Journal: Nature

Article Title: DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome

doi: 10.1038/s41586-019-1551-2

Figure Lengend Snippet: a, Co-immunoprecipitation (IP) of NLRP3 and DDX3X in BMDMs treated with LPS with or without nigericin. Representative blots (n= 3). b, Immunoblot analysis of CASP1 cleavage in wild-type (WT), Ddx3xfl/fl, Ddx3xfl/flLysMcre and Nlrp3−/− BMDMs treated with LPS and nigericin. Representative blots (n > 3). c, Immunoblots of DDX3X expression and CASP1 cleavage after siRNA-mediated knockdown of Ddx3x in BMDMs treated with LPS with or without nigericin. Representative blots (n = 2). d, Confocal microscopy imaging of ASC specks in BMDMs treated with LPS with or without nigericin, to visualize the subcellular localization of DDX3X, NLRP3 and ASC. Scale bars, 10 μm. Representative images (n = 3). e, Schematic of N-terminal Flag-tagged NLRP3 expression constructs: full-length NLRP3 (Flag-NLRP3-FL), the pyrin domain of NLRP3 (Flag-PYD; amino acids 1–90 of NLRP3), the NACHT domain of NLRP3 (Flag-NACHT; amino acids 91–710 of NLRP3) and the LRR domain of NLRP3 (Flag-LRR; amino acids 711–1034 of NLRP3). f, Immunoblot (IB) analysis of input lysates used for co-immunoprecipitation of Flag-tagged NLRP3 constructs and DDX3X-mCherry. Representative blots (n > 3). g, Immunoblot analysis of immunoprecipitation of DDX3X-mCherry and the indicated NLRP3 constructs. Red asterisk indicates the antibody light chain. Representative blots (n > 3).

Article Snippet: NLRP3 expression constructs pCDNA3-N-Flag-NLRP3, pCDNA3-N-Flag-NLRP3 1–90, pCDNA3-N-Flag-NLRP3 91–710 and pCDNA3-N-Flag-NLRP3 711–1034 were purchased from AddGene.

Techniques: Immunoprecipitation, Western Blot, Expressing, Confocal Microscopy, Imaging, Construct

Journal: Nature

Article Title: DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome

doi: 10.1038/s41586-019-1551-2

Figure Lengend Snippet: a, Immunoblot analysis of lysates fractionated by analytical ultra-centrifugation to test the oligomerization of NLRP3, DDX3X and ASC in BMDMs stimulated with LPS and treated either with nigericin or with arsenite and nigericin. Representative blots (n = 2). MW, molecular weight. b, Immunoblot analysis of ASC from the insoluble fraction of samples from a under non-reducing (without β-mercaptoethanol; – BME) and reducing (+BME) conditions. Representative blots (n = 2). c, STORM imaging of ASC speck assembly and its spatial organization with DDX3X over time. Scale bars, 5 μm (whole-cell images); 1 μm (magnified images). Representative images (n = 2). d, Plot of the r-index to show the extent of colocalization of ASC and DDX3X with the duration of nigericin treatment (n = 2). R2 was calculated using linear regression analysis; P = 0.0125 (for the significance of the slope being non-zero). Data are mean ± s.e.m. e, f, Immunoblot analysis of CASP1 cleavage in LPS-primed BMDMs to which arsenite was added at various time points (30 and 15 min before adding nigericin, simultaneously with nigericin and 15 and 30 min after adding nigericin), without (e) or with (f) pre-treatment with anisomycin. Representative blots (n = 3). g, h, Structured illumination microscopy of BMDMs to visualize the subcellular localization of DDX3X, G3BP1 and ASC in LPS-primed BMDMs treated with nigericin alone (g) or with arsenite and nigericin (h). Scale bars, 3 μm. Representative images (n = 3).

Article Snippet: NLRP3 expression constructs pCDNA3-N-Flag-NLRP3, pCDNA3-N-Flag-NLRP3 1–90, pCDNA3-N-Flag-NLRP3 91–710 and pCDNA3-N-Flag-NLRP3 711–1034 were purchased from AddGene.

Techniques: Western Blot, Centrifugation, Molecular Weight, Imaging, Microscopy

Journal: Nature

Article Title: DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome

doi: 10.1038/s41586-019-1551-2

Figure Lengend Snippet: a, Confocal microscopy imaging of peritoneal CD45+ cells to visualize in vivo stress granules in Ddx3xfl/fl and Ddx3xfl/flLysMcre mice that were treated with PBS or arsenite. Scale bars, 10 μm. Representative images (n = 2). b, Quantification of CD45+ cells that contain stress granules, from the peritoneal cavity of Ddx3xfl/fl and Ddx3xfl/flLysMcre mice that were injected with arsenite or PBS. ****P < 0.0001 (unpaired two-sided t-test). Data represent two biologically independent experiments (n = 10 frames). c, Quantification of the numbers and percentage of CD11b+ myeloid cells in the peritoneal cavity. P values (from left to right): ***P = 0.0001, ***P = 0.0005 (unpaired two-sided t-test; n = 7). d, e, Levels of IL-1β in the serum (d) and peritoneal fluid (PF) (e) of mice that were injected with LPS with or without prior arsenite injection in the peritoneum. P values: **P = 0.0026 (d), P = 0.09 (e) (unpaired two-sided t-test; n = 7). f, Levels of IL-1β in the serum and peritoneal fluid of Ddx3xfl/fl and Ddx3xfl/flLysMcre mice that were injected with LPS in the peritoneum. P values (from top to bottom): **P = 0.0073, *P = 0.0123 (unpaired two-sided t-test; n ≥ 6). Data are mean ± s.e.m. (b-f). g, Schematic of the interplay between the inflammasome and stress granules that is involved in cell-fate decisions. DDX3X promotes NLRP3 inflammasome activation and the pro-death cell-fate decision probably by interacting with the NLRP3 NACHT domain through its helicase (Heli) domain. Induction of stress granules causes the sequestration of DDX3X (along with 40S ribosomal subunits and translation initiation factors (eIFs)), thus making it unavailable for NLRP3 inflammasome activation and thereby allowing the cells to make a pro-survival cell-fate choice.

Article Snippet: NLRP3 expression constructs pCDNA3-N-Flag-NLRP3, pCDNA3-N-Flag-NLRP3 1–90, pCDNA3-N-Flag-NLRP3 91–710 and pCDNA3-N-Flag-NLRP3 711–1034 were purchased from AddGene.

Techniques: Confocal Microscopy, Imaging, In Vivo, Injection, Activation Assay

Journal: OncoTargets and therapy

Article Title: A negative regulation loop of long noncoding RNA HOTAIR and p53 in non-small-cell lung cancer

doi: 10.2147/OTT.S110219

Figure Lengend Snippet: The negative correlation between lncRNA HOTAIR and p53 expressions in NSCLC. Notes: ( A ) Western blot assay showed the p53 protein level in 12 NSCLC patients’ samples, and β-actin was used as an endogenous control. The relative expressions of p53 in different samples were standardized to the No 1 by gray scale scanning, and the number below each band showed its relative p53 expression. ( B ) The relative expressions of lncRNA HOTAIR in the same patients’ samples with ( A ) detected by real-time PCR assay. ( C ) Real-time PCR assay showed the expression of the lncRNA HOTAIR in the cancer adjacent NT or CT in all 48 patients. ( D ) The correlation between the expressions of HOTAIR and p53 was analyzed with regression analysis. ** P <0.01. Abbreviations: CT, cancer tissues; HOTAIR, HOX antisense intergenic RNA; lncRNA, long noncoding RNA; NSCLC, non-small-cell lung cancer; NT, normal tissues; PCR, polymerase chain reaction.

Article Snippet: The pcDNA3-flag-p53 (#10838) plasmid and the lncRNA HOTAIR expressing plasmid LZRS-HOTAIR (#26110) were purchased from Addgene (Cambridge, MA, USA); the pcDNA3-flag vector control and pGL3-basic vector were gifts from Dr Zhiqiang Liu, Department of Pathophysiology, Tianjin Medical University.

