Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases

doi: 10.3390/ijms241210031

Figure Lengend Snippet: The ubiquitination of GRK2 is involved in D2R β-arrestin pathway-mediated ERK activation. ( A ) CTRL-KD and Mdm2-KD cells were transfected with plasmids encoding [Gprot] D2R or [βarr] D2R. Serum-starved cells were stimulated with 10 μM DA for 2 min ( [Gprot] D2R producing) or 10 min ( [βarr] D2R producing). CTRL-KD and Mdm2-KD cell lysates were immunoblotted using p-ERK1/2 (1:1000 dilution) and ERK2 (1:1000 dilution) antibodies, respectively. Cell lysates were immunoblotted with Mdm2 or β-actin antibodies. About 86% of the Mdm2 levels in cells were diminished. ** p < 0.01 compared with the corresponding Veh group, # p < 0.05 compared with the DA stimulation group ( n = 3). ( B ) GRK2-KD cells were transfected with plasmids encoding [Gprot] D2R or [βarr] D2R, and co-transfected with plasmids encoding GRK2-WT or GRK2-4KR. Serum-starved cells were treated with 10 μM DA for 2 min ( [Gprot] D2R-producing) or 10 min ( [βarr] D2R-producing). Cells lysates were immunoblotted using p-ERK1/2 (1:1000 dilution) and ERK2 (1:1000 dilution) antibodies, respectively. We measured the levels of p-ERKs and total ERKs in the same sample and then divided the amount of pERKs by the amount of total ERKs to obtain the p-ERK/ERK ratio. The pERK/ERK ratio provided a normalized measure of ERK pathway activation. ** p < 0.01, * p < 0.05 compared with the corresponding Veh group, ## p < 0.01 compared with the DA/GRK2-WT/ [βarr] D2R expression group ( n = 3).

Article Snippet: GRK2 and Mdm2 shRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics, Activation Assay, Transfection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases

doi: 10.3390/ijms241210031

Figure Lengend Snippet: The activation of the D2R β-arrestin pathway promotes GRK2 ubiquitination. ( A ) HEK 293 cells producing [Gprot] D2R or [βarr] D2R were transfected with plasmids encoding GFP-GRK2 and FLAG-Mdm2. The cells were stimulated with either Veh or 10 μM DA for 2 min. FLAG beads were used to immunoprecipitate cell lysates. Co-IP/Lysate and IP were immunoblotted via the use of GFP (1:1000 dilution) and FLAG (1:1000 dilution) antibodies, respectively. The data represent the outcomes of three independent investigations with comparable results. ** p < 0.01 compared with the Veh group ( n = 3). ( B ) HEK 293 cells were transfected with plasmids encoding [Gprot] D2R or [βarr] D2R, HA-Ub, and FLAG-GRK2. The cells were treated for 2 min with either a vehicle or 10 μM DA. Immunoprecipitation of cell lysates using FLAG beads. Antibodies against HA (1:1000 dilution) and FLAG (1:1000 dilution) were used to immunoblot Co-IP and IP. ** p < 0.01 compared with the Veh group ( n = 3). ( C ) HEK 293 cells producing D2R were transfected with plasmids encoding HA-Ub and FLAG-GRK2. The cells were stimulated with either 10 μM MLS1547 or 1 μM UNC9994 for 2 min. Immunoprecipitation of cell lysates using FLAG beads. Antibodies against HA (1:1000 dilution) and FLAG (1:1000 dilution) were used to immunoblot Co-IP and IP. * p < 0.05 compared with the Veh group ( n = 3). ( D ) HEK 293 cells were transfected with plasmids encoding D2R and FLAG-GRK2. Cells were prestimulated with 50 μg/mL cycloheximide for 1 h, followed by 10 μM MLS1547 or 1 μM UNC9994 treatment for 0–2 h. Cell lysates were immunoblotted using FLAG (1:1000 dilution) or β-actin (1:2000 dilution) antibodies. “0 min” groups were normalized to 100%. * p < 0.05, *** p < 0.001 compared with the Veh group ( n = 3). ( E ) CTRL-KD and Mdm2-KD cells producing D2R were transfected with plasmids encoding GRK2. The pretreatment of cells with 50 μg/mL cycloheximide for 1 h was followed with treatment with 1 μM UNC9994 for 0–2 h. The immunoblotting of cell lysates with antibodies against GRK2 (1:2000 dilution) or β-actin (1:2000 dilution) was performed. “0 min” groups were normalized to 100%. * p < 0.05, *** p < 0.001 compared with the Veh/CTRL-KD group ( n = 3).

Article Snippet: GRK2 and Mdm2 shRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Activation Assay, Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases

doi: 10.3390/ijms241210031

Figure Lengend Snippet: The Mdm2-mediated ubiquitination of GRK2 occurs in the cytoplasm in response to UNC9994 stimulation. ( A ) HEK 293 cells were transfected with plasmids encoding GFP-GRK2 or GFP-β-arrestin2. Cells were exposed to a vehicle or 10 ng/mL LMB for 3 h. The horizontal bar represents 10 μm. One representative example from three independent investigations is depicted in the data. ( B ) HEK 293 cells were transfected with plasmids encoding FLAG-D2R and GFP-Mdm2. Cells were stimulated with either a vehicle or 1 μM UNC9994 for 10 min. Later, the cells were incubated with FLAG antibodies (1:1000 dilution) and Alexa-594-conjugated anti-rabbit secondary antibodies (1:500 dilution) in succession. The horizontal bar represents 10 μm. One representative example from three independent investigations is depicted in the data. ( C ) HEK 293 cells were transfected with plasmids encoding D2R, HA-Ub, and FLAG-GRK2. Cells were pretreated with 10 ng/mL LMB for 3 h, followed by 1 μM UNC9994 treatment for 2 min. Cell lysates were immunoprecipitated via the use of FLAG beads. Co-IP and IP were immunoblotted with HA (1:1000 dilution) and FLAG (1:1000 dilution) antibodies, respectively. *** p < 0.001 compared with the Veh/veh group ( n = 3). ( D ) HEK 293 cells were transfected with plasmids encoding D2R. Cells were stimulated with 1 μM UNC9994 for 2 min. The fractionation of cell lysates followed the protocol outlined in the “ ”. Nuclear and cytosolic fractions were utilized in ubiquitination assays. NF, nuclear fraction; CF, cytosolic fraction. ** p < 0.01 compared with the Veh/CF group ( n = 3).

