Journal: Nature Communications

Article Title: Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53

doi: 10.1038/s41467-024-51488-2

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 ( n = 6 samples) as indicated or ( B ) treatment with MEL23 (7 µM) for 24 h ( n = 4 samples). α-tubulin was used as a 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, n = 3 groups. E , F Representative images of the 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, n = 3 groups. Cells stained for actin (orange) and DNA via DAPI (blue). The cell area was quantified and plotted. Graphs show all three independent experimental replicates combined, for each independent experimental replicate a minimum of 100 cells were quantified summing a total of at least 300 cells/condition. G , H Quantification and representative micrographs showing attachment to different ECM components of cells ( G ) silenced for Mdm2, n = 3 samples or ( H ) treated with MEL23 for 24 h, n = 4 samples. The graphs shown represent the mean ± SD of independent experimental replicates. More details about the statistical tests used can be found in the Source Data file.

Article Snippet: Stable cell lines were established 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: Nature Communications

Article Title: Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53

doi: 10.1038/s41467-024-51488-2

Figure Lengend Snippet: A , B Tumor spheroid invasion in collagen matrix after ( A ) transfection of HT1080 p53KO cells with siRNAs against Mdm2 or ( B ) treatment with MEL23. Representative images above and quantification below of the area invaded and the number of cells invading 24 h after implantation. C , D Tumor spheroid invasion in the collagen-BME matrix after ( C ) transfection of HT1080 p53KO cells with siRNAs against Mdm2 or ( D ) treatment with MEL23. Representative images and quantification of the area invaded 24 h after implantation. Graphs show three pooled independent experimental replicates, the total number of spheroids in each condition is: siCtrl = 10, siMdm2#1 = 15, siMdm2#2 = 12 in panel ( A ); DMSO = 25, MEL23 = 24 in panel ( B ); siCtrl = 12, siMdm2#1 = 14, siMdm2#2 = 15 in panel ( C ), and DMSO = 8, MEL23 = 9 in panel ( D ). 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 ), n = 3 groups. The graph represents the average fold change of more circular morphology in Mdm2 silenced cells compared to control in three independent experimental replicates. More details about the quantification can be found in the method’s session. G – I HT1080 p53KO cells stably expressing shRNA scramble (shScramble) or a pool of Mdm2 shRNAs (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 a loading control. H , I Representative images above and quantification below of metastatic foci in the lungs after implantation of shScramble or shMdm2 cells using ( H ) orthotropic model, n = 4 mice/group or ( I ) tail-vein model, n = 8 mice/group. In all box and whisker plots the boxes extend from 25 to 75 percentiles and whiskers show min and max values, The line in the center of each box represents the median. Graphs shown in panels ( F , H , I ) represent the mean ± SD of independent experimental replicates. More details about the statistical tests used can be found in the Source Data file.

Article Snippet: Stable cell lines were established 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, Stable Transfection, Expressing, shRNA, Whisker Assay

Journal: Nature Communications

Article Title: Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53

doi: 10.1038/s41467-024-51488-2

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), cell surface was outlined by phalloidin staining (green), and nuclei (blue) detected by DAPI staining of DNA. Representative images are shown above, with quantification of FA parameters below. The graphs shown represent the mean ± SD of three independent experimental replicates. For each independent experimental replicate all parameters were quantified in at least 20 events/condition for a total of at least 60 events/condition. 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 a loading control, n = 3 samples. More details about the statistical tests used can be found in the Source Data file.

Article Snippet: Stable cell lines were established 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: Nature Communications

Article Title: Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53

doi: 10.1038/s41467-024-51488-2

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 proteins 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 the left of the 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, n = 3 samples. Black arrows point to the location of Spry4 in each Volcano plot. B Spry4 expression in HT1080 p53KO cells in response to Mdm2 knockdown or treatment with MEL23 for 24 h by immunoblotting. β-actin and α-tubulin were used as loading control for immunoblot, n = 6 samples. C Co-immunoprecipitation of Mdm2 and Spry4 in the presence or absence of MG132. β-actin was used as a loading control. Mdm2 was pulled down by using either a mix of antibodies against Mdm2 (4B11, 3G5, and 2A9) that recognize different domains within the protein, this condition was called “mix”, or by using a single monoclonal antibody D1V2Z, this condition was called “DIV”, n = 3 samples. D Quantification of Spry4, Mdm2, and MdmX protein levels after treatment with MG132 (or vehicle, DMSO) for 4 h, n = 3 samples. E Spry4 mRNA levels in response to Mdm2 knockdown ( n = 5 samples) or treatment with MEL23 for 24 h ( n = 4 samples). RPL32 was used as housekeeping. F Localization of Spry4 in HT1080 p53KO cells in response to Mdm2 knockdown. Immunofluorescence showed Spry4 staining (green), the cell surface was outlined by phalloidin staining (orange), and nuclei (blue) were detected by DAPI staining of DNA, n = 3 groups. Graphs shown in ( D , E ) represent the mean ± SD of independent experimental replicates. More details about the statistical tests used can be found in the Source Data file.

Article Snippet: Stable cell lines were established 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, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53

doi: 10.1038/s41467-024-51488-2

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 a loading control, n = 4 samples. B Cell migration assay. Representative images (top) and quantification (bottom) of wound scratch migration assay, n = 3 groups. C Quantification and representative micrographs showing attachment to ECM component, collagen I, n = 3 samples. D Immunofluorescence showing FA foci by vinculin staining (red), cell surface was outlined by phalloidin staining (green), and nuclei (blue) detected by DAPI staining of DNA, n = 3 groups. Representative images are shown on the left, and the quantification of FA parameters is shown on the right. E Protein levels of Spry4 as well as Mdm2, MdmX, and p53 after Mdm2 silencing using siRNAs. β-actin was used as a loading control, n = 4 samples. F mRNA levels of Spry4 after Mdm2 silencing using siRNAs, n = 3 samples. RPL32 was used as housekeeping. Graphs shown represent the mean ± SD of independent experimental replicates. More details about the statistical tests used can be found in the Source Data file.

Article Snippet: Stable cell lines were established 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: Nature Communications

Article Title: Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53

doi: 10.1038/s41467-024-51488-2

Figure Lengend Snippet: A – D 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, n = 3 samples. β-actin was used as a loading control. B Quantification of wound scratch migration assay in cells treated with the indicated siRNAs as in Fig. , n = 3 samples. C Quantification of cell area after attachment to collagen-coated coverslips as in Fig. , n = 3 samples. D Immunofluorescence showing FA foci by vinculin staining (red), cell surface was outlined by phalloidin staining (green), and nuclei (blue) as detected by DAPI staining, n = 3 groups. Representative images are shown on the left, and the quantification of FA parameters is shown on the right. In ( C , D ) the graphs shown represent mean ± SD of independent experimental replicates and in each replicate all parameters were quantified in at least 20 events/condition for total of at least 60 events/condition. E , F H1299 cells were transfected with siRNA against Mdm2 alone or both siRNA against Mdm2 and a pool of Spry4 siRNAs. E Protein levels of Mdm2, MdmX, and Spry4 after transfection with indicated siRNAs. β-actin was used as a loading control, n = 3 samples. F Quantification of wound scratch migration assay comparing migration into wound scratches in cells treated with the indicated siRNAs as in Fig. , n = 3 samples. G , H HT1080 p53KO cell lines were established stably expressing a pool of shRNAs against Mdm2 alone or Mdm2 and Spry4 together. G Protein levels of Mdm2, MdmX, and Spry4 in stable cell lines. β-actin was used as a loading control, n = 3 samples. H Analysis of metastatic burden in vivo using tail-vein injection model as in Fig. H, . Representative images above and quantification below of metastatic foci in the lungs after 8 weeks of injection, n = 7 mice/group. The graphs shown represent the mean ± SD of independent experimental replicates. More details about the statistical tests used can be found in the Source Data file.

Article Snippet: Stable cell lines were established 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, Stable Transfection, Expressing, In Vivo, Injection

Journal: Nature Communications

Article Title: Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53

doi: 10.1038/s41467-024-51488-2

Figure Lengend Snippet: A , B Effect of Spry4 modulation in the phosphorylation of ERKs in HT1080 p53KO cells. Protein levels of phospho-ERK and total ERK levels ( A ) after Spry4 silencing using a pool of siRNAs and ( B ) in response to silencing of Mdm2 alone or Mdm2 and Spry4 together. C , D RhoA modulation in HT1080 p53KO cells transfected with siRNAs against Mdm2 or treated with MEL23. C Protein levels and ( D ) mRNA levels of RhoA after transfection with indicated siRNAs ( n = 3 samples) or treatment with MEL23 ( n = 3 samples) for 24 h. β-actin was used as a loading control for immunoblots. RPL32 was used as a housekeeping control for qPCR. E Immunoprecipitation of Mdm2 in the presence of MG132. Lysates were probed for the presence of RhoA, and MdmX was used as a positive control. Mdm2 was pulled down by using a mix of antibodies against Mdm2 that recognize different domains within the protein. F RhoA protein levels in HT1080 p53KO cells transfected with siRNAs against Mdm2 alone or against Mdm2 and Spry4. α-tubulin was used as a loading control. G Quantification of wound scratch migration assay comparing cells transfected with siRNAs against Mdm2 alone or against Mdm2 and a pool of Spry4 siRNAs in the presence or absence of the RhoA inhibitor Rhosin (50 µM) for 24 h. The graph represents mean ± SD, n = 3 technical replicates. The graph for the other two independent experimental replicates can be found in the supplementary material. H Immunoblot 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. I Immunofluorescence showing F- (red) and G-actin (green) staining. Nuclei (blue) as detected by DAPI staining, n = 3 groups. Representative images (left) and quantification of F/G ratio (right). Graph represents mean ± SD of independent experimental replicates, in each replicate the F/G-actin ratio was quantified in at least 30 events/condition for a total of at least 90 events/condition. More details about the statistical tests used can be found in the Source Data file.

Article Snippet: Stable cell lines were established 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: Phospho-proteomics, Transfection, Control, Western Blot, Immunoprecipitation, Positive Control, Migration, Immunofluorescence, Staining

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: 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