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

Journal: OncoTargets and therapy

Article Title: A negative regulation loop of long noncoding RNA HOTAIR and p53 in non-small-cell lung cancer

doi: 10.2147/OTT.S110219

Figure Lengend Snippet: lncRNA HOTAIR negatively correlates with p53 functionality. Notes: ( A ) Western blot showed the p53 level in the A549 (p53 wild-type), NCI-H157 (p53 mutant), and H1299 (p53 deletion) cell lines, and the lncRNA HOTAIR level in these three cell lines were detected using real-time PCR ( B ). For all real-time PCR results, the formula 2 −ΔΔ Ct was used, and data here were represented as mean ± SD of three independent experiments. ** P <0.01. Abbreviations: HOTAIR, HOX antisense intergenic RNA; lncRNA, long noncoding RNA; PCR, polymerase chain reaction; SD, standard deviation.

Article Snippet: The pcDNA3-flag-p53 (#10838) plasmid and the lncRNA HOTAIR expressing plasmid LZRS-HOTAIR (#26110) were purchased from Addgene (Cambridge, MA, USA); the pcDNA3-flag vector control and pGL3-basic vector were gifts from Dr Zhiqiang Liu, Department of Pathophysiology, Tianjin Medical University.

Techniques: Western Blot, Mutagenesis, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation

Journal: OncoTargets and therapy

Article Title: A negative regulation loop of long noncoding RNA HOTAIR and p53 in non-small-cell lung cancer

doi: 10.2147/OTT.S110219

Figure Lengend Snippet: Modulation of p53 expression alternated the lncRNA HOTAIR expression and cell invasion in NSCLC cells. Notes: ( A ) pcDNA-flag-P53 expressing plasmid and the pcDNA3-flag vector control plasmids were transfected into A549 cells for 48 hours, and the expression of flag-tagged p53 was measured by Western blot assay. ( B ) The relative expression of lncRNA HOTAIR in A549 cells transfected with vector control or flag-p53 expressing plasmid was detected using real-time PCR 48 hours after transfection. ( C ) Cell proliferation of A549 cells transfected with vector control or flag-p53 expressing plasmid was measured by an MTT assay at days 0, 1, 3, 5, and 7 after transfection. ( D ) The cell invasion capability was measured by a transwell assay at 48 hours after p53 overexpression in A549 cells. Representative images were shown from five independent experiments. Data represent mean ± SD of three independent experiments. Magnification, 200×. * P <0.05; ** P <0.01. Abbreviations: HOTAIR, HOX antisense intergenic RNA; lncRNA, long noncoding RNA; NSCLC, non-small-cell lung cancer; PCR, polymerase chain reaction; SD, standard deviation.

Article Snippet: The pcDNA3-flag-p53 (#10838) plasmid and the lncRNA HOTAIR expressing plasmid LZRS-HOTAIR (#26110) were purchased from Addgene (Cambridge, MA, USA); the pcDNA3-flag vector control and pGL3-basic vector were gifts from Dr Zhiqiang Liu, Department of Pathophysiology, Tianjin Medical University.

Techniques: Expressing, Plasmid Preparation, Control, Transfection, Western Blot, Real-time Polymerase Chain Reaction, MTT Assay, Transwell Assay, Over Expression, Polymerase Chain Reaction, Standard Deviation

Journal: OncoTargets and therapy

Article Title: A negative regulation loop of long noncoding RNA HOTAIR and p53 in non-small-cell lung cancer

doi: 10.2147/OTT.S110219

Figure Lengend Snippet: The regulation of p53 on the HOTAIR promoter. Notes: ( A ) Two sequences shown at the top were two binding sites of p53 at the promoter of HOTAIR gene by bioinformatics prediction. Luciferase assay was carried out in A549 cells transfected with pGL3-basic or pGL-HOTAIR combined with pcDNA3-flag or flag-p53, respectively, for 48 hours. ( B ) The immunoprecipitation assay was performed to detect the activities of P53 binding to the promoter of lncRNA HOTAIR. The A549 cells were transfected with pcDNA-flag vector or flag-p53 expressing plasmids for 48 hours, and cell lysate was pulled down using IgG control or anti-p53 monoclonal antibody. Data represent mean ± SD of three independent experiments. ** P <0.001. Abbreviations: HOTAIR, HOX antisense intergenic RNA; IgG, immunoglobulin G; lncRNA, long noncoding RNA; SD, standard deviation; BS, binding sites.

