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Figure 2. The ubiquitination of GRK2 is involved in D2R β-arrestin pathway-mediated ERK activation. (A) CTRL-KD and <t>Mdm2-KD</t> 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).

Mdm2 Shrnas, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mdm2 shrnas - by Bioz Stars, 2026-09

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1) Product Images from "Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases."

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

Journal: International journal of molecular sciences

doi: 10.3390/ijms241210031

Figure 2. 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).

Figure Legend Snippet: Figure 2. 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).

Techniques Used: Ubiquitin Proteomics, Activation Assay, Transfection, Expressing

Figure 4. 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.

Figure Legend Snippet: Figure 4. 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.

Techniques Used: Ubiquitin Proteomics, Transfection

Figure 5. 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

Figure Legend Snippet: Figure 5. 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

Techniques Used: Phospho-proteomics, Ubiquitin Proteomics, Transfection

Figure 7. 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.

Figure Legend Snippet: Figure 7. 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.

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

Ubiquitin Proteomics:

Article Title: Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases.
Article Snippet: [βarr]D2R (A135R/M140D) and FLAG-tagged, [Gprot]D2R (L125N/Y133L) and FLAG-tagged, [D80A]D2R, FLAG-D2R, and GRK2 encoding plasmids (GFP, FLAG-tagged), and GRK2-4KR, GRK2-3YF, GRK2-K220R, and Mdm2 (GFP, FLAG-tagged) were generated via polymerase chain reaction (TL988/TianLong; China).[βarr]D2R (A135R/M140D) and FLAG-tagged, [Gprot]D2R (L125N/Y133L) and FLAG-tagged, [D80A]D2R, FLAG-D2R, and GRK2 encoding plasmids (GFP, FLAG-tagged), and GRK2-4KR, GRK2-3YF, GRK2-K220R, and Mdm2 (GFP, FLAG-tagged) were generated via polymerase chain reaction (TL988/TianLong; China).. GRK2 and Mdm2 shRNAs were obtained from Santa Cruz Biotechnology.

Article Title: Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases
Article Snippet: [βarr] D2R (A135R/M140D) and FLAG-tagged, [Gprot] D2R (L125N/Y133L) and FLAG-tagged, [D80A] D2R, FLAG-D2R, and GRK2 encoding plasmids (GFP, FLAG-tagged), and GRK2-4KR, GRK2-3YF, GRK2-K220R, and Mdm2 (GFP, FLAG-tagged) were generated via polymerase chain reaction (TL988/TianLong; China).[βarr] D2R (A135R/M140D) and FLAG-tagged, [Gprot] D2R (L125N/Y133L) and FLAG-tagged, [D80A] D2R, FLAG-D2R, and GRK2 encoding plasmids (GFP, FLAG-tagged), and GRK2-4KR, GRK2-3YF, GRK2-K220R, and Mdm2 (GFP, FLAG-tagged) were generated via polymerase chain reaction (TL988/TianLong; China).. GRK2 and Mdm2 shRNAs were obtained from Santa Cruz Biotechnology.

Activation Assay:

Transfection:

Expressing:

Phospho-proteomics:

Clinical Proteomics:

Membrane:

Transduction:

Co-Immunoprecipitation Assay:

Immunoprecipitation:

Western Blot:

Incubation:

Fractionation:

Image Search Results

Identification of METTL3 targets in podocytes. (A) Motif analysis of METTL3‐binding sites from RIP‐seq in AGEs‐treated podocytes and AGEs‐treated podocytes transfected with siMETTL3. (B) Peak distribution of gene regions in METTL3‐modified transcripts. (C) GO analysis of genes modified by METTL3. (D) M6A abundances in MDM2 transcripts in AGEs‐treated podocytes and AGEs‐treated podocytes transfected with siMETTL3. (E) Enriched m6A modification of MDM2 in BSA‐treated, AGEs‐treated and AGEs‐treated podocytes transfected with siMETTL3, determined by MeRIP‐qPCR assay. (F, G) Protein levels of MDM2 in WT‐Ctr, WT‐STZ, cKO‐Ctr, and cKO‐STZ groups, analysed by western blot with semi‐quantitative analysis. (H) MDM2 mRNA levels in WT‐Ctr, WT‐STZ, cKO‐Ctr and cKO‐STZ groups, determined by RT‐qPCR. (I) Protein levels of MDM2 in WT‐Ctr, WT‐STZ, cKO‐Ctr and cKO‐STZ groups, analysed by IHC assay; scale bar = 50 μm. (J, K) Direct interaction between MDM2 and METTL3 in podocytes, demonstrated by RIP‐PCR assay. (L) Mutations at the two putative m6A sites in MDM2 (A–G). (M) m6A levels of MDM2 in podocytes treated with AGEs, co‐expressing siMETTL3 and MDM2‐WT/Mutants, determined by MeRIP‐qPCR. Data represent mean ± SD of three independent experiments. ** p < 0.01 versus AGEs group (E), or WT‐STZ group (G) by one‐way ANOVA; ** p < 0.01 versus IgG group (K) by Student's t ‐test; ## p < 0.01 versus AGEs‐WT group; ++ p < 0.01 versus AGEs+siMETTL3‐WT group; ** p < 0.01 versus AGEs group by two‐way ANOVA.

Journal: Journal of Cellular and Molecular Medicine

Article Title: The Impact of METTL3 on MDM2 Promotes Podocytes Injury During Diabetic Kidney Disease

doi: 10.1111/jcmm.70627

Figure Lengend Snippet: Identification of METTL3 targets in podocytes. (A) Motif analysis of METTL3‐binding sites from RIP‐seq in AGEs‐treated podocytes and AGEs‐treated podocytes transfected with siMETTL3. (B) Peak distribution of gene regions in METTL3‐modified transcripts. (C) GO analysis of genes modified by METTL3. (D) M6A abundances in MDM2 transcripts in AGEs‐treated podocytes and AGEs‐treated podocytes transfected with siMETTL3. (E) Enriched m6A modification of MDM2 in BSA‐treated, AGEs‐treated and AGEs‐treated podocytes transfected with siMETTL3, determined by MeRIP‐qPCR assay. (F, G) Protein levels of MDM2 in WT‐Ctr, WT‐STZ, cKO‐Ctr, and cKO‐STZ groups, analysed by western blot with semi‐quantitative analysis. (H) MDM2 mRNA levels in WT‐Ctr, WT‐STZ, cKO‐Ctr and cKO‐STZ groups, determined by RT‐qPCR. (I) Protein levels of MDM2 in WT‐Ctr, WT‐STZ, cKO‐Ctr and cKO‐STZ groups, analysed by IHC assay; scale bar = 50 μm. (J, K) Direct interaction between MDM2 and METTL3 in podocytes, demonstrated by RIP‐PCR assay. (L) Mutations at the two putative m6A sites in MDM2 (A–G). (M) m6A levels of MDM2 in podocytes treated with AGEs, co‐expressing siMETTL3 and MDM2‐WT/Mutants, determined by MeRIP‐qPCR. Data represent mean ± SD of three independent experiments. ** p < 0.01 versus AGEs group (E), or WT‐STZ group (G) by one‐way ANOVA; ** p < 0.01 versus IgG group (K) by Student's t ‐test; ## p < 0.01 versus AGEs‐WT group; ++ p < 0.01 versus AGEs+siMETTL3‐WT group; ** p < 0.01 versus AGEs group by two‐way ANOVA.

Article Snippet: For targeted gene knockdown, an adeno‐associated virus (AAV9) carrying shRNA targeting MDM2 (shMDM2), under control of the nephrin promoter, was procured from GeneChem Company (Shanghai, China).

Techniques: Binding Assay, Transfection, Modification, Western Blot, Quantitative RT-PCR, Expressing

IGF2BP2 identifies m6A‐meditated MDM2 mRNA and participates in MDM2 mRNA stability. (A, B) Protein levels of IGF2BP2 in control and STZ‐treated groups, analysed by western blot with semi‐quantitative analysis. (C) Protein levels of IGF2BP2 in control and STZ‐treated groups, determined by IHC assay; scale bar = 50 μm. (D) MDM2 mRNA levels in podocytes treated with actinomycin D in AGEs‐treated and AGEs‐treated podocytes transfected with siIGF2BP2, determined by RT‐qPCR. (E, F) Direct interaction between MDM2 and IGF2BP2 in podocytes, demonstrated by RIP‐PCR assay. (G, H) Relative luciferase activity of the MDM2‐WT or MDM2‐Mut 3′UTR luciferase reporter in AGEs‐treated and AGEs‐treated podocytes transfected with siIGF2BP2. Data represent mean ± SD of three independent experiments. ** p < 0.01 versus STZ group (B), AGEs group (H), or IgG group (F) by Student's t‐test.

Journal: Journal of Cellular and Molecular Medicine

Article Title: The Impact of METTL3 on MDM2 Promotes Podocytes Injury During Diabetic Kidney Disease

doi: 10.1111/jcmm.70627

Figure Lengend Snippet: IGF2BP2 identifies m6A‐meditated MDM2 mRNA and participates in MDM2 mRNA stability. (A, B) Protein levels of IGF2BP2 in control and STZ‐treated groups, analysed by western blot with semi‐quantitative analysis. (C) Protein levels of IGF2BP2 in control and STZ‐treated groups, determined by IHC assay; scale bar = 50 μm. (D) MDM2 mRNA levels in podocytes treated with actinomycin D in AGEs‐treated and AGEs‐treated podocytes transfected with siIGF2BP2, determined by RT‐qPCR. (E, F) Direct interaction between MDM2 and IGF2BP2 in podocytes, demonstrated by RIP‐PCR assay. (G, H) Relative luciferase activity of the MDM2‐WT or MDM2‐Mut 3′UTR luciferase reporter in AGEs‐treated and AGEs‐treated podocytes transfected with siIGF2BP2. Data represent mean ± SD of three independent experiments. ** p < 0.01 versus STZ group (B), AGEs group (H), or IgG group (F) by Student's t‐test.

Techniques: Control, Western Blot, Transfection, Quantitative RT-PCR, Luciferase, Activity Assay

Inhibiting podocytes MDM2 expression alleviates renal pathological damage, and reduces podocytes dedifferentiation levels. (A) Representative images showing PAS and Masson staining (scale bar = 20 μm) in control, STZ, and shMDM2‐injected STZ groups. (B) IF staining of Ki‐67 (red), Cyclin B1 (green), Podocin (yellow), and MDM2 (pink), counterstained with DAPI (blue) in control, STZ and STZ injected with shMDM2 groups. (C) Protein levels of p21, Cyclin B1 and Podocin in control, STZ and STZ injected with shMDM2 groups, analysed by western blot. Data represent mean ± SD of three independent experiments.

Journal: Journal of Cellular and Molecular Medicine

Article Title: The Impact of METTL3 on MDM2 Promotes Podocytes Injury During Diabetic Kidney Disease

doi: 10.1111/jcmm.70627

Figure Lengend Snippet: Inhibiting podocytes MDM2 expression alleviates renal pathological damage, and reduces podocytes dedifferentiation levels. (A) Representative images showing PAS and Masson staining (scale bar = 20 μm) in control, STZ, and shMDM2‐injected STZ groups. (B) IF staining of Ki‐67 (red), Cyclin B1 (green), Podocin (yellow), and MDM2 (pink), counterstained with DAPI (blue) in control, STZ and STZ injected with shMDM2 groups. (C) Protein levels of p21, Cyclin B1 and Podocin in control, STZ and STZ injected with shMDM2 groups, analysed by western blot. Data represent mean ± SD of three independent experiments.

Techniques: Expressing, Staining, Control, Injection, Western Blot

MDM2 regulates the Notch1 signalling pathway in podocytes during AGE‐induced abnormal cell cycle regulation. (A, B) Western blot analysis and semi‐quantitative assessment of NICD and Hes1 protein levels in BSA‐treated, AGE‐treated, and siMDM2‐transfected AGE‐treated groups. (C, D) Western blot analysis and semi‐quantitative assessment of NICD and Hes1 protein levels in control, STZ‐treated, and shMDM2‐injected STZ‐treated groups. (E) Evaluation of NICD and Hes1 protein levels in control, STZ‐treated and shMDM2‐injected STZ‐treated groups by IHC assay (scale bar = 50 μm). (F, G) Western blot analysis and semi‐quantitative assessment of Hes1, Cyclin B1 and p‐H3 protein levels in AGE‐treated, AGE + siMDM2‐treated, and Jagged1‐treated AGE + siMDM2 groups. Data represent mean ± SD of three independent experiments. ** p < 0.01 versus AGE group (B), or STZ group (D), or AGE + siMDM2 group (G) by one‐way ANOVA.

Journal: Journal of Cellular and Molecular Medicine

Article Title: The Impact of METTL3 on MDM2 Promotes Podocytes Injury During Diabetic Kidney Disease

doi: 10.1111/jcmm.70627

Figure Lengend Snippet: MDM2 regulates the Notch1 signalling pathway in podocytes during AGE‐induced abnormal cell cycle regulation. (A, B) Western blot analysis and semi‐quantitative assessment of NICD and Hes1 protein levels in BSA‐treated, AGE‐treated, and siMDM2‐transfected AGE‐treated groups. (C, D) Western blot analysis and semi‐quantitative assessment of NICD and Hes1 protein levels in control, STZ‐treated, and shMDM2‐injected STZ‐treated groups. (E) Evaluation of NICD and Hes1 protein levels in control, STZ‐treated and shMDM2‐injected STZ‐treated groups by IHC assay (scale bar = 50 μm). (F, G) Western blot analysis and semi‐quantitative assessment of Hes1, Cyclin B1 and p‐H3 protein levels in AGE‐treated, AGE + siMDM2‐treated, and Jagged1‐treated AGE + siMDM2 groups. Data represent mean ± SD of three independent experiments. ** p < 0.01 versus AGE group (B), or STZ group (D), or AGE + siMDM2 group (G) by one‐way ANOVA.

Techniques: Western Blot, Transfection, Control, Injection

Primers sequences used for polymerase chain reaction (PCR).

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

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.

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.

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

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.

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.

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

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.

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.

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

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.

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.

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

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.

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.

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

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.

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.

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

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.

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: For the single Mdm2 knockdown, HT1080 p53KO cells were transduced with lentivirus carrying a pool of shRNAs against Mdm2 (4 different shRNAs purchased from OriGene, cat.#TL311529) or shScramble sequence as control (OriGene, cat.#TR30021).

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

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.

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.

Techniques: Transfection, Control, Stable Transfection, Expressing, shRNA, Whisker Assay

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.

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.

Techniques: Immunofluorescence, Staining, Control

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.

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.

Techniques: Transfection, Mass Spectrometry, Expressing, Knockdown, Western Blot, Control, Immunoprecipitation, Immunofluorescence, Staining

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.

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.

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

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.

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.

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

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.

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.

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: Figure 2. 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: Figure 4. 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.

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

Techniques: Ubiquitin Proteomics, Transfection

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: Figure 5. 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

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

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Transfection

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: Figure 7. 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

Review

mdm2 shrnas (Santa Cruz Biotechnology)

Structured Review

Images

1) Product Images from "Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases."

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Western Blot:

Incubation:

Fractionation:

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