLRP3, ASC, and procaspase-1) and pro-IL-1β along with Myc-vector or Myc-tagged WT/mutant human RIPK1 for 16 h. Porcine IL-1β levels from supernatants were measured by ELISA. Protein expression was analyzed by Western blotting. ( E ) HEK293T cells were transfected as in ( D ) along with the Myc-vector or Myc-tagged porcine RIPK1 WT/mutant for 16 h. ELISA and Western blotting were done as in ( D ). Statistical analysis was done with one-way ANOVA except for the knockdown efficiency data done with an unpaired t -test. * p < 0.05; ** p < 0.01; *** p < 0.001. Results are representative of three independent experiments.

Article Snippet: Myc-tagged human RIPK1 (#44159, Addgene, Cambridge, MA, USA) was a gift from Xin Lin [ ].

Techniques: Activity Assay, Transfection, Negative Control, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Knockdown, Real-time Polymerase Chain Reaction, Reconstitution Assay, Plasmid Preparation, Mutagenesis

Journal: Viruses

Article Title: Swine Influenza Virus Induces RIPK1/DRP1-Mediated Interleukin-1 Beta Production

doi: 10.3390/v10080419

Figure Lengend Snippet: RIPK1 interacts with DRP1. HEK293T cells were co-transfected with the Flag-vector or Flag-RIPK1 construct along with the DRP1-Myc construct for 24 h. Cell lysates were subjected to co-IP with Flag antibody, and the expression of Flag-RIPK1 and DRP1-Myc in input and IP samples was analyzed by Western blotting. Results are representative of three independent experiments.

Article Snippet: Myc-tagged human RIPK1 (#44159, Addgene, Cambridge, MA, USA) was a gift from Xin Lin [ ].

Techniques: Transfection, Plasmid Preparation, Construct, Co-Immunoprecipitation Assay, Expressing, Western Blot

Journal: Viruses

Article Title: Swine Influenza Virus Induces RIPK1/DRP1-Mediated Interleukin-1 Beta Production

doi: 10.3390/v10080419

Figure Lengend Snippet: A proposed model of RIPK1/DRP1-mediated IL-1β production in SIV-infected PAMs. Recognition of IAV RNA by endosomal TLRs or RIG-I is known to induce pro-IL-1β synthesis. SIV infection turns on the RIPK1/DRP1 signaling for the NLRP3 inflammasome activation in PAMs. SIV seems to utilize the RIPK1 function, while how RIPK1 is activated by SIV or whether an upstream sensor plays a role in its activation is unclear. By interacting with DRP1 and through its kinase activity, RIPK1 induces the phosphorylation of porcine DRP1 at S579. Upon DRP1 translocation to mitochondria, mitochondrial fission occurs and ROS is generated. This promotes the activation of NLRP3 that is required for the NLRP3 inflammasome assembly and caspase-1 activation. Active caspase-1 converts pro-IL-1β into mature IL-1β, which is critical for SIV-induced lung inflammation. Sensing LPS by TLR4 is known to induce the pro-IL-1β expression, and this also induces the DRP1 phosphorylation leading to the NLRP3 inflammasome activation in PAMs, although it is unclear whether LPS activates any upstream regulator of RIPK1.

Article Snippet: Myc-tagged human RIPK1 (#44159, Addgene, Cambridge, MA, USA) was a gift from Xin Lin [ ].

Techniques: Infection, Activation Assay, Activity Assay, Phospho-proteomics, Translocation Assay, Generated, Expressing

Journal: Journal of Biological Chemistry

Article Title: The Kinase Activity of Rip1 Is Not Required for Tumor Necrosis Factor-α-induced IκB Kinase or p38 MAP Kinase Activation or for the Ubiquitination of Rip1 by Traf2

