human mdmx  (Addgene inc)

Journal: The Journal of experimental medicine

Article Title: Targeting mechanosensitive MDM4 promotes lung fibrosis resolution in aged mice.

doi: 10.1084/jem.20202033

Figure Lengend Snippet: Figure 1. MDM4 expression is increased in human IPF and bleomycin-induced lung fibrosis in aged mice. (A and B) IHC analysis of MDM4 expression in formalin-fixed, paraffin-embedded lung tissue sections derived from patients with IPF and normal (Norm) hu- man donors (A; n = 3 per group) and from aged mice treated with bleomycin (Bleo) or saline (Sal; B; n = 3 per group). Representative human IHC images were derived from a 63-yr-old white male (IPF) and a 52-yr-old white male (Norm). Scale bar = 50 µm (bottom two images) or 200 µm (top four images). (C) Immunoblot analysis of MDM4 expression in primary lung (myo)fibroblasts iso- lated from patients with IPF or normal control (Ctrl) subjects (n = 5 per group). (D) Immunoblot analysis of Mdm4 expression in primary Pdgfrα+ lung fibroblasts isolated from aged (n = 6) and young mice (n = 7) by flow cytometry. Relative levels of MDM4 protein were de- termined by scanning densitometry of the blots and normalized to GAPDH expression. Results are the means ± SD of three separate experiments. *, P < 0.05; **, P < 0.01 (ANOVA). FB, fibroblasts; MFB, myofibroblasts; k, kilodalton.

Article Snippet: Validated MDM4 siRNAs were from OriGene.

Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Derivative Assay, Saline, Western Blot, Control, Isolation, Flow Cytometry

Journal: The Journal of experimental medicine

Article Title: Targeting mechanosensitive MDM4 promotes lung fibrosis resolution in aged mice.

doi: 10.1084/jem.20202033

Figure Lengend Snippet: Figure 2. MDM4 is a matrix stiffness–regulated me- chanosensitive inhibitor of p53. Primary human lung fi- broblasts were cultured on 1–20-kPa PA gels. (A) mRNA levels of MDM4 and MDM2 were determined by qPCR. (B) Protein levels of MDM4 and MDM2 were determined by immunoblot. (C) Levels of acetylated p53 (Ac-p53) and total p53 were determined by immunoblot. (D) Human lung fi- broblasts were cultured on 1- or 20-kPa PA gels in the presence of 50 nM MDM4 (M4) siRNAs (siR) or control siRNAs. Levels of MDM4, Ac-p53, and total p53 were de- termined by immunoblot. (E) Human lung fibroblasts in- fected with MDM4-expressing lentivirus (Lenti-M4) or the control lentivirus (Lenti-Ctrl) were cultured on 1- or 20-kPa PA gels. Levels of MDM4, Ac-p53, and total p53 were de- termined by immunoblot. GAPDH was used as internal reference control in qPCR analysis and as loading control in immunoblot analysis. Results are the means ± SD of three or four separate experiments. **, P < 0.01 (ANOVA). k, kilodalton.

Article Snippet: Validated MDM4 siRNAs were from OriGene.

Techniques: Cell Culture, Western Blot, Control, Expressing

Journal: The Journal of experimental medicine

Article Title: Targeting mechanosensitive MDM4 promotes lung fibrosis resolution in aged mice.

doi: 10.1084/jem.20202033

Figure Lengend Snippet: Figure 3. ELK1 mediates matrix stiffness–regulated MDM4 expression. (A) The 1-kb proximal regulatory region of MDM4 was cloned into a promoter reporter (PGL3). PGL3-M4 and the empty vector were transfected into human lung fibroblasts by electroporation. Cells were then cultured on 1–20-kPa PA gels for 48 h. Promoter activity was determined by luciferase-based assay. (B) Schematic depiction of two EBMs in the proximal promoter of human MDM4 and mouse Mdm4. (C) Human lung fibroblasts were cultured on 1–20-kPa PA gels. Levels of phospho-ELK1 (pELK1) and total ELK1 were determined by immunoblot. (D) Human lung fibroblasts were cultured on 1- or 20-kPa PA gels in the presence of 50 nM ELK1 siRNAs (siR) or the control siRNAs. Levels of ELK1 and MDM4 were determined by immunoblot. GAPDH was used for loading control. (E) Nuclear extracts derived from human lung fibroblasts cultured on 1–20-kPa PA gels were incubated with immobilized oligonucleotides containing EBMs. ELK1-specific antibodies were added to the reactions followed by incubation with a secondary HRP-conjugated antibody. EBM binding activity was quantified by colorimetric ELISA. (F) IF analysis of ELK1 subcellular localization in human lung fibroblasts cultured on 1- or 20-kPa PA gels. Nuclei were stained by DAPI. Scale bars = 50 µm. (G) Subcellular fractionation and immunoblot analysis of ELK1 localization in human lung fibroblasts cultured on 1- or 20-kPa PA gels. GAPDH was used as loading control for cytoplasmic protein and lamin B as loading control for nuclear protein. Results are the means ± SD of three or four separate experiments. **, P < 0.01 (ANOVA). k, kilodalton.

Article Snippet: Validated MDM4 siRNAs were from OriGene.

Techniques: Expressing, Clone Assay, Plasmid Preparation, Transfection, Electroporation, Cell Culture, Activity Assay, Luciferase, Western Blot, Control, Derivative Assay, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, Fractionation

Journal: The Journal of experimental medicine

Article Title: Targeting mechanosensitive MDM4 promotes lung fibrosis resolution in aged mice.

doi: 10.1084/jem.20202033

Figure Lengend Snippet: Figure 4. Reducing matrix stiffness sensitizes lung myofibroblasts to apoptosis by promoting MDM4–p53-dependent Fas expression. (A) Human IPF lung myofibroblasts were cultured on 1–20-kPa PA gels. Levels of Fas mRNA were determined by qPCR. (B) Levels of Fas protein were determined by im- munoblot. (C–E) IPF lung myofibroblasts were cultured on 1- or 20-kPa PA gels in the presence of 50 nM MDM4 siRNA or the control siRNA (C), MDM4- expressing lentiviral vector or the control lentiviral vector (D), and 25 µM C646 or the vehicle (Vehi) control (E). Levels of Fas protein were determined by immunoblot. (F) IPF lung myofibroblasts were cultured on 1–20-kPa PA gels. Cell apoptosis was evaluated by TUNEL staining and IF/differential interference contrast (DIC) microscopy. Nuclei were stained by DAPI. A representative image shows cell apoptosis (arrowheads) under 20-kPa gel conditions. (G) IPF lung myofibroblasts were cultured on 1–20-kPa PA gels in the presence or absence of CH11 (100–500 ng/ml), MDM4-expressing lentiviruses or the control len- tiviruses, C646, and MDM4 siRNA (siR) or the control siRNA. Cell apoptosis was evaluated by TUNEL staining and confocal IF microscopy. Nuclei were stained by DAPI. Representative images showed cell apoptosis (arrowheads) under 1-kPa gel conditions in the presence of 250 ng/ml CH11. (H) qPCR analysis for transcription of BAX, BID, NOXA, and PUMA in IPF lung myofibroblasts cultured on 1- and 20-kPa PA gels. GAPDH was used as internal reference control in qPCR analysis and as loading control in immunoblot analysis. Results are the means ± SD of three to five separate experiments. Apoptosis was evaluated in at least 200 lung myofibroblasts per experiment. Scale bar = 250 µm (F) and 50 µm (G). *, P < 0.05; **, P < 0.01 (ANOVA). k, kilodalton.

Article Snippet: Validated MDM4 siRNAs were from OriGene.