Article Snippet: The pcDNA3-flag-p53 (#10838) plasmid and the lncRNA HOTAIR expressing plasmid LZRS-HOTAIR (#26110) were purchased from Addgene (Cambridge, MA, USA); the pcDNA3-flag vector control and pGL3-basic vector were gifts from Dr Zhiqiang Liu, Department of Pathophysiology, Tianjin Medical University.

Techniques: Binding Assay, Luciferase, Transfection, Immunoprecipitation, Plasmid Preparation, Expressing, Control, Standard Deviation

Journal: OncoTargets and therapy

Article Title: A negative regulation loop of long noncoding RNA HOTAIR and p53 in non-small-cell lung cancer

doi: 10.2147/OTT.S110219

Figure Lengend Snippet: The epigenetic regulation of HOTAIR on the promoter of p53 gene. Notes: ( A ) Western blot showed the p53 protein level in A549 cells transfected with vector control or LZRS-HOTAIR plasmid for 48 hours. β -Actin was used as the endogenous control. ( B ) p53 mRNA level in A549 cells transfected with vector or LZRS-HOTAIR plasmid for 48 hours. ( C ) The activities of H3K27me3 level were determined with ChIP assay in A549 cells transfected with LZRS-HOTAIR plasmid or vector control for 48 hours. Data represent mean ± SD of three independent experiments. ** P <0.001. Abbreviations: ChIP, chromatin immunoprecipitation; HOTAIR, HOX antisense intergenic RNA; IgG, immunoglobulin G; SD, standard deviation.

Article Snippet: The pcDNA3-flag-p53 (#10838) plasmid and the lncRNA HOTAIR expressing plasmid LZRS-HOTAIR (#26110) were purchased from Addgene (Cambridge, MA, USA); the pcDNA3-flag vector control and pGL3-basic vector were gifts from Dr Zhiqiang Liu, Department of Pathophysiology, Tianjin Medical University.

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Chromatin Immunoprecipitation, Standard Deviation

Journal: Oncotarget

Article Title: AKT regulates NPM dependent ARF localization and p53mut stability in tumors.

doi: 10.18632/oncotarget.2178

Figure Lengend Snippet: Fig.4: Inhibition of AKT promotes enhanced MDM2 activity via the increased association between NPM and p14ARF. (A) Npm-/-, p53-/-double null MEF were infected with pBABE retrovirus empty vector and pBABE expressing FLAG-tagged-NPM-WT, NPM-S48A or S48E as indicated. Immunopurification of NPM was done by pulling down with the Flag tag (middle panel) followed by elution of complexes by the Flag peptide and subsequent immunopurification of endogenous MDM2 (lower panel). (B) Nuclear immunoprecipitates of MDM2 from T24 cells treated with MK-2206 (5 μM, 24 hrs). Immunoprecipitates and lysates were blotted with the indicated antibodies. (C) T24 cells were treated with MK-2206 (5 μM) as indicated. p14ARF was immunoprecipitated from whole cell lysates and nuclear extracts and the association with NPM and MDM2 determined by western blot. Immunoprecipitates and lysates were blotted with the indicated antibodies. (D) MDM2 and (E) p53 ubiquinitation assay in H1299 cells transfected with wild type p53, HA-tagged ubiquitin and treated for 16 hrs with DMSO, MK-2206 (5 μM) or Nutlin3A (5 μM) as indicated. Immunoprecipitates and whole cell lysates were probed with the indicated antibodies.

Article Snippet: The following plasmids were purchased from Addgene; pBABE puro-myr-FLAGAKT1 (Addgene plasmid 15294), [104], pBABE PuroL myr-HAAKT2 (Addgene plasmid 9018), pBABE-puro-KRas V12 (Addgene plasmid 9052) and pcDNA3 MDM2 S166D S186D (Addgene plasmid 16236).

Techniques: Inhibition, Activity Assay, Infection, Plasmid Preparation, Expressing, Immu-Puri, FLAG-tag, Immunoprecipitation, Western Blot, Transfection, Ubiquitin Proteomics