doi: 10.1074/jbc.m404206200

Figure Lengend Snippet: FIG. 2. The kinase activity of Rip1 is not essential for TNF-induced Ikk or p38 MAP kinase activation or for TNF--induced IL-6 production. A, TNF--induced Ikk activation is observed in cells expressing a kinase-inactive Rip1D138N. rip1/ MEFs were infected with vector alone (MSCV) or with retroviruses containing wild type Rip1 or a kinase-inactive Rip1 (rip1/(D138N)). Wild type (wt), rip1/, rip1/ (rip1), and rip1/(D138N) were left untreated or stimulated with TNF- for the time periods indicated. Cells were subsequently lysed and immunoprecipitated with -IKK- antibody, and the kinase activity of Ikk was measured by an in vitro kinase assay using glutathione S-transferase-IB (GST-IB) as a substrate. The amount of Ikk- and Rip in each cell type was determined by immunoblotting with -IKK- Ab (Santa Cruz Biotechnology) and -RIP Ab, respectively. B, normal p38 MAP kinase response to TNF in rip1/(D138N) cells. Wild type, rip1/, rip1/ (MSCV), rip1/ (rip1), and rip1/(D138N) cells were left untreated or stimulated with TNF- for the time periods indicated, and p38 MAP kinase activity was measured by immunoblotting with an -phospho-p38 MAP kinase antibody (catalog no. 9211S, Cell Signaling Technology). The Rip1 and p38- expression was determined by immunoblotting with anti-RIP and anti-p38- antibodies. C, TNF--induced IL-6 production is restored in rip1/ cells infected with rip1 or kinase-inactive rip1 retrovirus. Wild type, rip1/ MEFs, rip1/ MEFs infected with vector (MSCV), rip1 retrovirus (rip1/(rip1)) or a kinase-inactive rip1 retrovirus (rip1/(D138N)) were plated at 3 104 cells/well on 24-well plates and left untreated or treated with 10 ng/ml TNF- for 24 h. The supernatants were then analyzed for IL-6 levels using the OptEIA Mouse IL-6 enzyme-linked immunosorbent assay kit (BD Biosciences, catalog no. 2653KI). The amount of IL-6 is presented as the mean S.D. of triplicate observations.

Article Snippet: Wild type, rip1 / , rip1 / (MSCV), rip1 / (rip1), and rip1 / (D138N) cells were left untreated or stimulated with TNF- for the time periods indicated, and p38 MAP kinase activity was measured by immunoblotting with an -phospho-p38 MAP kinase antibody (catalog no. 9211S, Cell Signaling Technology).

Techniques: Activity Assay, Activation Assay, Expressing, Infection, Plasmid Preparation, Immunoprecipitation, In Vitro, Kinase Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: The Kinase Activity of Rip1 Is Not Required for Tumor Necrosis Factor-α-induced IκB Kinase or p38 MAP Kinase Activation or for the Ubiquitination of Rip1 by Traf2

doi: 10.1074/jbc.m404206200

Figure Lengend Snippet: FIG. 2. The kinase activity of Rip1 is not essential for TNF-induced Ikk or p38 MAP kinase activation or for TNF--induced IL-6 production. A, TNF--induced Ikk activation is observed in cells expressing a kinase-inactive Rip1D138N. rip1/ MEFs were infected with vector alone (MSCV) or with retroviruses containing wild type Rip1 or a kinase-inactive Rip1 (rip1/(D138N)). Wild type (wt), rip1/, rip1/ (rip1), and rip1/(D138N) were left untreated or stimulated with TNF- for the time periods indicated. Cells were subsequently lysed and immunoprecipitated with -IKK- antibody, and the kinase activity of Ikk was measured by an in vitro kinase assay using glutathione S-transferase-IB (GST-IB) as a substrate. The amount of Ikk- and Rip in each cell type was determined by immunoblotting with -IKK- Ab (Santa Cruz Biotechnology) and -RIP Ab, respectively. B, normal p38 MAP kinase response to TNF in rip1/(D138N) cells. Wild type, rip1/, rip1/ (MSCV), rip1/ (rip1), and rip1/(D138N) cells were left untreated or stimulated with TNF- for the time periods indicated, and p38 MAP kinase activity was measured by immunoblotting with an -phospho-p38 MAP kinase antibody (catalog no. 9211S, Cell Signaling Technology). The Rip1 and p38- expression was determined by immunoblotting with anti-RIP and anti-p38- antibodies. C, TNF--induced IL-6 production is restored in rip1/ cells infected with rip1 or kinase-inactive rip1 retrovirus. Wild type, rip1/ MEFs, rip1/ MEFs infected with vector (MSCV), rip1 retrovirus (rip1/(rip1)) or a kinase-inactive rip1 retrovirus (rip1/(D138N)) were plated at 3 104 cells/well on 24-well plates and left untreated or treated with 10 ng/ml TNF- for 24 h. The supernatants were then analyzed for IL-6 levels using the OptEIA Mouse IL-6 enzyme-linked immunosorbent assay kit (BD Biosciences, catalog no. 2653KI). The amount of IL-6 is presented as the mean S.D. of triplicate observations.