Article Snippet: GRK2 and Mdm2 shRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics, Transfection, Incubation, Immunoprecipitation, Co-Immunoprecipitation Assay, Fractionation

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases

doi: 10.3390/ijms241210031

Figure Lengend Snippet: The tyrosine phosphorylation of GRK2 is required for Mdm2-mediated GKR2 ubiquitination upon the stimulation of the D2R β-arrestin-dependent pathway. ( A ) HEK 293 cells were transfected with plasmids encoding [βarr] D2R, GRK2, and FLAG-Mdm2. The pretreatment of cells with 10 μM PP2 for 30 min was followed by a 2-min treatment with 10 μM DA. The immunoprecipitation of cell lysates using FLAG beads. Co-IP/Lysate and IP were immunoblotted, respectively, with antibodies against GRK2 (1:2000) and FLAG (1:1000). ( B ) HEK 293 cells were transfected with plasmids encoding [βarr] D2R, HA-Ub, and FLAG-GRK2. Cells were pretreated with 10 μM PP2 for 30 min, followed by 10 μM DA treatment for 2 min. The immunoprecipitation of cell lysates using FLAG beads. Co-IP/Lysate and IP were immunoblotted, respectively, with antibodies against HA (1:1000 dilution) and FLAG (1:1000 dilution). ** p < 0.01 compared with the Veh group ( n = 3). ( C ) HEK 293 cells were transfected with plasmids encoding [βarr] D2R, HA-Ub, and FLAG-GRK2-WT or FLAG-GRK2-3YF. The cells were treated with either a vehicle or 10 μM DA for 2 min. Immunoprecipitation of cell lysates using FLAG beads. Co-IP/Lysate and IP were immunoblotted with antibodies against HA (1:1000 dilution) and FLAG (1:1000 dilution), respectively. ** p < 0.01 compared with the Veh group ( n = 3). ( D ) HEK 293 cells were transfected with plasmids encoding [βarr] D2R, HA-Src, and FLAG-GRK2-WT or FLAG-GRK2-4KR. Cells were treated with either a vehicle or 10 μM DA for 2 min. The immunoprecipitation of cell lysates using FLAG beads. HA (1:1000 dilution) and FLAG (1:1000 dilution) antibodies were used to immunoblot Co-IP/Lysate and IP, respectively. ** p < 0.01, *** p < 0.001 compared with corresponding Veh group (n = 3). ( E ) GRK2-KD cells producing [βarr] D 2 R were transfected with plasmids encoding GRK2-WT or GRK2-3YF. Serum-starved cells were incubated in the presence of 10 μM DA for 10 min. Antibodies against p-ERK1/2 (1:1000 dilution) and ERK2 (1:1000 dilution) were used to immunoblot lysates. ** p < 0.01 compared with the corresponding Veh/WT group ( n = 3).

Article Snippet: GRK2 and Mdm2 shRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases

doi: 10.3390/ijms241210031

Figure Lengend Snippet: Diagram showing the mechanisms involved in D2R β-arrestin-dependent pathway-mediated ERK activation. After stimulation with an agonist to activate the D2R β-arrestin signaling pathway, Mdm2 moves out of the nucleus to ubiquitinate GRK2, which is in an Src-dependent tyrosine phosphorylation state. Ubiquitinated GRK2 then translocates to the plasma membrane and interacts with activated D2R, followed by the phosphorylation of D2R and recruiting β-arrestin to mediate downstream ERK signal transduction.

Article Snippet: GRK2 and Mdm2 shRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Clinical Proteomics, Membrane, Transduction

Journal: Oncotarget

Article Title: Opposite regulation of MDM2 and MDMX expression in acquisition of mesenchymal phenotype in benign and cancer cells

doi:

Figure Lengend Snippet: Western blotting and qRT-PCR analysis of MDM2, MDMX, epithelial and mesenchymal markers, and cell migration of human benign prostate BPH-1 cells and two independent tumorigenic clones, CAFTD01 and CAFTD03. A. Images of representative blots are shown; the full length MDM2 protein product is marked by an arrow, a faster-migrating product is marked by an asterisk . Relative protein expression was evaluated by measuring optical density (OD). B–C. PCR data represent mean ± SEM. (B) ** P < 1.10 −3 to BPH-1 cells, n = 3 (C) * P < 0.05, ** P < 1.10 −6 to BPH-1 cells, n = 7. D. Migration results through an uncoated 8 μm-pore transwell represent the average number (mean ± SD) of migrating cells in five independent viewing fields after 6 h of migration, from 3 independent experiments in technical duplicate; * P < 0.01 to BPH-1 cells. Photographs show results of a representative experiment at 40× magnification. E. Viability of BPH-1 and CAFTD03 cells treated with docetaxel for 72 h was analyzed using a luminescence-based ATP assay. Graphs show data from a representative experiment in technical triplicate. IC50 values were calculated from 3 independent experiments.

Article Snippet: BPH-1 cells stably knocked down for MDM2 were generated after transduction with lentiviruses expressing shRNA against MDM2 (sc-29394-V, Santa Cruz Biotechnology), 1 μg/ml puromycin selection, and single-cell cloning.

Techniques: Western Blot, Quantitative RT-PCR, Migration, Clone Assay, Expressing, ATP Assay

Journal: Oncotarget

Article Title: Opposite regulation of MDM2 and MDMX expression in acquisition of mesenchymal phenotype in benign and cancer cells

doi:

Figure Lengend Snippet: Western blotting and qRT-PCR analysis of MDM2, MDMX, epithelial and mesenchymal markers, and cell migration in A–D. human breast MCF10A cells stably transfected with empty vector (EV) or oncogenic K-RasV12; E–H. murine prostate cancer cell lines; and I. BPH-1 cells. (A, E, I) In representative blots, the full-length MDM2 protein product is marked by an arrow, a faster-migrating product observed in human cells is marked by an asterisk; relative protein expression was evaluated by measuring OD. (B-C, F-G) PCR data represent mean ± SEM. (B–C) * P < 0.05; ** P < 1.10 −3 to MCF10A EV, n = 4; (F–G) ** P < 1.10 −3 to cE2 cells, n = 4. (D, H) Migration results through an uncoated 8 μm-pore transwell represent the average number (mean ± SD) of migrating cells in five independent viewing fields after 6 h of migration, from 3 independent experiments in technical duplicate; * P < 0.01. Photographs show results of a representative experiment at 40× magnification. (I) Both TGF-β1-induced EMT and MDM2 downregulation are reversible. Cells were treated with vehicle or TGF-β1 for 96 h, passaged and cultivated in the presence or absence of TGF-β1 for another 96 h. The graph shows the fold-over control of normalized protein expression quantified by measuring the OD, * P < 1.10 −2 to untreated cells, n = 3.