Techniques: Expressing, Cell Culture, Control, Plasmid Preparation, Western Blot, TUNEL Assay, Staining, Microscopy

Journal: The Journal of experimental medicine

Article Title: Targeting mechanosensitive MDM4 promotes lung fibrosis resolution in aged mice.

doi: 10.1084/jem.20202033

Figure Lengend Snippet: Figure 5. Reducing matrix stiffness pro- motes MDM4–p53-dependent DD1α expres- sion by lung myofibroblasts, facilitating macrophage phagocytosis of apoptotic lung myofibroblasts. (A) Human IPF lung myofibro- blasts were cultured on 1–20-kPa PA gels. Levels of DD1α mRNA were determined by qPCR. (B) Levels of DD1α protein were determined by immunoblot. (C) Levels of DD1α expression on the cell surface were evaluated by flow cytom- etry. Cells incubated with nonimmune control IgG and cells alone were included as negative controls. (D–F) IPF lung myofibroblasts were cultured on 1- or 20-kPa PA gels in the presence of 50 nM MDM4 siRNA (siR) or the control siRNA (D), MDM4-expressing lentiviral vector or the control lentiviral vector (E), and 25 µM C646 or the vehicle (Vehi) control (F). Levels of DD1α protein were determined by immunoblot. (G) IPF lung myofibroblasts were cultured on 1- or 20- kPa PA gels in the presence or absence of 10 µg/ ml DD1a neutralizing antibody (DDab), MDM4- expressing lentiviral vector, or 25 µM C646. Af- ter 24 h, cells were treated with 500 ng/ml CH11 to induce apoptosis. Apoptotic cells were collected by flow cytometry cell sorting, labeled by pHrodo Red, and incubated with THP1 at 1:10 to 1:15 ratio (THP1/myofibroblasts). Phagocytosis was ob- served were under a fluorescence microscope using bright field or Texas Red filter set. The phagocytic index was calculated as described in Materials and methods. A representative image shows macrophage phagocytosis (arrowheads) of pHrodo Red–labeled apoptotic IPF lung myofi- broblasts derived from 1-kPa gel conditions. GAPDH was used as internal reference control in qPCR analysis and as loading control in immuno- blot analysis. Results are the means ± SD of three to five separate experiments. Phagocytosis was evaluated in at least 500 macrophages per ex- periment. Scale bar = 20 µm. *, P < 0.05; **, P < 0.01 (ANOVA). k, kilodalton.

Article Snippet: Validated MDM4 siRNAs were from OriGene.

Techniques: Cell Culture, Western Blot, Expressing, Incubation, Control, Plasmid Preparation, Flow Cytometry, FACS, Labeling, Fluorescence, Microscopy, Derivative Assay

Journal: The Journal of experimental medicine

Article Title: Targeting mechanosensitive MDM4 promotes lung fibrosis resolution in aged mice.

doi: 10.1084/jem.20202033

Figure Lengend Snippet: Figure 6. Reducing matrix stiffness promotes macrophage chemotaxis by a MDM4–p53-dependent CX3CL1 paracrine signal derived from lung myofibroblasts. (A) An equal number of IPF lung myofibroblasts were cultured on 0.5–20-kPa PA gels or plastic surface (P). CM was collected and incubated with 2 × 105 THP1 or mBMDMs in 96-Transwell plates. Migrating macrophages were stained by calcein-AM. Chemotaxis index was calculated as a ratio of quantitative fluorescence of cells incubated with the CM to cells incubated with plain DMEM. Mϕ, macrophage. (B) Relative levels of CX3CL1 and CXCL10 in the CM collected from IPF lung myofibroblasts cultured on 1–20-kPa PA gels were determined by ELISA. (C) IPF lung myofibroblasts were cultured on 1-kPa PA gels. CM were collected and incubated with THP1 or mBMDMs in the presence of increasing concentrations (0, 0.1, 1, and 10 µg/ml) of CX3CL1 neutralizing antibody (3CL Ab), nonimmune goat IgG (gIgG), CXCL10 neutralizing Ab (CL10 Ab), or mouse IgG (mIgG). Macrophage chemotaxis index was determined as described above. (D) IPF lung myofibroblasts were cultured on 1- or 20-kPa PA gels in the presence or absence of MDM4-expressing lentiviral vector or 25 µM C646. Levels of CX3CL1 in the CM were determined by ELISA. (E and F) CM collected from cells treated in D were incubated with THP1 or mBMDMs. Effects of MDM4 overexpression (E) and C646 treatment (F) on macrophage chemotaxis were determined as described above. Results are the means ± SD of three separate experiments. *, P < 0.05; **, P < 0.01 (ANOVA).

Article Snippet: Validated MDM4 siRNAs were from OriGene.

Techniques: Chemotaxis Assay, Derivative Assay, Cell Culture, Incubation, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Expressing, Plasmid Preparation, Over Expression

Journal: The Journal of experimental medicine

Article Title: Targeting mechanosensitive MDM4 promotes lung fibrosis resolution in aged mice.

doi: 10.1084/jem.20202033

Figure Lengend Snippet: Figure 8. Matrix destiffening activates the MDM4–p53 pathway and promotes the clearance of lung myofibroblasts by macrophages in the fibrotic lungs of aged mice. (A) IHC analysis of Mdm4 expression in aged fibrotic mice treated by CA or vehicle at 4 mo (n = 5 per group). Mdm4-positive signals were quantified by ImageJ. Scale bars = 50 µm. (B) Confocal IF microscopy to evaluate Ac-p53 expression in αSMA-expressing myofibroblasts in the fibrotic lungs of aged mice treated with CA or vehicle. Scale bars = 50 µm. (C) Levels of CX3CL1 in the BAL fluids of aged fibrotic mice treated with CA or vehicle were quantified by ELISA (n = 5 per group). (D) TUNEL staining and confocal IF microscopy to evaluate lung myofibroblast apoptosis in the fibrotic lungs of aged mice treated with CA or vehicle. Scale bars = 50 µm. (E) Confocal IF microscopy to evaluate macrophage (CD68) engulfment of myofibroblasts (αSMA) in the fibrotic lungs of aged mice treated with CA or vehicle. Scale bars = 50 µm. Quantitative IF analysis was performed in 5–10 randomly selected lung areas. Results are the means ± SD from a total of five mice per treatment group, each performed in triplicate. Nuclei were stained with DAPI. **, P < 0.01 (ANOVA).

Article Snippet: Validated MDM4 siRNAs were from OriGene.

Techniques: Expressing, Microscopy, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining

Journal: The Journal of experimental medicine

Article Title: Targeting mechanosensitive MDM4 promotes lung fibrosis resolution in aged mice.

doi: 10.1084/jem.20202033

Figure Lengend Snippet: Figure 10. Genetic deletion of Mdm4 in mesenchymal cells promotes lung fibrosis resolution in aged mice. (A) Schematic depiction of the course of animal experiments. ELI, ELISA; Hyp, Hyp content assay; IB, immunoblot; I.P., intraperitoneal; q.d., quaque die; Tam, tamoxifen; Tch, trichrome staining. (B) Primary lung (myo)fibroblasts were isolated from the fibrotic Col1α2-CreERT2;Mdm4fl/fl mice treated by tamoxifen or corn oil at week 4 after bleomycin administration. Protein levels of Mdm4 (M4), Ac-p53, and total p53 were determined by immunoblot. GAPDH was used as loading control. (C) Levels of CX3CL1 in the BAL fluids of M4+/+ and M4−/−mice were quantified by ELISA (n = 5 per group). (D) Ac-p53 expression in αSMA-expressing myofibroblasts in the fibrotic lungs of M4+/+ and M4−/−mice were evaluated by confocal IF microscopy. Scale bars = 50 µm. (E) Apoptosis of lung myofibroblasts (arrows) were evaluated by TUNEL staining and confocal IF microscopy. Scale bars = 50 µm. (F) Engulfments of myofibroblasts (αSMA+) by macrophage (CD68+) were evaluated by confocal IF microscopy. Scale bars = 50 µm. (G) Representative images show trichrome staining of lung collagen in paraffin-embedded lung sections harvested from saline- (Sal) or bleomycin- (Bleo) treated M4−/−and M4+/+ mice at 3 wk and 4 mo. Scale bars = 150 µm. (H) Protein levels of collagen I (Col I), fibronectin

Article Snippet: Validated MDM4 siRNAs were from OriGene.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Isolation, Control, Expressing, Microscopy, TUNEL Assay, Saline

Journal: Frontiers in Pharmacology

Article Title: Targeting the NFAT1-MDM2-MDMX Network Inhibits the Proliferation and Invasion of Prostate Cancer Cells, Independent of p53 and Androgen

doi: 10.3389/fphar.2017.00917

Figure Lengend Snippet: Inulanolide A (InuA) suppresses prostate cancer cell proliferation, migration and invasion, and inhibits the protein expression of MDM2 and MDMX, regardless of the p53 status. (A) The chemical structure of InuA. (B) IMR90, LNCaP, PC3, and DU145 cells were treated with InuA at the indicated concentrations for 72 h, followed by MTT assays. (C) LNCaP, PC3, and DU145 cells were treated with InuA at the indicated concentrations for 24 h, followed by BrdU cell proliferation assays. (D) LNCaP, PC3, and DU145 cells were treated with InuA at the indicated concentrations for 24 h, followed by 10-day colony formation assays. (E) LNCaP, PC3, and DU145 cells were treated with InuA at the indicated concentrations for 24 h then the cell invasion was determined by transwell invasion assays ( # P < 0.01). (F) LNCaP, PC3, and DU145 cells were treated with InuA at the indicated concentrations and cell migration was evaluated by 48-h wound-healing assays. (G) LNCaP, PC3, and DU145 were treated with InuA at the indicated concentrations for 24 h, and the levels of various proteins were detected using specific antibodies by Western blotting. The data are representative of three or more experiments.

Article Snippet: The siRNAs against human NFAT1, MDM2, and MDMX were purchased from Thermo Fisher Scientific (Rockford, IL, United States).

Techniques: Migration, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Targeting the NFAT1-MDM2-MDMX Network Inhibits the Proliferation and Invasion of Prostate Cancer Cells, Independent of p53 and Androgen

doi: 10.3389/fphar.2017.00917

Figure Lengend Snippet: Inulanolide A directly binds to the RING domains of MDM2 and MDMX. (A,B) Computational modeling of InuA binding to the RING domains of MDM2 (A) and MDMX (B) . InuA was rendered in yellow, with the atoms important for binding highlighted in red. The key residues interacting with InuA were rendered as sticks. The hydrogen bonds were shown by red dotted lines. (C) The chemical structure of biotinylated InuA (Biotin-InuA). (D) Biotin-InuA-conjugated avidin beads were incubated with various concentrations of purified GST-MDM2 RING or GST-MDMX RING in the presence or absence of non-biotinylated InuA. Purified GST was used as a negative control. The bound proteins were detected using anti-GST antibodies. (E) Biotin-InuA-conjugated avidin beads were incubated with LNCaP and PC3 cell lysates in the presence or absence of a 2-, 10-, or 20-fold excess of non-biotinylated InuA. The mixtures were blotted for bound MDM2 and MDMX proteins. (F,G) Plots of the k obs values for the binding of the MDM2 RING domain (F) and the MDMX RING domain (G) as a function of the InuA concentration. Data are representative of three or more experiments.

Article Snippet: The siRNAs against human NFAT1, MDM2, and MDMX were purchased from Thermo Fisher Scientific (Rockford, IL, United States).