Article Snippet: Wild type, rip1 / , rip1 / (MSCV), rip1 / (rip1), and rip1 / (D138N) cells were left untreated or stimulated with TNF- for the time periods indicated, and p38 MAP kinase activity was measured by immunoblotting with an -phospho-p38 MAP kinase antibody (catalog no. 9211S, Cell Signaling Technology).

Techniques: Activity Assay, Activation Assay, Expressing, Infection, Plasmid Preparation, Immunoprecipitation, In Vitro, Kinase Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Blood Cancer Journal

Article Title: MYC is a positive regulator of choline metabolism and impedes mitophagy-dependent necroptosis in diffuse large B-cell lymphoma

doi: 10.1038/bcj.2017.61

Figure Lengend Snippet: MYC acted on lymphoma choline metabolism by regulating PCYT1A expression. ( a ) Schematic description of choline metabolism. PCYT1A , phosphate cytidylyltransferase 1 choline-α PLA2G4C , phospholipase A; PC, phosphatidylcholine; LPC, lysophoaphatidylcholine. ( b ) HEK-293T cells were transfected with pCMV6- MYC plasmid or control plasmid (pCMV6-ct). The efficiency of transfection was confirmed by western blot (left panel). Expression of PCYT1A and PLA2G4C were assessed by quantitative real-time PCR (middle panel). HEK-293T cells were transfected with promoter-driven luciferase reporter of PCYT1A and PLA2G4C . Luciferase reporter activity was measured at 24 h after transfection (right panel). ( c ) Schematic diagram represented the regulatory region of PCYT1A promoter (upper panel). The activity of MYC binding to the PCYT1A promoter was measured by chromatin immunoprecipitation assay in DB cells either treated with 10058-F4 (40 μ M ) for 48 h or transfected with MYC siRNA. DNA–protein complexes from DB cells were precipitated with anti-MYC antibody and amplified with primers amplifying MYC binding sites on PCYT1A promoter using PCR and quantitative real-time PCR (lower panel). Antibody against RNA Polymerase II was referred as positive control. Nonspecific IgG was referred as negative control. ( d ) Schematic diagram represented the PCYT1A promoter reporters with or without mutation of MYC binding site (left panel). HEK-293T cells were transfected with mutated promoter-driven luciferase reporter of PCYT1A . Luciferase reporter activity was measured at 24 h after transfection (right panel). ( e ) Co-immunoprecipitation showed increased formation of RIP1/RIP3/MLKL complex in DB and Ramos cells, either treated with 10058-F4 (DB, 40 μ M ; Ramos, 30 μ M ) for 48 h or transfected with MYC siRNA. ( f ) Transmission electron microscopy showed typical necroptotic cells after 10058-F4 treatment or MYC siRNA transfection. Data in b , c and d were represented as mean±s.e.m. Assays in b , c and d were set up in triplicate.

Article Snippet: Anti-RIP1 monoclonal antibody (BD Pharmingen, San Diego, CA, USA, 1:1000), anti-MLKL monoclonal antibody (Cell Signaling, 1:1000) and anti-RIP3 monoclonal antibody (Cell Signaling, 1:1000) were used.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Binding Assay, Chromatin Immunoprecipitation, Amplification, Positive Control, Negative Control, Mutagenesis, Immunoprecipitation, Transmission Assay, Electron Microscopy

Journal:

Article Title: The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-κB signalling

doi: 10.1038/emboj.2008.285

Figure Lengend Snippet: Dynamic interactions between RNF11 and the A20 ubiquitin-editing complex. (A) 293T cells were transfected with plasmids encoding Flag-tagged TAX1BP1, A20 and STAT1 in the absence or presence of Myc–RNF11. After 36 h, lysates were immunoprecipitated with anti-Myc followed by immunoblotting with anti-Flag. Lysates were examined for the expression of ectopic proteins by immunoblotting with anti-Flag and Myc. IgH, immunoglobulin heavy chain. (B) MEFs were untreated or treated with TNF (10 ng/ml) for 30 min. Cells were lysed in RIPA buffer and immunoprecipitated with anti-RNF11 or isotype control immunoglobulin (control Ig) and immunoblotted with anti-TAX1BP1, A20, RIP1 and RNF11. (C) BMDMs or (D) MEFs were untreated or treated with TNF (10 ng/ml) for the indicated times. Lysates were immunoprecipitated with anti-RNF11 or control Ig and immunoblotted with anti-TAX1BP1, A20 and RIP1.

Article Snippet: The following antibodies were used in this study: IκBα (C-21; Santa Cruz Biotechnology), TRAF6 (H-274; Santa Cruz Biotechnology), β-actin (AC-15; Abcam), TAX1BP1 (ab22049; Abcam), JNK (56G8; Cell Signaling), phospho-JNK (9251; Cell Signaling), phospho-IκBα (14D4; Cell Signaling), A20 (clone E5-1619; BD Biosciences Pharmingen), Itch (clone 32; BD Biosciences Pharmingen), RIP1 (clone 38; BD Biosciences Pharmingen), Flag (M2; Sigma), HA (12CA5; Roche), RNF11 (A01; Abnova) and Ubiquitin (SPA-200F; Assay Designs).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing

Journal:

Article Title: The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-κB signalling

doi: 10.1038/emboj.2008.285

Figure Lengend Snippet: RNF11 is essential for the termination of NF-κB signalling. (A) THP-1 cells were transfected on consecutive days with control scrambled or RNF11 siRNAs. At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK and RNF11. (B) THP-1 cells were transfected on consecutive days with control scrambled, RNF11 or A20 siRNAs. At 2 days after the second transfection, cells were treated with LPS (1 μg/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK, A20, RNF11 and β-actin. (C) THP-1 cells were transfected with control scrambled or RNF11 siRNA as described in (A). At 2 days after the second transfection, cells were stimulated with TNF (10 ng/ml) for various times and RNA was subjected to real-time PCR for IκBα and A20 expression. This experiment was repeated twice with similar results. (D) THP-1 cells were transfected with siRNAs as described in (A). Supernatants were subjected to an IL-6 ELISA. Error bars indicate s.e.m. of triplicate samples. Statistical analysis was performed by one-way ANOVA, followed by the Tukey–Kramer test for multiple comparisons. *P<0.01 (compared with control IL-1-treated samples); **P<0.001 (compared with control TNF-treated samples).

Article Snippet: The following antibodies were used in this study: IκBα (C-21; Santa Cruz Biotechnology), TRAF6 (H-274; Santa Cruz Biotechnology), β-actin (AC-15; Abcam), TAX1BP1 (ab22049; Abcam), JNK (56G8; Cell Signaling), phospho-JNK (9251; Cell Signaling), phospho-IκBα (14D4; Cell Signaling), A20 (clone E5-1619; BD Biosciences Pharmingen), Itch (clone 32; BD Biosciences Pharmingen), RIP1 (clone 38; BD Biosciences Pharmingen), Flag (M2; Sigma), HA (12CA5; Roche), RNF11 (A01; Abnova) and Ubiquitin (SPA-200F; Assay Designs).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-κB signalling