Article Snippet: BPH-1 cells stably knocked down for MDM2 were generated after transduction with lentiviruses expressing shRNA against MDM2 (sc-29394-V, Santa Cruz Biotechnology), 1 μg/ml puromycin selection, and single-cell cloning.

Techniques: Western Blot, Quantitative RT-PCR, Migration, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Control

Journal: Oncotarget

Article Title: Opposite regulation of MDM2 and MDMX expression in acquisition of mesenchymal phenotype in benign and cancer cells

doi:

Figure Lengend Snippet: Changes in MDM2 and MDMX expression clustered by changes in E-cadherin expression in paired CaP and BrCa samples and respective LN metastases

Article Snippet: BPH-1 cells stably knocked down for MDM2 were generated after transduction with lentiviruses expressing shRNA against MDM2 (sc-29394-V, Santa Cruz Biotechnology), 1 μg/ml puromycin selection, and single-cell cloning.

Techniques: Expressing

Journal: Oncotarget

Article Title: Opposite regulation of MDM2 and MDMX expression in acquisition of mesenchymal phenotype in benign and cancer cells

doi:

Figure Lengend Snippet: A. BPH-1 cells were transiently transfected with control or MDM2-specific siRNA and cell migration was evaluated by monitoring the impedance signal using a fibronectin-coated CIM module on an xCELLigence DP system. The graph shows the average Cell Index values (mean ± SD) from three independent experiments, * P < 0.05. B. Migration results through an uncoated 8 μm-pore transwell from three independent BPH-1 clones stably transfected with control or MDM2-specific shRNA. C, D. Transwell migration of a pool of CAFTD03 cells transiently overexpressing (C) wt, GFP-tagged or the C464A mutant of MDM2 or (D) MDMX. Graphs represent the relative numbers of migrating cells (mean ± SD) from 3 independent experiments in technical duplicate; * P < 0.05; n.s., P > 0.05. EV, empty vector. E. Viability of stable shRNA-expressing clones from BPH-1 cells treated with docetaxel for 72 h was analyzed using a luminescence-based ATP assay. Graphs show the mean ± SD from 3 independent experiments; IC50 values were calculated from 3 independent experiments.

Article Snippet: BPH-1 cells stably knocked down for MDM2 were generated after transduction with lentiviruses expressing shRNA against MDM2 (sc-29394-V, Santa Cruz Biotechnology), 1 μg/ml puromycin selection, and single-cell cloning.

Techniques: Transfection, Control, Migration, Clone Assay, Stable Transfection, shRNA, Mutagenesis, Plasmid Preparation, Expressing, ATP Assay

Journal: Oncotarget

Article Title: Opposite regulation of MDM2 and MDMX expression in acquisition of mesenchymal phenotype in benign and cancer cells

doi:

Figure Lengend Snippet: A. qRT-PCR analysis of MDM2 transcripts from untreated BPH-1 cells and two independent CAFTD clones. The assay design represented by black bars detects mRNA products originating from both P1 and P2 promoters, while the assay design represented by grey bars detects only the product of the P1 promoter. ** P < 1.10 −3 to BPH-1 cells, n = 4 B. Expression of MDM2 transcripts from the P1 and P2 promoter in BPH-1 cells and tumorigenic CAFTD clones was analyzed by semi-quantitative RT-PCR. Sizes of the PCR products are 452 bp for Exon 1-Exon 6 of MDM2, 398 bp for Exon2-Exon 6 of MDM2, and 372 bp for GAPDH, an internal normalization control. The number of amplification cycles is indicated at the bottom of the gel. The graph represents average relative amounts of respective MDM2 transcripts normalized to GAPDH from four independent experiments. C.01 and C.03 refer to the CAFTD clones. C. The activity of the MDM2 P2 promoter was evaluated using a luciferase reporter assay. The cells were transfected with a construct bearing the MDM2 P2 promoter sequence upstream of the firefly luciferase gene, along with a Renilla luciferase internal control (pCIneo RL). The graph represents the mean ± SD of normalized luciferase activity, ** P < 1.10 −4 to BPH-1 cells, n = 5.

Article Snippet: BPH-1 cells stably knocked down for MDM2 were generated after transduction with lentiviruses expressing shRNA against MDM2 (sc-29394-V, Santa Cruz Biotechnology), 1 μg/ml puromycin selection, and single-cell cloning.

Techniques: Quantitative RT-PCR, Clone Assay, Expressing, Control, Amplification, Activity Assay, Luciferase, Reporter Assay, Transfection, Construct, Sequencing

Journal: Oncotarget

Article Title: Opposite regulation of MDM2 and MDMX expression in acquisition of mesenchymal phenotype in benign and cancer cells

doi:

Figure Lengend Snippet: A–C. Western blotting (A) and qRT-PCR (B–C) analysis of EMT-driving transcription factors in BPH-1 and CAFTD cells. PCR data represent mean ± SEM of at least three independent experiments, * P < 0.05 to BPH-1 cells. D–E. BPH-1 and MCF10A cells were transfected with (D) MDM2-specific siRNA or (E) increasing concentrations of MDM2 overexpressing plasmid and expression of MDM2, Slug and Twist was evaluated by western blotting 48 h after transfection. F–H. CAFTD03 cells were transfected with siRNA against Slug, Twist, or both. Protein expression was evaluated by (F) western blotting and (G) mRNA expression by qRT-PCR 48 h after transfection. The full-length MDM2 protein product is marked by an arrow; a faster-migrating product is marked by an asterisk. (H) Transwell migration of CAFTD03 cells transfected with 20 nM siRNA. Bars show the mean ± SD values for relative cell migration of three independent experiments, * P < 0.05; ** P < 0.01.

Article Snippet: BPH-1 cells stably knocked down for MDM2 were generated after transduction with lentiviruses expressing shRNA against MDM2 (sc-29394-V, Santa Cruz Biotechnology), 1 μg/ml puromycin selection, and single-cell cloning.

Techniques: Western Blot, Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, Migration

Journal: Oncotarget

Article Title: Opposite regulation of MDM2 and MDMX expression in acquisition of mesenchymal phenotype in benign and cancer cells

doi:

Figure Lengend Snippet: A. In case of intact p53 function, MDM2 cooperates with p53 in the degradation of Slug, thereby preserving the epithelial phenotype. B. When p53 function is impaired, elevated expression of MDM2 correlates with the epithelial phenotype. Slug and Twist inhibit expression of MDM2 and promote EMT, which is associated with upregulated expression of MDMX. Solid lines delineate direct effects, dotted lines represent indirect mechanisms.