Techniques: Binding Assay, Avidin-Biotin Assay, Incubation, Purification, Negative Control, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Targeting the NFAT1-MDM2-MDMX Network Inhibits the Proliferation and Invasion of Prostate Cancer Cells, Independent of p53 and Androgen

doi: 10.3389/fphar.2017.00917

Figure Lengend Snippet: Inulanolide A disrupts the MDM2–MDMX interaction and induces the ubiquitination and degradation of MDM2 and MDMX. (A) LNCaP and PC3 cells were treated with InuA at the indicated concentrations for 2 h, followed by the co-immunoprecipitation of the MDM2-MDMX complex with an MDM2 antibody. The protein levels of MDM2 and MDMX were determined by Western blotting. Band intensity ratios were obtained using the IMAGE J software program, and were normalized to those of vehicle controls, and the binding ratios were also normalized to the intensity of the input. (B) The His-MDM2 and GST-MDMX RING domain proteins were incubated with InuA at the indicated concentrations overnight at 4°C. The complexes were immobilized on glutathione-sepharose beads for 2 h at 4°C and blotted for the binding of His-MDM2 to GST-MDMX RING using anti-His antibody. (C) LNCaP and PC3 cells were treated with InuA (2 μM) for 24 h, followed by exposure to a protein synthesis inhibitor, cycloheximide (CHX, 15 μg/mL). The protein expression levels of MDM2 and MDMX were detected by Western blotting at indicated times after exposure to CHX. Graphs (on the right) show the quantification of the immunoblotting data. (D) LNCaP and PC3 cells were co-transfected with MDM2 and ubiquitin plasmids, followed by treatment with InuA at the indicated concentrations for 24 h. Cell lysates were subjected to immunoprecipitation with an MDM2 antibody. The ubiquitinated MDM2 was detected using an anti-ubiquitin antibody. (E) LNCaP and PC3 cells were co-transfected with MDMX and ubiquitin plasmids, followed by treatment with InuA at the indicated concentrations for 24 h. Cell lysates were subjected to immunoprecipitation with an MDMX antibody. The ubiquitinated MDMX was detected using an anti-ubiquitin antibody. Data are representative of at least three experiments.

Article Snippet: The siRNAs against human NFAT1, MDM2, and MDMX were purchased from Thermo Fisher Scientific (Rockford, IL, United States).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Software, Binding Assay, Incubation, Expressing, Transfection

Journal: Frontiers in Pharmacology

Article Title: Targeting the NFAT1-MDM2-MDMX Network Inhibits the Proliferation and Invasion of Prostate Cancer Cells, Independent of p53 and Androgen

doi: 10.3389/fphar.2017.00917

Figure Lengend Snippet: MDMX is important for the InuA-induced MDM2 degradation. (A) MEF MDMX -/- p53 -/- cells were transfected with a Myc-MDMX plasmid or the empty vector for 24 h, followed by a 24-h exposure to InuA at the indicated concentrations. The expression levels of Myc-MDMX and MDM2 were determined by a Western blot analysis. (B) MEF MDM2 -/- p53 -/- cells were transfected with a Myc-MDM2 plasmid or the empty vector for 24 h, followed by a 24-h exposure to InuA at the indicated concentrations. The expression levels of Myc-MDM2 and MDMX were determined by Western blotting. Band intensity ratios were obtained using the IMAGE J software program and were normalized to those of vehicle controls. (C) LNCaP and PC3 cells were transfected with MDMX siRNA (siMDMX) or the respective control siRNA (siCtrl) for 36 h, followed by exposure to InuA at the indicated concentrations for 24 h. Cell lysates were subjected to immunoprecipitation with an MDM2 antibody. The ubiquitinated MDM2 was detected using an anti-ubiquitin antibody. Data are representative of three or more experiments.

Article Snippet: The siRNAs against human NFAT1, MDM2, and MDMX were purchased from Thermo Fisher Scientific (Rockford, IL, United States).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Software, Control, Immunoprecipitation, Ubiquitin Proteomics