doi: 10.1038/emboj.2008.285

Figure Lengend Snippet: RNF11 is essential for A20 to interact with and inactivate RIP1. (A) THP-1 cells were transfected on consecutive days with control scrambled, RNF11 or TAX1BP1 siRNAs. At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for 30 min. IPs were performed with anti-RIP1 or isotype control (cont. Ig) followed by immunoblotting with anti-A20 or RIP1. Lysates were immunoblotted with anti-RNF11, TAX1BP1 and β-actin. (B) THP-1 cells were transfected on consecutive days with control scrambled or RNF11 siRNAs. At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for 30 min where indicated. IPs were performed with anti-Itch or isotype control (cont. Ig) followed by immunoblotting with anti-RIP1 or Itch. Lysates were immunoblotted with anti-RNF11 and β-actin. (C) Itch+/+ and Itch−/− MEFs were treated with TNF (10 ng/ml) for 30 min as indicated. Cells were lysed and immunoprecipitated with anti-RNF11 or isotype control (cont. Ig) followed by immunoblotting with anti-RIP1 or RNF11. (D) Tax1bp1+/− and Tax1bp1−/− MEFs were treated and subjected to co-IPs as in (C). (E) MEFs were transfected with control scrambled or RNF11 siRNAs. The following day, the cells were transfected with 0.25, 0.5 or 1 μg of Flag A20 (wedges in lanes 3–5 and 6–8). After 2 days, the cells were lysed and endogenous TRAF6 was immunoprecipitated with anti-TRAF6 followed by immunoblotting with anti-Ub. Molecular sizes are shown to the left of the Ub blot panel. The expression of ectopic A20 was examined by immunoblotting with anti-Flag. The efficiency of RNF11 knockdown was determined by immunoblotting with anti-RNF11. (F) 293T cells were transfected with control scrambled or RNF11 siRNAs. The following day, the cells were transfected with pRL-tk, κB-TATA-Luc and 0, 0.25, 0.5 or 1 μg of Flag–A20 or Flag–A20 C103A as indicated (wedges). At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for 8 h and lysates were subjected to dual luciferase assays. Error bars indicate s.e.m. of triplicate samples. (G) MEFs were transfected with control scrambled or RNF11 siRNAs. The following day, the cells were transfected with 1 μg of empty vector (lanes 1–4) or Flag A20 (lanes 5–12). At 2 days after the second transfection, cells were treated with CHX for the indicated times. Cells were lysed and the stability of endogenous RIP1 was examined by immunoblotting with anti-RIP1. Lysates were also immunoblotted with anti-Flag, RNF11 and β-actin.

Article Snippet: The following antibodies were used in this study: IκBα (C-21; Santa Cruz Biotechnology), TRAF6 (H-274; Santa Cruz Biotechnology), β-actin (AC-15; Abcam), TAX1BP1 (ab22049; Abcam), JNK (56G8; Cell Signaling), phospho-JNK (9251; Cell Signaling), phospho-IκBα (14D4; Cell Signaling), A20 (clone E5-1619; BD Biosciences Pharmingen), Itch (clone 32; BD Biosciences Pharmingen), RIP1 (clone 38; BD Biosciences Pharmingen), Flag (M2; Sigma), HA (12CA5; Roche), RNF11 (A01; Abnova) and Ubiquitin (SPA-200F; Assay Designs).

Techniques: Transfection, Western Blot, Immunoprecipitation, Expressing, Luciferase, Plasmid Preparation

Journal:

Article Title: The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-κB signalling

doi: 10.1038/emboj.2008.285

Figure Lengend Snippet: RNF11 requires the RING domain and PPXY motif to interact with the A20 ubiquitin-editing complex and to terminate NF-κB signalling. (A) Schematic diagram of RNF11 domain organization and mutants used in this figure. The PPXY motif is located between amino acids 37 and 40. The RING finger domain is located between amino acids 99 and 140. The RNF11 C99A mutant has a non-functional RING domain. The RNF11 Y40A mutant has a disrupted PPXY motif. (B) THP-1 cells were transfected with a control scrambled or a single duplex RNF11 siRNA. The following day, the cells were transfected with siRNA-resistant forms of Flag–RNF11 (WTR), Flag–RNF11 C99A (C99AR) and Flag–RNF11 Y40A (Y40AR). After two more days, cells were treated with TNF (10 ng/ml) for the indicated times and lysates were immunoblotted with anti-pIκBα, IκBα, pJNK, JNK, Flag, RNF11 and β-actin. (C) THP-1 cells were transfected with siRNA and siRNA-resistant forms of RNF11 as described in (B). At 2 days after DNA transfections, cells were treated with TNF for 30 min where indicated. Lysates were subjected to immunoprecipitations with anti-RNF11 followed by immunoblotting with A20, TAX1BP1 and RIP1. The lysates were also used for immunoblotting using anti-IκBα, Flag, RNF11 and β-actin.