Article Snippet: BPH-1 cells stably knocked down for MDM2 were generated after transduction with lentiviruses expressing shRNA against MDM2 (sc-29394-V, Santa Cruz Biotechnology), 1 μg/ml puromycin selection, and single-cell cloning.

Techniques: Preserving, Expressing

Journal: Journal of Biological Chemistry

Article Title: Mdm2 Promotes Myogenesis through the Ubiquitination and Degradation of CCAAT/Enhancer-binding Protein β

doi: 10.1074/jbc.m115.638577

Figure Lengend Snippet: FIGURE 2. Mdm2 expression is regulated during myogenesis. A, C/EBP, Mdm2, and myogenic marker protein expression in C2C12 myoblasts cul- tured in GM or induced in DM for 1–9 days. -Tubulin is a loading control. B, Western analysis of p53 expression in C2C12 myoblasts in growth medium (GM) or in differentiation medium (DM) for the time points indicated. -Tubu- lin is used as a loading control. C, C/EBP, Mdm2, and myogenic marker pro- tein expression in primary myoblasts cultured in GM or DM for the indicated time points in hours.

Article Snippet: Lentivirus containing a shRNA against mouse Mdm2 (shMdm2, sc-37263-V) or a scrambled shRNA (shScr, sc-108080) (Santa Cruz Biotechnology) were added to C2C12 cells or primary myoblasts at 30 – 40% confluency in GM according to manufacturer’s instructions.

Techniques: Expressing, Marker, Control, Western Blot, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Mdm2 Promotes Myogenesis through the Ubiquitination and Degradation of CCAAT/Enhancer-binding Protein β

doi: 10.1074/jbc.m115.638577

Figure Lengend Snippet: FIGURE 3. Mdm2 interacts with C/EBP. A, coimmunoprecipitation assay using anti-C/EBP (IP:) antibody, or nonspecific type matched IgG to pull- down Mdm2. Input (In) is 10% of the material used for immunoprecipitation. B, coimmunoprecipitation assay using anti-Mdm2 (IP:M) antibody or nonspe- cific type matched IgG to pulldown C/EBP. C, GST pulldown assay using full-length GST-C/EBP (amino acids 1–296; FL), GST-C/EBP1–151 (C) GST-C/ EBP152–296 (N), or GST alone as bait to precipitate in vitro translated Mdm2 detected by Western blotting. Migration of the GST constructs is indicated by Coomassie Blue staining (bottom).

Article Snippet: Lentivirus containing a shRNA against mouse Mdm2 (shMdm2, sc-37263-V) or a scrambled shRNA (shScr, sc-108080) (Santa Cruz Biotechnology) were added to C2C12 cells or primary myoblasts at 30 – 40% confluency in GM according to manufacturer’s instructions.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, GST Pulldown Assay, In Vitro, Western Blot, Migration, Construct, Staining

Journal: Journal of Biological Chemistry

Article Title: Mdm2 Promotes Myogenesis through the Ubiquitination and Degradation of CCAAT/Enhancer-binding Protein β

doi: 10.1074/jbc.m115.638577

Figure Lengend Snippet: FIGURE 4. Mdm2 ubiquitinates and targets C/EBP for degradation. A, C/EBP and Mdm2 protein expression in C2C12 cells lentivirally transduced to express shRNA against Mdm2 (shMdm2) or a scrambled shRNA (shScr) and induced to differentiate for 24 h during which the last 6 h included MG132 or vehicle as indicated. -Tubulin is a loading control. B, in vitro ubiquitination assay in which biotinylated-ubiquitin and GST-C/EBP (target) was added to C2C12 cell extract from cells expressing shMdm2 or shScr. GST-C/EBP precipitates were analyzed for the addition of biotin-ubiquitin using strepavidin-HRP. C, C/EBP, Mdm2, Pax7, and MyoD protein expression in C2C12 myoblasts expressing shMdm2 or shScr in GM or DM for 24 h. D, C/EBP, Mdm2, Pax7, and MyoD protein expression in primary myoblasts expressing shMdm2 or shScr in GM or DM for 12 h. E, Western analysis of p53 expression in C2C12 myoblasts lentivirally transduced to express a shRNA against Mdm2 (shMdm2) or a scrambled shRNA control (shScr) cultured in GM or induced to differentiate in DM for the time points indicated.

Article Snippet: Lentivirus containing a shRNA against mouse Mdm2 (shMdm2, sc-37263-V) or a scrambled shRNA (shScr, sc-108080) (Santa Cruz Biotechnology) were added to C2C12 cells or primary myoblasts at 30 – 40% confluency in GM according to manufacturer’s instructions.

Techniques: Expressing, shRNA, Control, In Vitro, Ubiquitin Proteomics, Western Blot, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Mdm2 Promotes Myogenesis through the Ubiquitination and Degradation of CCAAT/Enhancer-binding Protein β

doi: 10.1074/jbc.m115.638577

Figure Lengend Snippet: FIGURE 5. Loss of Mdm2 blocks myogenic differentiation and regenera- tion. A, immunocytochemistry for myosin heavy chain (MyHC) expression in C2C12 cells transduced to express shMdm2 or shScr and induced to differen- tiate for 7 days. DAPI stain reveals nuclei. B, differentiation index (no. nuclei in MyHC cells/total nuclei) for cells transduced and differentiated as in A. **, p 0.01, n 6. C, cell cycle analysis by flow cytometry in C2C12 cells forced into G1 arrest, or transduced as in A. Graph indicates the mean percentage of cells in each phase of the cell cycle S.D. The % cells in S-phase in shMdm2 cultures are significantly different (p 0.044) from shScr controls. All other comparisons are not significantly different. D, average size of myotubes (no. nuclei in MyHC cells with nuclei 2/no. myotubes) for cells transduced and differentiated as in A. *, p 0.05, n 6. E, Mdm2, C/EBP, and myogenic marker protein expression in cells transduced and induced to differentiate as in A. F, percent of GFP fibers in regenerating muscle 2 weeks after injury with cardiotoxin and transplantation of primary myoblasts isolated from a GFP transgenic mouse transduced to express shMdm2 or shScr. ***, p 0.001, n 6. G, representative images of TA muscle from F. DAPI stains the nuclei. Scale bar, 25 m.

Article Snippet: Lentivirus containing a shRNA against mouse Mdm2 (shMdm2, sc-37263-V) or a scrambled shRNA (shScr, sc-108080) (Santa Cruz Biotechnology) were added to C2C12 cells or primary myoblasts at 30 – 40% confluency in GM according to manufacturer’s instructions.