Article Snippet: The following antibodies were used in this study: IκBα (C-21; Santa Cruz Biotechnology), TRAF6 (H-274; Santa Cruz Biotechnology), β-actin (AC-15; Abcam), TAX1BP1 (ab22049; Abcam), JNK (56G8; Cell Signaling), phospho-JNK (9251; Cell Signaling), phospho-IκBα (14D4; Cell Signaling), A20 (clone E5-1619; BD Biosciences Pharmingen), Itch (clone 32; BD Biosciences Pharmingen), RIP1 (clone 38; BD Biosciences Pharmingen), Flag (M2; Sigma), HA (12CA5; Roche), RNF11 (A01; Abnova) and Ubiquitin (SPA-200F; Assay Designs).

Techniques: Mutagenesis, Functional Assay, Transfection, Western Blot

Journal: Cell Death and Differentiation

Article Title: A novel role for the apoptosis inhibitor ARC in suppressing TNF α -induced regulated necrosis

doi: 10.1038/cdd.2013.195

Figure Lengend Snippet: ARC inhibits TNFα-induced NF-κB activation in a CARD-dependent manner. (a) The abundance of p65 in nuclear extracts was quantified using an ELISA that detects p65 binding to its DNA consensus sequence. Nuclear fractions were obtained from TNFα-treated L929 cells stably transduced with empty vector (Φ), ARC-HA, or ARC CARD mutant-HA (DM-HA). Data shown as mean±S.E. from n=3. ****P-value<0.0001 versus empty vector. (b–f) Levels of NF-κB target gene transcripts normalized to those of 18S in the same cell lines as in panel a as determined by quantitative real-time PCR using transcript-specific primers. Data shown as mean±S.E. N=3 in panels b, e and f; n=7 in panels c and d. *P-value<0.05 compared with empty vector; **P-values<0.01 versus empty vector. FLIP, FLICE-like inhibitory protein; IL, interleukin; iNOS, inducible nitric oxide synthase; RLU, relative luminescence unit

Article Snippet: Primary antibodies include: ARC (Cayman Chemical, Ann Arbor, MI, USA); β -actin (Sigma-Aldrich, St. Louis, MO, USA); HA (Roche Applied Science, Indianapolis, IN, USA); BSA, HMGB1, His (Abcam, Cambridge, MA, USA); histone H3, PARP, p65 (Cell Signaling Technology, Danvers, MA, USA); RhoGDI α (A-20) (Santa Cruz Biotechnology, Dallas, TX, USA); mouse RIP3 (Prosci, Poway, CA, USA); human RIP3 (Thermo Scientific); RIP1 (BD Biosciences, San Jose, CA, USA); and FADD (Enzo Life Sciences, Farmingdale, NY, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Sequencing, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis, Real-time Polymerase Chain Reaction

Journal: Cell Death and Differentiation

Article Title: A novel role for the apoptosis inhibitor ARC in suppressing TNF α -induced regulated necrosis

doi: 10.1038/cdd.2013.195

Figure Lengend Snippet: The knockdown of ARC increased TNFα-induced NF-κB activation. (a) L929 cells stably transduced with scrambled shRNA control (Scr) or ARC shRNA (ARC KD) were subjected to TNFα treatments, and p65 abundance in nuclear extracts was quantified using ELISA. Mean±S.E. from n=3 are shown. *P-value<0.05 versus scrambled control. (b–f) Levels of NF-κB target gene transcripts normalized to those of 18S in the same cell lines as in panel a as determined by quantitative real-time PCR using transcript-specific primers. Data shown as mean±S.E. from n=3. *P-value<0.05; ***P-values<0.001, compared with scrambled control. FLIP, FLICE-like inhibitory protein; IL, interleukin; iNOS, inducible nitric oxide synthase; RLU, relative luminescence unit

Article Snippet: Primary antibodies include: ARC (Cayman Chemical, Ann Arbor, MI, USA); β -actin (Sigma-Aldrich, St. Louis, MO, USA); HA (Roche Applied Science, Indianapolis, IN, USA); BSA, HMGB1, His (Abcam, Cambridge, MA, USA); histone H3, PARP, p65 (Cell Signaling Technology, Danvers, MA, USA); RhoGDI α (A-20) (Santa Cruz Biotechnology, Dallas, TX, USA); mouse RIP3 (Prosci, Poway, CA, USA); human RIP3 (Thermo Scientific); RIP1 (BD Biosciences, San Jose, CA, USA); and FADD (Enzo Life Sciences, Farmingdale, NY, USA).