Techniques: Immunocytochemistry, Expressing, Staining, Cell Cycle Assay, Flow Cytometry, Marker, Transplantation Assay, Isolation, Transgenic Assay

Journal: Journal of Biological Chemistry

Article Title: Mdm2 Promotes Myogenesis through the Ubiquitination and Degradation of CCAAT/Enhancer-binding Protein β

doi: 10.1074/jbc.m115.638577

Figure Lengend Snippet: FIGURE 6. Loss of C/EBP rescues differentiation in cells lacking Mdm2. A, Mdm2, C/EBP, and myogenic marker protein expression in primary myoblasts from C/EBP conditional null mice (/) or control littermates (WT) transduced to express shMdm2 or shScr and induced to differentiate for 3 days. B, immunocytochemistry for MyHC expression in differentiated cultures as in A. C, differentiation index for cells transduced and differentiated as in A. Means indicated with different letters are significantly different from one another, meeting a minimum cut-off of p 0.05, n 3. D, average myotube size for cells transduced and differentiated as in A.

Article Snippet: Lentivirus containing a shRNA against mouse Mdm2 (shMdm2, sc-37263-V) or a scrambled shRNA (shScr, sc-108080) (Santa Cruz Biotechnology) were added to C2C12 cells or primary myoblasts at 30 – 40% confluency in GM according to manufacturer’s instructions.

Techniques: Marker, Expressing, Control, Immunocytochemistry

Journal: bioRxiv

Article Title: Sprouty4 is required for Mdm2 regulation of invasion, focal adhesion formation and metastasis in cells lacking p53

doi: 10.1101/2023.05.08.539890

Figure Lengend Snippet: HT1080 p53KO cells were transfected with siRNAs against Mdm2 and siCtrl or treated with the Mdm2 inhibitor, MEL23. (A-B) Protein levels of Mdm2 and MdmX after (A) transfection using siRNAs as indicated or (B) treatment with MEL23 (7 µM) for 24 h. β-actin was used as loading control. (C-D) Cell migration assay. Representative images (top) and quantification (bottom) of wound scratch migration assay with cells (C) silenced for Mdm2 or (D) treated with MEL23 for 24 h. Scale bar equivalent to 1 mm. (E-F) Representative images of morphology of cells attached to collagen coated coverslips and quantification of cell area after (E) silencing of Mdm2 or (F) treatment with MEL23 for 24 h. Cells stained for actin (orange) and DNA via DAPI (blue). Scale bar equivalent to 50 µm. (G-H) Quantification and representative micrographs showing attachment to different ECM components of cells (G) silenced for Mdm2 or (H) treated with MEL23 for 24 h. Scale bar equivalent to 1 mm. Experiments shown represent mean ±SD of at least 3 biological replicates, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Stable cell lines were stablished transducing cells with a set of 4 shRNAs against Mdm2 (Origene, cat.#TL311529) or shRNA negative control (Origene, cat.#TR30021) and using the adequate drug for cell selection.

Techniques: Transfection, Control, Cell Migration Assay, Migration, Staining

Journal: bioRxiv

Article Title: Sprouty4 is required for Mdm2 regulation of invasion, focal adhesion formation and metastasis in cells lacking p53

doi: 10.1101/2023.05.08.539890

Figure Lengend Snippet: (A-B) Tumor spheroid invasion in collagen I matrix after (A) transfection of HT1080 p53KO cells with siRNAs against Mdm2 or (B) treatment with MEL23. Representative images above and quantification below of area invaded and number of cells invading 24 h after implantation. (C-D) Tumor spheroid invasion in collagen I-BME matrix after (C) transfection of HT1080 p53KO cells with siRNAs against Mdm2 or (D) treatment with MEL23. Representative images and quantification of area invaded 24 h after implantation. (E) Representative images of cell morphology of HT1080 p53KO cells transfected with siRNAs against Mdm2 or siCtrl in collagen matrix, 6 h after implantation. (F) Quantification of more circular cells (circularity of 0.75 or higher) in each condition shown in (E). (G-I) HT1080 p53KO cells stably expressing shRNA scramble (shScramble) or a pool of shRNAs against Mdm2 (shMdm2) were used to analyze metastatic burden using mouse models. (G) Protein levels of Mdm2 and MdmX in HT1080 shScramble and shMdm2 stable cell lines. β-actin was used as loading control. (H) Representative images above and quantification below of metastatic foci in the lungs after implantation of shScramble or shMdm2 cells using orthotropic model. (I) Representative images above and quantification below of metastatic foci in the lungs after injection of shScramble or shMdm2 cells using tail-vein model. Scale bars equivalent to 200 µm. Experiments shown represent mean ±SD of at least 3 biological replicates, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Stable cell lines were stablished transducing cells with a set of 4 shRNAs against Mdm2 (Origene, cat.#TL311529) or shRNA negative control (Origene, cat.#TR30021) and using the adequate drug for cell selection.

Techniques: Transfection, Stable Transfection, Expressing, shRNA, Control, Injection

Journal: bioRxiv

Article Title: Sprouty4 is required for Mdm2 regulation of invasion, focal adhesion formation and metastasis in cells lacking p53

doi: 10.1101/2023.05.08.539890

Figure Lengend Snippet: HT1080 p53KO cells were (A) silenced for Mdm2 using siRNAs or (B) treated with 7 µM MEL23 for 24 h. (A-B) Immunofluorescence showing FA foci by vinculin staining (red), stress fiber formation by phalloidin staining of F-actin (green), and nuclei (blue) detected by DAPI staining of DNA. Representative images shown above with quantification of FA parameters below. Scale bar equivalent to 20 µm. (C-D) Protein levels of FA related proteins in HT1080 p53KO cells (C) silenced for Mdm2 or (D) treated with MEL23 for 24 h. α-tubulin was used as loading control. Experiments shown represent mean ±SD of at least 3 biological replicates, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Stable cell lines were stablished transducing cells with a set of 4 shRNAs against Mdm2 (Origene, cat.#TL311529) or shRNA negative control (Origene, cat.#TR30021) and using the adequate drug for cell selection.

Techniques: Immunofluorescence, Staining, Control

Journal: bioRxiv

Article Title: Sprouty4 is required for Mdm2 regulation of invasion, focal adhesion formation and metastasis in cells lacking p53

doi: 10.1101/2023.05.08.539890

Figure Lengend Snippet: HT1080 p53KO cells were transfected with siRNAs against Mdm2 or siCtrl for 24 h; or treated with 7 µM MEL23 or DMSO (vehicle) for 24 h. (A) Volcano plots show all peptides identified by mass spectrometry. Colored dots represent significantly differentially expressed proteins that were downregulated (blue dots) and upregulated (red dots) in each condition shown at left of plot. Gray dots represent non-significant changes. MEL23 treated cells were compared to DMSO treated cells. Cells transfected with siMdm2#1 or #2 were compared to siCtrl-transfected cells. (B-C) Spry4 expression in HT1080 p53KO cells in response to Mdm2 knockdown or treatment with MEL23 for 24 h by (B) mass spectrometry or (C) immunoblotting. β-actin and α-tubulin were used as loading control for immunoblot. (D) Co-immunoprecipitation of Mdm2 and Spry4 in the presence or absence of MG132. β-actin was used as loading control. (E) Quantification of Spry4, Mdm2 and MdmX protein levels after treatment with MG132 (or vehicle, DMSO) for 4 h. (F) Spry4 mRNA levels in response to Mdm2 knockdown or treatment with MEL23 for 24 h. RPL32 was used as housekeeping. (G) Spry4 luciferase promoter assay in cells silenced for Mdm2 using siRNA. (H) Spry4 mRNA levels in cells treated with MEL23 or vehicle at the indicated times after treatment with DMSO, ActinomycinD (10 µg/ml), or DRB (100 µM) as indicated. Experiments shown represent mean ±SD of at least 3 biological replicates, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Stable cell lines were stablished transducing cells with a set of 4 shRNAs against Mdm2 (Origene, cat.#TL311529) or shRNA negative control (Origene, cat.#TR30021) and using the adequate drug for cell selection.

Techniques: Transfection, Mass Spectrometry, Expressing, Knockdown, Western Blot, Control, Immunoprecipitation, Luciferase, Promoter Assay

Journal: bioRxiv

Article Title: Sprouty4 is required for Mdm2 regulation of invasion, focal adhesion formation and metastasis in cells lacking p53

doi: 10.1101/2023.05.08.539890

Figure Lengend Snippet: H1299 cells were transfected with siRNAs against Mdm2 and siCtrl . (A) Protein levels of Mdm2, MdmX and p53 after. β-actin was used as loading control. (B) Cell migration assay. Representative images (top) and quantification (bottom) of wound scratch migration assay. Scale bar equivalent to 1 mm. (C) Quantification and representative micrographs showing attachment to ECM component, collagen I. Scale bar equivalent to 1 mm. (D) Representative images of immunofluorescence showing FA foci by vinculin staining (red), stress fiber formation by phalloidin staining of F-actin (green), and nuclei (blue) detected by DAPI staining of DNA. Scale bar equivalent to 20 µm. (E) Protein levels of Spry4 as well as Mdm2, MdmX and p53 after Mdm2 silencing using siRNAs. β-actin was used as loading control. (F) mRNA levels of Spry4 after Mdm2 silencing using siRNAs. RPL32 was used as housekeeping. Experiments shown represent mean ±SD of at least 3 biological replicates, *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: Stable cell lines were stablished transducing cells with a set of 4 shRNAs against Mdm2 (Origene, cat.#TL311529) or shRNA negative control (Origene, cat.#TR30021) and using the adequate drug for cell selection.

Techniques: Transfection, Control, Cell Migration Assay, Migration, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Sprouty4 is required for Mdm2 regulation of invasion, focal adhesion formation and metastasis in cells lacking p53

doi: 10.1101/2023.05.08.539890

Figure Lengend Snippet: (A-E) HT1080 p53KO cells were transfected with siRNA against Mdm2 alone or both siRNA against Mdm2 and a pool of Spry4 siRNAs. (A) Protein levels of Mdm2, MdmX and Spry4 after transfection with indicated siRNAs. β-actin was used as loading control. (B) Quantification of wound scratch migration assay comparing migration into wound scratches in cells treated with the indicated siRNAs as in . (C) Quantification of cell area after attachment to collagen coated coverslips as in . (D) Immunofluorescence showing FA foci by vinculin staining (red), stress fiber formation by phalloidin staining of F-actin (green), and nuclei (blue) as detected by DAPI staining of DNA. Representative images shown at left and quantification of FA parameters shown at right. Scale bar equivalent to 20 µm. (E) Immunoblot (left) and densitometry (right) of levels of total and phospho-cofilin-1(Ser3) in HT1080 p53KO cells silenced for Mdm2 alone or with double KD of Mdm2 and Spry4. (F-H) H1299 cells were transfected with siRNA against Mdm2 alone or both siRNA against Mdm2 and a pool of Spry4 siRNAs. (F) Protein levels of Mdm2, MdmX and Spry4 after transfection with indicated siRNAs. β-actin was used as loading control. (G) Quantification of wound scratch migration assay comparing migration into wound scratches in cells treated with the indicated siRNAs as in . (H) Immunoblot of levels of total and phospho-cofilin-1(Ser3) in H1299 cells silenced for Mdm2 alone or with double KD of Mdm2 and Spry4. (I-J) HT1080 p53KO cell lines were established stably expressing a pool of shRNAs against Mdm2 alone or Mdm2 and Spry4 together. (I) Protein levels of Mdm2, MdmX and Spry4 in stable cells lines. β-actin was used as loading control. (J) Analysis of metastatic burden in vivo using tail-vein injection model as in and . Representative images above and quantification below of metastatic foci in the lungs after 8 weeks of injection. Experiments shown represent mean ±SD of at least 3 biological replicates, *p<0.05, **p<0.01, ****p<0.0001, n.s.: not significant.

Article Snippet: Stable cell lines were stablished transducing cells with a set of 4 shRNAs against Mdm2 (Origene, cat.#TL311529) or shRNA negative control (Origene, cat.#TR30021) and using the adequate drug for cell selection.

Techniques: Transfection, Control, Migration, Immunofluorescence, Staining, Western Blot, Stable Transfection, Expressing, In Vivo, Injection

Journal: Cancer research

Article Title: MDM2 derived from dedifferentiated liposarcoma extracellular vesicles induces MMP2 production from preadipocytes

doi: 10.1158/0008-5472.CAN-19-0203

Figure Lengend Snippet: (A) RT-PCR representing the number of molecules of MDM2 in DDLPS patient serum EVs (n=16) compared to normal healthy controls (n=6) (p≤ 0.001). (B) MDM2 copy number variation (CNV) measured in DDLPS tissues (n=14) compared to normal adjacent tissues (n=5) (p≤0.0045). (C) EVs derived from DDLPS patient serum contain MDM2 by DNA Sequencing on the entire exons 1, 6, 10 of MDM2. (D) ROC curve analysis to estimate the sensitivity and specificity for circulating EV-MDM2 in discriminate DDLPS patients from controls. Results are presented as average ±SEM. Statistical analysis were performed using unpaired t-test with Welch’s correction. ** 0.001 ≤p ≤0.01; *** ≤ 0.001.

Article Snippet: DNA plasmids, virus production and transduction Non-targeting control vector plasmid and shRNA targeting endogenous human MDM2 transcript were obtained from Origene.

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, DNA Sequencing

Journal: Cancer research

Article Title: MDM2 derived from dedifferentiated liposarcoma extracellular vesicles induces MMP2 production from preadipocytes

doi: 10.1158/0008-5472.CAN-19-0203

Figure Lengend Snippet: (A) EVs derived from Lipo246 contain MDM2 by DNA Sequencing on the entire exons 1, 6, 10 of MDM2. (B) Level of MDM2 (calculated by RT-PCR) in DDLPS secreted vesicles (Lipo863, Lipo246, Lipo224) is consistently and significantly up regulated compared to the level of MDM2 DNA in P-a secreted EVs (p≤0.01). The level of MDM2 in EVs is proportional to the level of MDM2 in the originating cells. (C) Level of MDM2 in different LPS cell lines calculated by CNV (p≤0.0001). Results are presented as average ± SD. Statistical analysis were performed using t-test. ** 0.001 ≤p ≤0.01; *** ≤ 0.001.

Article Snippet: DNA plasmids, virus production and transduction Non-targeting control vector plasmid and shRNA targeting endogenous human MDM2 transcript were obtained from Origene.

Techniques: Derivative Assay, DNA Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer research

Article Title: MDM2 derived from dedifferentiated liposarcoma extracellular vesicles induces MMP2 production from preadipocytes

doi: 10.1158/0008-5472.CAN-19-0203

Figure Lengend Snippet: (A) Level of MDM2 (measured by RT-PCR) in recipient P-a increases at mRNA level, when P-a are treated with Lipo246 EVs for 72h (p≤0.04). (B) Recipient P-a treated with Lipo246 EVs for 72h, show an increased level of MDM2-mRNA in a dose-response manner (p≤0.002). (C) When P-a are treated with Lipo246 EVs, the protein level of MDM2 increases 3 fold after 96h. Results are presented as average ± SD. Statistical analysis were performed using t-test. ** 0.001 ≤p ≤0.01; *** ≤ 0.001. (D) When P-a are treated with Lipo246-EVs (for 96h) they show a decreased level of p53 and p21 compared to untreated P-a (lane three). When P-a are treated with Lipo246-EVs together with MDM2 inhibitor (SAR405838, 0.2 μM), the inhibitory effect of EVs on p53 and p21 is rescued (lane four). Treatment of P-a with EVs isolated from Lipo863 (whose MDM2 levels are lower compared to Lipo246), produces results analogous to the treatment of P-a with SAR405838 (lane five). When P-a are treated with EVs isolated from Lipo863 where MDM2 is overexpressed (OE), the level of p53 and p21 decreases again (lane six).

Article Snippet: DNA plasmids, virus production and transduction Non-targeting control vector plasmid and shRNA targeting endogenous human MDM2 transcript were obtained from Origene.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Cancer research

Article Title: MDM2 derived from dedifferentiated liposarcoma extracellular vesicles induces MMP2 production from preadipocytes

doi: 10.1158/0008-5472.CAN-19-0203

Figure Lengend Snippet: (A) EVs increase P-a proliferation. When P-a are treated with Lipo246 EVs, for 72h, they show increased proliferation (p≤0.0001) compared to P-a treated with EV depleted medium (Pa+CM) and media alone (P-a). (B) Lipo246 EVs promote migration in recipient P-a. When P-a are treated with Lipo246-EVs they show an increased migration compared to P-a treated with EV depleted medium (Pa+CM) and media alone (P-a). Furthermore, P-a treated with Lipo246-EVs together with MDM2 inhibitor (SAR405838, 0.2 μM), show a decrease migration compared to P-a treated with Lipo246-EVs without drug (p≤0.05). P-a treated with drug alone don’t have a significant change on migration compared to P-a treated with EV depleted medium. P-a treated with Lipo863 EVs (whose MDM2 levels are lower compared to Lipo246) have decresed migration compared to P-a treated with Lipo246 EVs. P-a treated with EVs isolated from a pool of serum derived from DDLPS patients (N=8), have increased proliferation (p≤0.0001) (C) and migration (p≤0.005) (D) compared to P-a treated with EVs isolated from a pool of normal serum (N=3) and compared to P-a treated with media alone. (E) Cell cycle analysis of P-a by FACS. Treatment with Lipo246 EVs induces increase in S phase compared to normal medium or EV-depleted medium. Results are presented as average ± SD. Statistical analysis were performed using t-test. ** 0.001 ≤p ≤0.01; *** ≤ 0.001.

Article Snippet: DNA plasmids, virus production and transduction Non-targeting control vector plasmid and shRNA targeting endogenous human MDM2 transcript were obtained from Origene.

Techniques: Migration, Isolation, Derivative Assay, Cell Cycle Assay

Journal: Cancer research

Article Title: MDM2 derived from dedifferentiated liposarcoma extracellular vesicles induces MMP2 production from preadipocytes

doi: 10.1158/0008-5472.CAN-19-0203

Figure Lengend Snippet: Zymography showing increase of active MMP-2 released by P-a after EV incubation for 96h. MMP-2 activity (active MMP-2, 62 kDa) was significantly enhanced in the medium of Lipo246-EVs and Lipo224-treated P-a compared to P-a treated with EV depleted medium (P-a+CM) and P-a alone (P-a). The treatment with the MDM2 inhibitor SAR405838 (0.2 μM) impaired the release of active MMP-2. When P-a are treated with Lipo246-EVs after incubation of DDLPS cells with GW4869 (a drug that blocks EVs generation), the active MMP-2 released decreases as well as when P-a are treated with EVs isolated from Lipo863 (whose MDM2 level are lower compared to Lipo246). The level of active MMP-2 are rescued when P-a are treated with Lipo863 EVs where MDM2 is overexpressed. Representative images, experiments performed at least 3 times. In B results are presented as average ± SD. Statistical analysis were performed using a one-way ANOVA with Dunnett’s Multiple Comparisons Test. **** p ≤ 0.0001 and ** p ≤ 0.01.

Article Snippet: DNA plasmids, virus production and transduction Non-targeting control vector plasmid and shRNA targeting endogenous human MDM2 transcript were obtained from Origene.

Techniques: Zymography, Incubation, Activity Assay, Isolation

Journal: Cancer research

Article Title: MDM2 derived from dedifferentiated liposarcoma extracellular vesicles induces MMP2 production from preadipocytes

doi: 10.1158/0008-5472.CAN-19-0203

Figure Lengend Snippet: Our study establish the presence of high level of MDM2 DNA in DDLPS EVs derived from both DDLPS cell lines and patient serum samples. We also show that DDLPS EV MDM2-cargo can be transfer to recipient P-a in the DDLPS TME. This DDLPS EV-origin MDM2 leads to downregulated P-a p53 activity conferring oncogenic feature in normal recipient cells. Importantly, MDM2-cargo promote the release of active MMP-2 in normal recipient P-a thereby possibly contributing to subsequent loco-regional multifocal DDLPS dissemination.

Article Snippet: DNA plasmids, virus production and transduction Non-targeting control vector plasmid and shRNA targeting endogenous human MDM2 transcript were obtained from Origene.

Techniques: Derivative Assay, Activity Assay

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Primers sequences used for polymerase chain reaction (PCR).

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Polymerase Chain Reaction

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) was highly expressed in non‐small cell lung cancer (NSCLC) tissues and cells. (a) USP22 expression in NSCLC tissues was evaluated by immunohistochemistry (IHC) assay. (b, c) USP22 mRNA and protein levels in NSCLC tissues and adjacent normal tissues were measured by qRT‐PCR and western blot assays. (d, e) The mRNA and protein levels of USP22 in 16HBE, PC9, and HCC827 cells were determined by qRT‐PCR assay and western blot assay, respectively. * p < 0.05.

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Ubiquitin Proteomics, Expressing, Immunohistochemistry, Quantitative RT-PCR, Western Blot

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Deficiency of ubiquitin‐specific peptidase 22 (USP22) repressed non‐small cell lung cancer (NSCLC) cell migration and aggravated apoptosis and ferroptosis. (a) The protein level of USP22 in sh‐NC or sh‐USP22‐transfected PC9 and HCC827 cells was measured by western blot. (b) The migration of PC9 and HCC827 cells with sh‐NC or sh‐USP22 transfection was evaluated by transwell assay. (c) The apoptosis of PC9 and HCC827 cells transfected with sh‐NC or sh‐USP22 was analyzed by flow cytometry analysis. (d–g) The levels of reactive oxygen species (ROS), MDA, Fe 2+ , and GSH in sh‐NC or sh‐USP22 transfected PC9 and HCC827 cells were examined by relevant commercial kits. * p < 0.05.

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Ubiquitin Proteomics, Migration, Transfection, Western Blot, Transwell Assay, Flow Cytometry

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) overexpression promoted gefitinib resistance and repressed ferroptosis in gefitinib‐treated non‐small cell lung cancer (NSCLC) cells. (a) USP22 protein level in gefitinib or dimethyl sulfoxide (DMSO)‐treated PC9 and HCC827 cells was measured by western blot. (b) The effect of USP22 on gefitinib resistance was assessed by CCK‐8 assay. (c–i) PC9 and HCC827 cells were treated with DMSO+pcDNA, gefitinib+pcDNA, or gefitinib+USP22. (c, d) The migration and apoptosis of PC9 and HCC827 cells were evaluated by transwell assay and flow cytometry analysis, respectively. (e, f) The oxidized C11‐BODIPY fluorescence intensity in PC9 and HCC827 cells was examined by C11‐BODIPY staining. (g–i) The levels of MDA, Fe 2+ , and glutathione (GSH )in PC9 and HCC827 cells were examined by relevant commercial kits. * p < 0.05.

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Ubiquitin Proteomics, Over Expression, Western Blot, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry, Fluorescence, Staining

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) stabilized murine double minute 2 (MDM2) and modulated MDM2 expression. (a) Ubibrowser software predicted the relation between USP22 and MDM2. (b) Pull‐down assay was conducted to analyze the level of MDM2 ubiquitination. (c) Co‐IP assay was performed to explore the interaction between USP22 and MDM2. (d) The expression of MDM2 in non‐small cell lung cancer (NSCLC) tissues and normal tissues was estimated through IHC assay. (e, f) The mRNA and protein levels of MDM2 in NSCLC tissues and normal tissues were measured by qRT‐PCR and western blot, respectively. (g, h) The mRNA and protein levels of MDM2 in 16HBE, PC9, and HCC827 cells were examined by qRT‐PCR and western blot, respectively. (i) After PC9 and HCC827 cells were treated with DMSO, gefitinib, gefitinib+USP22, or gefitinib+USP22 + sh‐MDM2, MDM2 protein level was measured by western blot. * p < 0.05.

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Ubiquitin Proteomics, Expressing, Software, Pull Down Assay, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Western Blot

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Murine double minute 2 (MDM2) knockdown reversed the effects of Ubiquitin‐specific peptidase 22 (USP22) overexpression on gefitinib sensitivity and ferroptosis in gefitinib‐treated NSCLC cells. (a) IC50 of gefitinib in PC9 and HCC827 cells transfected with pcDNA, USP22, or USP22 + sh‐MDM2 was estimated through CCK‐8 assay. (b–h) PC9 and HCC827 cells were treated with dimethyl sulfoxide (DMSO), gefitinib, gefitinib+USP22, gefitinib+USP22 + sh‐NC, or gefitinib+USP22 + sh‐MDM2. (b, c) The migration and apoptosis of PC9 and HCC827 cells were explored by transwell assay and flow cytometry analysis. (d, e) The oxidized C11‐BODIPY fluorescence intensity in PC9 and HCC827 cells was examined by C11‐BODIPY staining. (f–h) The levels of MDA, Fe 2+ , and glutathione (GSH) in PC9 and HCC827 cells were determined with commercial kits. * p < 0.05.

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Knockdown, Ubiquitin Proteomics, Over Expression, Transfection, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry, Fluorescence, Staining

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) enhanced gefitinib resistance of non‐small cell lung cancer (NSCLC) cells in vivo. (a) The protein levels of USP22 and murine double minute 2 (MDM2) in PBS, gefitinib, and gefitinib+USP22 treated PC9 cells were measured by western blot. (b) Xenograft tumor volume was monitored every 5 days. (c) Xenograft tumor weight was examined after 25 days. (d) The expression of USP22 and MDM2 in xenograft tumors was examined through immunohistochemistry (IHC) assay. * p < 0.05.

Article Snippet: RIBOBIO (Guangzhou, China) synthesized the short hairpin RNA (shRNA) targeting USP22 (sh‐USP22), shRNA targeting MDM2 (sh‐MDM2) and their control sh‐NC, USP22 overexpression vector (USP22) and empty control (pcDNA).

Techniques: Ubiquitin Proteomics, In Vivo, Western Blot, Expressing, Immunohistochemistry