Techniques: Knockdown, Activation Assay, Stable Transfection, Transduction, shRNA, Control, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Cell Death and Differentiation

Article Title: A novel role for the apoptosis inhibitor ARC in suppressing TNF α -induced regulated necrosis

doi: 10.1038/cdd.2013.195

Figure Lengend Snippet: ARC inhibits TNFα-induced NF-κB activation in a CARD-dependent manner. (a) The abundance of p65 in nuclear extracts was quantified using an ELISA that detects p65 binding to its DNA consensus sequence. Nuclear fractions were obtained from TNFα-treated L929 cells stably transduced with empty vector (Φ), ARC-HA, or ARC CARD mutant-HA (DM-HA). Data shown as mean±S.E. from n=3. ****P-value<0.0001 versus empty vector. (b–f) Levels of NF-κB target gene transcripts normalized to those of 18S in the same cell lines as in panel a as determined by quantitative real-time PCR using transcript-specific primers. Data shown as mean±S.E. N=3 in panels b, e and f; n=7 in panels c and d. *P-value<0.05 compared with empty vector; **P-values<0.01 versus empty vector. FLIP, FLICE-like inhibitory protein; IL, interleukin; iNOS, inducible nitric oxide synthase; RLU, relative luminescence unit

Article Snippet: Antibodies and reagents Primary antibodies include: ARC (Cayman Chemical, Ann Arbor, MI, USA); β -actin (Sigma-Aldrich, St. Louis, MO, USA); HA (Roche Applied Science, Indianapolis, IN, USA); BSA, HMGB1, His (Abcam, Cambridge, MA, USA); histone H3, PARP, p65 (Cell Signaling Technology, Danvers, MA, USA); RhoGDI α (A-20) (Santa Cruz Biotechnology, Dallas, TX, USA); mouse RIP3 (Prosci, Poway, CA, USA); human RIP3 (Thermo Scientific); RIP1 (BD Biosciences, San Jose, CA, USA); and FADD (Enzo Life Sciences, Farmingdale, NY, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Sequencing, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis, Real-time Polymerase Chain Reaction

Journal: Cell Death and Differentiation

Article Title: A novel role for the apoptosis inhibitor ARC in suppressing TNF α -induced regulated necrosis

doi: 10.1038/cdd.2013.195

Figure Lengend Snippet: The knockdown of ARC increased TNFα-induced NF-κB activation. (a) L929 cells stably transduced with scrambled shRNA control (Scr) or ARC shRNA (ARC KD) were subjected to TNFα treatments, and p65 abundance in nuclear extracts was quantified using ELISA. Mean±S.E. from n=3 are shown. *P-value<0.05 versus scrambled control. (b–f) Levels of NF-κB target gene transcripts normalized to those of 18S in the same cell lines as in panel a as determined by quantitative real-time PCR using transcript-specific primers. Data shown as mean±S.E. from n=3. *P-value<0.05; ***P-values<0.001, compared with scrambled control. FLIP, FLICE-like inhibitory protein; IL, interleukin; iNOS, inducible nitric oxide synthase; RLU, relative luminescence unit

Article Snippet: Antibodies and reagents Primary antibodies include: ARC (Cayman Chemical, Ann Arbor, MI, USA); β -actin (Sigma-Aldrich, St. Louis, MO, USA); HA (Roche Applied Science, Indianapolis, IN, USA); BSA, HMGB1, His (Abcam, Cambridge, MA, USA); histone H3, PARP, p65 (Cell Signaling Technology, Danvers, MA, USA); RhoGDI α (A-20) (Santa Cruz Biotechnology, Dallas, TX, USA); mouse RIP3 (Prosci, Poway, CA, USA); human RIP3 (Thermo Scientific); RIP1 (BD Biosciences, San Jose, CA, USA); and FADD (Enzo Life Sciences, Farmingdale, NY, USA).

Techniques: Activation Assay, Stable Transfection, Transduction, shRNA, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction