Journal: Cell Death & Disease

Article Title: USP7 mediates pathological hepatic de novo lipogenesis through promoting stabilization and transcription of ZNF638

doi: 10.1038/s41419-020-03075-8

Figure Lengend Snippet: A , B ZNF638 protein level was down-regulated with the treatment of P22077 in SK-Hep1 and Huh-7 cells. C The knockdown efficiency of USP7 by two specific SgRNA (Sg1, Sg2) and their effects on ZNF638 expression were determined by immunoblotting in SK-Hep1 cells; proteasome inhibitor Bortezomib (100 nM 8 h) partially rescued declined expression of ZNF638 in USP7-deficient cells. D P22077 (20 μM 24 h) induced ZNF638 protein inhibition was partially rescued by Bortezomib (100 nM 8 h) in SK-Hep1 and Huh-7 cells. E The half-life of ZNF638 protein in normal and USP7-deficient SK-Hep1 cells was determined by using CHX (100 μg/ml) at indicated time points. F Genetic inhibition of USP7 in SK-Hep1 cells increased ubiquitination level of ZNF638. G The enhanced polyubiquitination of ZNF638 in SK-Hep1 cells according to USP7 knockdown was mainly Lys48 but not Lys 63 linked polyubiquitination. H K48-resistant ubiquitin (Lys48 to Arg48) could reverse ZNF638 expression in USP7-deficient SK-Hep1 cells. I ZNF638 and USP7 were endogenously interacted with each other in SK-Hep1 cells as determined by Co-IP assay. J USP7 and ZNF638 were mainly co-localized in cell nucleus as determined by immunofluorescence in Huh-7 and SK-Hep1 cells. K Schematic illustration of the protein structure of USP7. L Three of flag tagged truncated mutant plasmids (Flag-TRAF, Flag-CD, and Flag-HUBL) and wild-type USP7 (Flag-USP7) were transfected into SK-Hep1 cells, followed by immunoprecipitation using flag antibody and immunoblotting with ZNF638 antibody. Each immunoblotting assay was performed at least three times from independent studies.

Article Snippet: For the wound healing test, cells with stable ZNF638/USP7 knockdown, P22077 (Selleck, 10 μM) or C75 (C5490, Sigma, 2.5 mg/ml) treatment were pre-scratched with sterilized 10 μl pipette tips and cultured with or without fructose (8 mM) for 24 h in 6-well plates.

Techniques: Knockdown, Expressing, Western Blot, Inhibition, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Immunofluorescence, Mutagenesis, Transfection, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: USP7 mediates pathological hepatic de novo lipogenesis through promoting stabilization and transcription of ZNF638

doi: 10.1038/s41419-020-03075-8

Figure Lengend Snippet: A P22077 treatment caused concentration-dependent reduction of ZNF638 mRNA in SK-Hep1 cells and Huh-7 cells. B USP7 knockdown or overexpression resulted in paralleled decrease or increase of ZNF638 mRNA in SK-Hep1 cells. C The mRNA of USP7 and ZNF638 were positively correlated in HCC patients based on the data from TCGA; the correlation Rho values differed among the different BMI and etiology groups. D CREB protein was suppressed via 20 μM of P22077 induction in SK-Hep1 cells. E CREB protein was reduced in USP7-deficient SK-Hep1 cells, while proteasome inhibitor Bortezomib (100 nM 8 h) rescued the effects. F Overexpression of USP7 in SK-Hep1 cells resulted in the enhanced protein level of CREB. G Flag tagged USP7 wild-type, TRAF domain, CD domain, HUBL domain were transfected in SK-Hep1 cells, followed by immunoprecipitation to determine the binding site of USP7 to CREB. H The half-life of CREB protein in normal and USP7-deficient SK-Hep1 cells was determined by using CHX (100 μg/ml) at indicated time intervals. I The mRNA levels of CREB in SK-Hep1 cells were unchanged neither by USP7 knockdown nor overexpression. The representative statistical results were performed using one-way ANOVA or unpaired t- test and shown as means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the wound healing test, cells with stable ZNF638/USP7 knockdown, P22077 (Selleck, 10 μM) or C75 (C5490, Sigma, 2.5 mg/ml) treatment were pre-scratched with sterilized 10 μl pipette tips and cultured with or without fructose (8 mM) for 24 h in 6-well plates.

Techniques: Concentration Assay, Knockdown, Over Expression, Transfection, Immunoprecipitation, Binding Assay

Journal: Cell Death & Disease

Article Title: USP7 mediates pathological hepatic de novo lipogenesis through promoting stabilization and transcription of ZNF638

doi: 10.1038/s41419-020-03075-8

Figure Lengend Snippet: A Grouped SK-Hep1 cells as normal, USP7 knockdown, ZNF638 knockdown, and P22077 induction (10 μM pretreated for 24 h) were treated with or without palmitic acid (0.5 mM) for 8 h and applied to oil-red o assay; quantitative analysis of the staining area was revealed by ImageJ software. B The relative TG content of SK-Hep1 cells in different groups were measured by TG kit. C The alterations of USP7 and ZNF638 protein levels after PA (0.5 mM) induction at different time intervals in SK-Hep1 cells were examined by immunoblotting; quantification of protein levels relative to GAPDH was performed using ImageJ. D The alterations of USP7 and ZNF638 mRNA levels upon PA induction at different time intervals were examined by RT-PCR. E Grouped SK-Hep1 cells were treated with or without fructose (8 mM) for 48 h and applied to oil-red o assay; quantitative analysis of the staining area was revealed by ImageJ software. F The relative TG content of cells in different groups was measured by TG kit. G The alterations of USP7 and ZNF638 protein levels within different fructose inducing were assessed by immunoblotting and quantified by ImageJ. H The alterations of USP7 and ZNF638 mRNA levels upon different fructose induction were detected by RT-PCR. Representative statistical results were performed using one or two-way ANOVA test and shown as means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the wound healing test, cells with stable ZNF638/USP7 knockdown, P22077 (Selleck, 10 μM) or C75 (C5490, Sigma, 2.5 mg/ml) treatment were pre-scratched with sterilized 10 μl pipette tips and cultured with or without fructose (8 mM) for 24 h in 6-well plates.

Techniques: Knockdown, Staining, Software, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: USP7 mediates pathological hepatic de novo lipogenesis through promoting stabilization and transcription of ZNF638

doi: 10.1038/s41419-020-03075-8

Figure Lengend Snippet: A The relative mRNA levels of ACACA, FASN, SCD in normal, USP7-deficient, ZNF638-deficient SK-Hep1 cells were determined by real-time PCR. B The protein levels of ACACA, FASN, SCD and SREBP1C in normal, USP7-deficient, ZNF638-deficient SK-Hep1 cells were assessed by immunoblotting. C Pharmacological inhibition of USP7 reduced protein levels of ACACA, FASN, and SCD in SK-Hep1 cells. D The promoter activity of ACACA, FASN, and SCD relating to ZNF638, USP7 knockdown or P22077 treatment was determined by luciferase reporter assay. The representative statistical results were performed using one-way ANOVA test and shown as means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the wound healing test, cells with stable ZNF638/USP7 knockdown, P22077 (Selleck, 10 μM) or C75 (C5490, Sigma, 2.5 mg/ml) treatment were pre-scratched with sterilized 10 μl pipette tips and cultured with or without fructose (8 mM) for 24 h in 6-well plates.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Inhibition, Activity Assay, Knockdown, Luciferase, Reporter Assay

Journal: Cell Death & Disease

Article Title: USP7 mediates pathological hepatic de novo lipogenesis through promoting stabilization and transcription of ZNF638

doi: 10.1038/s41419-020-03075-8

Figure Lengend Snippet: A , B C57BL/6 mice were fed with standard chow diet or diet added with 30% fructose in drinking water. The fructose group mice were subclassified and disposed to intraperitoneal injection of P22077 (10 mg/kg BW) and GalNAc-ZNF638-SiRNA (2 μg/g BW) every week. The body weight of the mice was weighed every week, and after 4 weeks induction, the liver weight and the TG content of the liver were assessed. C The pathological alterations among different groups were detected by HE staining using paraffin sections; the liver lipids accumulation was determined by oil-red o assay; the expression of USP7, ZNF638, ACACA, FASN, SCD, and SREBP1C among different groups were observed by immunochemistry. D Alterations of USP7, ZNF638, ACACA, FASN, SCD, and cleaved-SREBP1C among different groups were determined by immunoblotting, followed by quantitative analysis. The representative statistical results were performed using one-way ANOVA test and shown as means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the wound healing test, cells with stable ZNF638/USP7 knockdown, P22077 (Selleck, 10 μM) or C75 (C5490, Sigma, 2.5 mg/ml) treatment were pre-scratched with sterilized 10 μl pipette tips and cultured with or without fructose (8 mM) for 24 h in 6-well plates.

Techniques: Injection, Staining, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: USP7 mediates pathological hepatic de novo lipogenesis through promoting stabilization and transcription of ZNF638

doi: 10.1038/s41419-020-03075-8

Figure Lengend Snippet: A SK-Hep1 cells with or without stable ZNF638 knockdown, stable USP7 knockdown, P22077 treatment (5 μM), and C75 treatment (1 μg/ml) were cultured with or without fructose (8 mM) for 14 days; after that, the cell clones were harvested and calculated by ImageJ. B Seventy percent of different SK-Hep1 cells (ZNF638 knockdown, USP7 knockdown, treatment of 5 μM P22077, treatment of 1 μg/ml C75) and 30% of LXR cells were mixed and glued with Collagen I (5 μg/ml); the culture medium with or without fructose (8 mM) was added with 20 ng/ml b-FGF, 20 ng/ml HGF to promote the growth of both cell lines. At the time points of 3 days and 5 days, organoids volumes of different groups were analyzed. C SK-Hep1 cells with or without stable ZNF638 knockdown, stable USP7 knockdown, P22077 treatment (10 μM), and C75 treatment (2.5 μg/ml) were pre-scratched and cultured with or without fructose (8 mM) for 24 h, the migration area (area% of per field of view) of different groups was evaluated by ImageJ. D SK-Hep1 cells with or without stable ZNF638 knockdown, stable USP7 knockdown, P22077 treatment (10 μM), and C75 treatment (2.5 μg/ml) were cultured with or without fructose (8 mM) for 48 h in transwell chambers; the invaded cells were calculated and analyzed by ImageJ. E The overall survival difference between high and low expression of USP7 or ZNF638 in HCC patients was analyzed using the transcriptome data from TCGA; the cutoff value was computed by survminer R package. F The discrepancy in the ratio of HCC patients with different USP7/ZNF638 expression from different etiologies was analyzed based on the data from TCGA. G USP7 and ZNF638 expression were evaluated in four paired HCC paraffin sections (2 of steatosis, 2 of non-steatosis) by immunochemistry. The representative statistical results were performed using one- or two-way ANOVA test and shown as means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the wound healing test, cells with stable ZNF638/USP7 knockdown, P22077 (Selleck, 10 μM) or C75 (C5490, Sigma, 2.5 mg/ml) treatment were pre-scratched with sterilized 10 μl pipette tips and cultured with or without fructose (8 mM) for 24 h in 6-well plates.

Techniques: Knockdown, Cell Culture, Clone Assay, Migration, Expressing

Journal: bioRxiv

Article Title: HAUSP Stabilizes SOX2 through Deubiquitination to Maintain Self-renewal and Tumorigenic Potential of Glioma Stem Cells

doi: 10.1101/2021.06.09.447550

Figure Lengend Snippet: (A) Representative images of tumorspheres derived from GSCs treated with the HAUSP inhibitor P22077 or vehicle control (DMSO). T387 GSCs were treated with P22077 (10 µM or 20 µM) or DMSO for 7 days. Pharmacological inhibition of HAUSP by P22077 reduced GSC tumorsphere formation in a dose-dependent manner. Scale bar, 180 µm. (B) Immunoblot analyses of cleaved PARP, total PARP, HAUSP, SOX2 and GAPDH (loading control) in GSCs treated with the increased doses of the HAUSP inhibitor P22077. T387 GSCs were treated with indicated doses (0, 5, 10, 15, 20 µM) of P22077 for 48 hours and then harvested for the immunoblot analyses. ( C and D ) In vivo bioluminescent imaging (IVIS) of GSC-derived orthotopic xenografts in immunocompromised mice treated with the HAUSP inhibitor P22077 or vehicle control. 7 days after GSC transplantation, the mice were treated with P22077 (20 mg/kg/daily) or vehicle control (DMSO) through tail vein injection. Representative IVIS images ( C ) on day 7 (before treatment) and day 20 (after treatment) are shown. Quantifications of luciferase intensities ( D ) shows that treatment with P22077 significantly inhibited GBM tumor growth in mouse brains. Data are means ± SD. Student’s t test was used to assess the significance. n =5 mice/group. Ns, no significant difference; ***, P < 0.001. ( E ) Kaplan-Meier survival curves of mice intracranially transplanted with GSCs (T387) and treated with P22077 or vehicle control (DMSO). n =5 mice/group. Log-rank analysis was used. **, p<0.01. ( F and G ) Immunofluorescent staining of the GSC marker SOX2 in GSC-derived xenografts treated with the HAUSP inhibitor P22077 or vehicle control. Tumor sections were immunostained with a specific antibody against SOX2 (in red) and counterstained with DAPI (blue) to mark nuclei ( F ). Quantification of SOX2+ cells ( G ) indicated that inhibiting HAUSP by P22077 significantly reduced GSC population. Scale bar, 20 µm. Student’s t test was used to assess the significance. Data are means ± SD. n =3 tumors (200 cells per arm). ***, p<0.001. ( H and I ) Immunofluorescent staining of the cell apoptotic marker cleaved caspase 3 in GSC-derived xenografts treated with the HAUSP inhibitor P22077 or vehicle control. Tumor sections were immunostained with a specific antibody against cleaved caspase 3 (in green) and counterstained with DAPI (blue) to mark nuclei ( H ). Quantification of cleaved caspase 3 intensity ( I ) indicated that inhibition of HAUSP with P22077 significantly promoted cell apoptosis in GBM tumors. Scale bar, 20 µm. Student’s t test was used to assess significance. Data are means ± SD. n =3 tumors (200 cells per arm). ***, p<0.001. ( J and K ) Immunofluorescent staining of the cell proliferation marker Ki67 in GSC-derived xenografts treated with P22077 or vehicle control (DMSO). Tumor sections were immunostained with a specific antibody against Ki67 (in red) and counterstained with DAPI (blue) to mark nuclei ( J ). Quantifications of Ki67 signal ( K ) indicated that targeting HAUSP by P22077 significantly reduced cell proliferation in GBM xenografts. Scale bar, 20 µm. Student’s t test was used to assess significance. Data are means ± SD. n =3 tumors (200 cells per arm). **, p<0.001.

Article Snippet: To examine the effect of HAUSP inhibition with P22077 (BOC Sciences, B0084-462597) on tumor growth, mice were treated with P22077 (20mg/Kg/daily) or vehicle control through tail vein injection.

Techniques: Derivative Assay, Control, Inhibition, Western Blot, In Vivo, Imaging, Transplantation Assay, Injection, Luciferase, Staining, Marker

Journal: bioRxiv

Article Title: HAUSP Stabilizes SOX2 through Deubiquitination to Maintain Self-renewal and Tumorigenic Potential of Glioma Stem Cells

doi: 10.1101/2021.06.09.447550

Figure Lengend Snippet: (A) A schematic illustration showing the treatment schedule. GSCs (T4121) expressing luciferase were transplanted into brains of immunocompromised NSG mice. 7 days after GSC transplantation, mice were treated with the HAUSP inhibitor P22077 (20 mg/kg/daily) or DMSO (control) through tail vein injection in combination with or without irradiation (IR). IR (2 x 2 Gy) was performed on mouse heads on Day 9 after GSC transplantation in two groups of mice. (B) Representative In vivo bioluminescent imaging (IVIS) images of intracranial GBM xenografts in the four groups of mice (Control, IR, P22077, IR plus P222077) on Day 7 (before treatment) and Day 20 (after treatment) after GSC transplantation are shown. (C) Quantifications of luciferase intensities of intracranial GBM xenografts in the four groups of mice (control, IR, P22077, IR plus P222077) on Day 7 (before treatment) and Day 20 (after treatment) after GSC transplantation. Data are means ± SD. n =5. Student’s t test was used to assess the significance. **, P < 0.01; ***, p<0.001. (D) Kaplan-Meier survival curves of mice intracranially transplanted with GSCs (T4121) and treated with the HAUSP inhibitor P22077, irradiation (IR), P22077 plus IR, or DMSO (control). The combined treatment with P2077 and IR significantly extended the survival of mice bearing the GSC-derived tumors relative the single treatment with P22077 or IR alone. n =5 mice per group. P22077 vs control, p<0.003; IR vs control, p<0.01; P22077 plus IR vs control, p<0.001; P22077 vs P22077 plus IR, p<0.01; IR vs P22077 plus IR, p<0.01. Log-rank analysis was used. ( E and F ) Immunofluorescent staining of the cell apoptotic marker cleaved caspase 3 in GSC-derived xenografts treated with the HAUSP inhibitor P22077, irradiation (IR), P22077 plus IR, or DMSO (control). Tumor sections were immunostained with a specific antibody against cleaved caspase 3 (in green) and counterstained with DAPI (blue) to mark nuclei ( E ). Quantifications of cleaved caspase 3 intensities ( I ) in GSC-derived xenografts treated with P22077, IR, P22077 plus IR, or DMSO. Scale bar, 15 µm. Student’s t test was used to assess the significance. Data are means ± SD. n =3 tumors (200 cells per arm). **, p<0.01; ***, p<0.001. ( G and H ) Immunofluorescent staining of SOX2 in GSC-derived xenografts treated with the HAUSP inhibitor P22077, irradiation (IR), P22077 plus IR, or DMSO (control). Tumor sections were immunostained with a specific antibody against SOX2 (in red) and counterstained with DAPI (in blue) to mark nuclei ( G ). Quantifications of SOX2+ cells ( H ) in GSC-derived xenografts treated with P22077, IR, P22077 plus IR, or DMSO. Scale bar, 15 µm. Student’s t test was used to assess the significance. Data are means ± SD. n =3 tumors (200 cells per arm). **, p<0.01; ***, p<0.001. ( I and J ) Immunofluorescent staining of the cell proliferation marker Ki67 in GSC-derived xenografts treated with the HAUSP inhibitor P22077, irradiation (IR), P22077 plus IR, or DMSO (control). Tumor sections were immunostained with a specific antibody against Ki67 (in green) and counterstained with DAPI (in blue) to mark nuclei ( I ). Quantifications of Ki67+ cells ( J ) in GSC-derived xenografts treated with P22077, IR, P22077 plus IR, or DMSO. Scale bar, 15 µm. Student’s t test was used to assess the significance. Data are means ± SD. n =3 tumors (200 cells per arm). **, p<0.01; ***, p<0.001.

Article Snippet: To examine the effect of HAUSP inhibition with P22077 (BOC Sciences, B0084-462597) on tumor growth, mice were treated with P22077 (20mg/Kg/daily) or vehicle control through tail vein injection.

Techniques: Expressing, Luciferase, Transplantation Assay, Control, Injection, Irradiation, In Vivo, Imaging, Derivative Assay, Staining, Marker

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: LC-MS/MS-based identification of new PRC1.1 subunits (A) LC-MS/MS analysis of KDM2B-GFP, GFP-RING1B, and PCGF1-GFP pull outs in K562 cells showing 20 overlapping interaction partners including PRC1.1 subunits (in red) and new interaction partners such as USP7 and TRIM27 (in blue). (B) Schematic overview of PRC1.1 subunit composition. PRC1.1 preferentially binds to non-methylated CpG islands via the CxxC domain of KDM2B. The ubiquitination of histone H2A on lysine 119 is mediated by RING1A/B. (C) Schematic representation of KDM2B deletion mutants and the endogenously expressed long form (LF) and short form (SF; lacking the JmjC domain) of KDM2B. (D) Western blots showing expression of GFP-fusions at near to endogenous KDM2B expression levels. (E) Table showing LC-MS/MS data of PRC1.1 subunits and other PcG proteins co-precipitating with wild-type and mutant forms of KDM2B-GFP. Numbers indicate the average total spectra counts, as measured in triplicate, corrected for maximally identifiable peptides based on in silico protein digests. (F) Volcano plot showing MaxQuant-based label-free quantification of triplicate measurements of KDM2B LF-GFP and KDM2B Δ(LRR)-GFP pull outs. PRC1.1 subunits are indicated in red and USP7 and TRIM27 in blue. (G) Western blot analysis of independent pull outs of (mutant) KDM2B-GFP with input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B, 1/3 rd ) fractions loaded and stained with antibodies against GFP, PCGF1, RING1B, USP7, and TRIM27. (H) PCGF1-GFP pull outs with input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B, 1/3 rd ) and stained with antibodies directed against GFP, KDM2B, RING1B, USP7, and TRIM27. See also Figure S1 and , , and .

Article Snippet: USP7 inhibitor P22077 , Merck , Cat# 662142.

Techniques: Liquid Chromatography with Mass Spectroscopy, Methylation, Western Blot, Expressing, Mutagenesis, In Silico, Staining

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: GFP-USP7 predominantly interacts with PRC1.1 (A) Confocal images of fixed K562 GFP-USP7 cells stained with DAPI. Scale bars represent 25 μm. (B) Western blots showing relative expression of GFP-USP7 versus endogenous USP7 in K562 cells and efficient precipitation of GFP-USP7 using GFP-Trap beads. Input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B, 1/3 rd ) are indicated. (C) Volcano plot showing enrichment of GFP-USP7-specific interaction partners in GFP and GFP-USP7 pull outs. Pull outs were performed in triplicate on K562 GFP and GFP-USP7 cells, and samples were analyzed using LC-MS/MS, followed by data analysis using MaxQuant and Perseus software. Statistical analysis was performed using Student's t test (false discovery rate (FDR) < 0.01; fold change (FC) > 10). PRC1.1, PRC1.2/1.4, and PRC1.6 subunits are indicated in red, blue, and purple respectively. (D) Volcano plot showing enrichment of previously identified USP7 interaction partners (orange), including the MRN-MDC1 complex (green). Statistical analysis was performed using Student's t test (false discovery rate (FDR) < 0.01; fold change (FC) > 10). (E) Intensity-based absolute quantification (iBAQ) value-based calculation of relative stoichiometry values relative to BCOR. (F) Western blot analysis of independent pull outs of GFP, PCGF1-GFP, PCGF2-GFP, PCGF4-GFP, GFP-RING1B, and GFP-CBX2 where input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B1/3 rd ) fractions are loaded and stained with USP7 and RING1B antibodies. See also .

Article Snippet: USP7 inhibitor P22077 , Merck , Cat# 662142.

Techniques: Staining, Western Blot, Expressing, Liquid Chromatography with Mass Spectroscopy, Software

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: USP7 deubiquitinase activity is essential for PRC1.1 integrity (A) Purification of His-tagged ubiquitinated proteins under denaturing conditions in GFP-RING1B and PCGF1-GFP cells treated with DMSO or P22077 for 24 h (30 μM) followed by western blot analysis. (B) Volcano plot showing differential interaction of GFP-RING1B (left) and PCGF1-GFP (right; both indicated in green) with PRC1.1 subunits (highlighted in orange) as measured by label-free quantification (LFQ) of LC-MS/MS data in triplicate. The UBB protein is indicated in blue. Statistical analysis was performed using Student's t test (false discovery rate (FDR) < 0.1; fold change (FC) > 2). (C) Intensity-based absolute quantification (iBAQ) value ratios of several identified PRC1.1 proteins as identified in GFP-RING1B (left) and PCGF1-GFP (right) pull outs from P22077- or DMSO-treated cells. Data are shown as mean ± SD (n = 2 or 3). (D) Western blot of GFP pull outs on PCGF1-GFP and GFP-RING1B in the absence (−) or presence (+) of P22077 (72 h, 30 μM) probed with antibodies for GFP, RING1B, and PCGF1. Input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B, 1/3 rd ) fractions are shown. (E) Mean fluorescent intensity (MFI) analysis of K562 cells expressing either GFP-RING1B, PCGF1-GFP, or KDM2B-GFP treated with DMSO or P22077 for 72 h. See also .

Article Snippet: USP7 inhibitor P22077 , Merck , Cat# 662142.

Techniques: Activity Assay, Purification, Western Blot, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: USP7 inhibition induces loss of PRC1.1 occupancy and H2AK119ub at target loci (A) ChIP-qPCRs on K562, K562 GFP-RING1B, and K562 PCGF1-GFP cells, treated with DMSO or P22077 (72 h, 30 μM), using antibodies against KDM2B or GFP. (B and C) ChIP-qPCRs on K562 cells using antibodies directed against H2AK119ub (B), H3K4me3 (C), on several PRC1.1 loci. Error bars represent SD of technical qPCR replicates. (D and E) ChIP-qPCRs on DMSO- or P22077-treated K562 cells for 4 h, 8 h, and 16 h using antibodies against H2AK119ub (D), GFP (reading out KDM2B-GFP and GFP-RING1B), and H3K27ac (E) on various PRC1.1 loci. Error bars represent SD of technical qPCR replicates. (F) ChIP-qPCRs on K562 cells treated with either DMSO or FT671 (48 h, 10 μM) using antibodies against KDM2B and H2AK119ub. Error bars represent SD of three biological replicate experiments. See also Figure S2 .

Article Snippet: USP7 inhibitor P22077 , Merck , Cat# 662142.

Techniques: Inhibition

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: Genome-wide loss of H2AK119ub upon USP7 inhibition (A–D) ChIP-seq on K562 cells treated with either DMSO or FT671 (24 h, 10 μM) using antibodies against H2AK119ub, H3K4me3, H3K36me3, or H3K27me3. (A) Venn diagram depicting overlapping peaks −/+ 5kb from the transcription start site (TSS). (B) Density plots displaying epimarks around the TSS or across the gene body until + 5kb after the transcription end site (TES). (C) Heat maps of the H2AK119ub signal around the TSS. (D) Representative screenshots of epimarks at four PRC1.1 loci. (E) Non-canonical PRC1.1 or canonical PRC1 peaks shared between six primary AML CD34 + patient samples ( van den Boom et al., 2016 ) were overlaid with USP7 peaks from CUTTL1 cells ( Jin et al., 2019 ). (F) Representative screens shots of PRC1.1 and PRC1 loci depicting USP7 and KDM2B binding and H3K27me3 levels. See also Figure S3 .

Article Snippet: USP7 inhibitor P22077 , Merck , Cat# 662142.

Techniques: Genome Wide, Inhibition, ChIP-sequencing, Binding Assay

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: Loss of TRIM27 partially rescues USP7 inhibitor sensitivity (A) Knockdown efficiencies of two independent TRIM27 shRNAs in K562 cells. Error bars represent SD from technical triplicates. (B) KDM2B-GFP and H2AK119ub ChIP-qPCRs with error bars representing SD based on three independent experiments, statistical analysis was performed using Student's t test; ∗p < 0.05 and ∗∗p < 0.01. (C) Western blot analysis of KDM2B-GFP pull outs on K562 cells expressing SCR or TRIM27 (#1) shRNAs using the indicated antibodies. Input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B, 1/3 rd ) as indicated. (D) H2AK119ub ChIP-qPCR on K562 cells expressing SCR or TRIM27 (#2) shRNAs and treated with DMSO or FT671 (48 h, 10μM). Error bars represent SD based on three independent experiments; statistical analysis was performed using Student's t test; ∗p < 0.05 and ∗∗p < 0.01. (E) Cumulative cell proliferation of K562 cells expressing SCR or TRIM27 (#1 or #2) shRNAs and treated with DMSO or FT671. Error bars represent SD based on two independent experiments. (F) Knockdown efficiencies of two independent USP7 shRNAs in K562 cells. Error bars represent SD from technical triplicates. (G) Western blot analysis of KDM2B-GFP pull outs on K562 cells expressing SCR or USP7 (#1 and #2) shRNAs using the indicated antibodies. Input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B, 1/3 rd ) as indicated. (H) Endogenous KDM2B and H2AK119ub ChIP-qPCRs on K562 cells expressing SCR of USP7 (#1 and #2) shRNAs. Error bars represent SD based on three independent experiments; statistical analysis was performed using Student's t test; ∗p < 0.05 and ∗∗p < 0.01. (I) Western blot analysis of THP-1 doxycycline-inducible CRISPR/Cas9 USP7 knockout cells treated with doxycycline for 3 days and subsequent culture for 4 days and stained with the indicated antibodies. (J) Endogenous KDM2B and H2AK119ub ChIP-qPCRs on doxycycline-treated cells (day 6). Error bars represent SD based on three independent experiments; statistical analysis was performed using Student's t test; ∗p < 0.05 and ∗∗p < 0.01. (K) Graphical abstract of effects of USP7 inhibition versus USP7 or TRIM27 knockdown. See also Figure S4 .

Article Snippet: USP7 inhibitor P22077 , Merck , Cat# 662142.

Techniques: Western Blot, Expressing, CRISPR, Knock-Out, Staining, Inhibition

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: USP7 inhibition leads to transcriptional changes of PRC1.1 target genes (A) Unsupervised clustering of RNA-seq data from three independent experiments where K562 cells were treated with DMSO or FT671 (10μM, 48 h) samples. (B) Venn diagram showing overlap of significantly up- and downregulated genes (Student's t test, p < 1x10 −6 ) with previously identified PRC1.1 target genes. (C) GSEA analysis showing enrichment score against ranked RNA-seq data. RNA-seq data were compared with indicated gene sets. (D) Gene ontology analyses of regulatory processes (RP) of all up- and downregulated genes in FT671-treated cells. (E) Gene ontology analyses of regulatory processes (RP) of up- and downregulated genes in FT671-treated cells that are also targeted by PRC1.1. (F) Screens shots of our ChIP-seq tracks ( van den Boom et al., 2016 ) for H2AK119ub, H3K27me3, PCGF1, PCGF2, PCGF4, CBX2, RING1A, RING1B, and KDM2B (all GFP-fusions) in K562 cells. ChIP-seq tracks for H3K4me3, H3K36me3, RNAPII, H3K27ac, EZH2, SUZ12 (all K562), and USP7 (CUTLL1) were downloaded from ENCODE/Broad. In addition, endogenous KDM2B, H2AK119ub, H3K27me3, and H3K4me3 in two primary AML patient cell samples are shown. See also Figure S5 .

Article Snippet: USP7 inhibitor P22077 , Merck , Cat# 662142.

Techniques: Inhibition, RNA Sequencing Assay, ChIP-sequencing

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: Sensitivity of AML cells toward USP7 inhibition (A) Cumulative cell growth of various AML cell lines treated with DMSO or various concentrations of P22077. (B) Cumulative cell growth of various AML cell lines treated with DMSO or various concentrations of FT671. Error bars represent SD based on measurement of biological triplicates. (C) Cumulative cell growth of primary AML patient cells (n = 3) grown on MS5 stromal cells treated with DMSO (control) or P22077. Error bars represent SD based on measurement of biological duplicates. (D) Cumulative cell growth of primary AML patient cells (n = 3) grown on MS5 stromal cells treated with DMSO or FT671. Error bars represent SD based on measurement of biological duplicates. (E) Viable cell numbers after 8 days of treatment with DMSO or increasing concentrations of FT671. Representative examples of primary AML patient cells and cord blood CD34 + cell samples are shown. Error bars represent SD based on measurement of biological triplicates. (F) IC50 curves and data for 11 independent primary AML samples, cord blood CD34 + cells, and mobilized peripheral blood stem cells (PBSCs), cocultured on MS5 stromal cells for 8 days in the presence of increasing amounts of FT671. Asterisk indicates TP53-mutant AMLs. (G) Experimental setup of our human CB MLL-AF9 xenograft mouse model. Here 5 x 10 4 MLL-AF9 GFP + cells from a primary leukemic mouse were IV injected into secondary recipients (n = 11). (H) Peripheral blood analysis of MLL-AF9 GFP/CD45 + cells, three weeks after injection prior to treatment (left) and 2.5 weeks following treatment (right). Mice were treated daily with either DMSO as control (n = 5) or 20 mg/kg P22077 (n = 6). (I) Peripheral blood chimerism levels of control and P22077 (20 mg/kg)-treated mice over the course of the experiment. Treatment was started at day 28 (4 weeks post-transplant) as indicated with an arrow. See also Figure S6 .

Article Snippet: USP7 inhibitor P22077 , Merck , Cat# 662142.

Techniques: Inhibition, Mutagenesis, Injection

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet:

Article Snippet: USP7 inhibitor P22077 , Merck , Cat# 662142.

Techniques: Recombinant, Blocking Assay, Plasmid Preparation, Software, Purification, SYBR Green Assay

Journal: International Journal of Molecular Sciences

Article Title: Combination of Inhibitors of USP7 and PLK1 has a Strong Synergism against Paclitaxel Resistance

doi: 10.3390/ijms21228629

Figure Lengend Snippet: Combination of the USP7 inhibitor P22077 and PLK1 inhibitor volasertib shows synergic anticancer effects in paclitaxel-resistant lung cancer. ( a – c ) NCI-H460 and NCI-H460 TXR cells were treated with paclitaxel ( a ), volasertib ( b ), or P22077 ( c ) for 48 h in a concentration-dependent manner. The percentages of viable cells were measured by a cell viability assay. The bar graph presents the mean values of half maximal growth inhibitory concentration (GI 50 ). ( d ) Combination treatment with volasertib and P22077 was performed in NCI-H460 and NCI-H460 TXR cells. Cells were grown for 48 h in the presence of 0, 13.2, or 17.6 µM P22077 and volasertib at the indicated concentrations. The percentages of viable cells were measured by a cell viability assay. ( e ) QRT-PCR was performed for ABCB1 , PLK1 , and USP7 in NCI-H460 and NCI-H460 TXR cells treated with 14 nM volasertib (Vol) and 17.6 µM P22077 (P2) for 48 h. Three independent experiments were performed and comparisons within groups were presented as the mean ± SDs. (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: Volasertib and P22077 were from Selleckchem (Houston, TX, USA) and Lifesensors (Malvern, PA, USA), respectively.

Techniques: Concentration Assay, Viability Assay, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Combination of Inhibitors of USP7 and PLK1 has a Strong Synergism against Paclitaxel Resistance

doi: 10.3390/ijms21228629

Figure Lengend Snippet: The half maximal growth inhibitory concentration (GI 50 ) values for P22077 and volasertib and their combination index (CI) in H460 and H460TXR cells. CI < 1 represents synergism, CI = 1 represents additive effect, CI > 1 represents antagonism.

Article Snippet: Volasertib and P22077 were from Selleckchem (Houston, TX, USA) and Lifesensors (Malvern, PA, USA), respectively.

Techniques: Concentration Assay

Journal: Nature Communications

Article Title: Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

doi: 10.1038/s41467-020-20259-0

Figure Lengend Snippet: a – e Peripheral blood mononuclear cells (PBMC) from normal donors ( a ) ( n = 3 samples examined over three independent experiments), CML cell lines ( b ) ( n = 3 cells examined over three independent experiments), and BM mononuclear cells from IM-sensitive ( c ) ( n = 5 samples examined over three independent experiments) and IM-resistant ( d ) ( n = 7 samples examined over three independent experiments) CML patients were treated with different concentrations of P22077 or IM for 48 h. Cell viability was measured by CCK8 assay. e BM mononuclear cells resistance to the second-generation TKIs (dasatinib, nilotinib, bosutinib) from CML patients were treated with P22077 and IM for 48 h ( n = 3 samples examined over three independent experiments). Cell viability was measured by CCK8 assay. Data are presented as mean ± s.d. p- values were analyzed by one-way analysis of variance (ANOVA), * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001. f Primary CML (IM-sensitive), CML-R (IM-resistant) BM mononuclear cells and KBM5 T315I cells were treated with P22077 for 48 h. Cell lysates from the indicated cells were extracted and subjected to immunoblotting with indicated antibodies. g P22077 (30 mg/kg/day), IM (50 mg/kg/day), P22077 (30 mg/kg/day) in combination with IM (50 mg/kg/day) or vehicle were given to mice transplanted with KBM5 T315I cells by intraperitoneal injection ( n = 6 biologically independent samples per group) from day 15 to day 30. ** p < 0.01, by Mantel-Cox-log-rank test. h , i Human CD45 cells from CML BM (patient-derived xenograft model) were transplanted into two groups of B-NDG mice. The survival time of the mice ( h ) ( n = 7 biologically independent samples per group) was determined, and the percentages of CD34 + CD38 − cells in human CD45 cells ( i ) ( n = 3 biologically independent samples per group) from BM were measured. Data are mean ± s.d. p- values were analyzed by Mantel-Cox-log-rank test ( h ) and two-sided Student’s t -test ( i ). ** p < 0.01; *** p < 0.001. j CD34 + cells derived from normal ( n = 3 samples examined over three independent experiments), primary IM-sensitive CML ( n = 3 samples examined over three independent experiments), and primary IM-resistant CML BM mononuclear cells ( n = 3 samples examined over three independent experiments) were treated with different concentrations of P22077. The colonies were counted on day 14. Scale bars, 100 μm. Data are mean ± s.d. p- values were analyzed by one-way analysis of variance (ANOVA). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns no significant. k Wild-type mice received BCR-ABL retroviral transplantation for 21 days, and the mice were treated with control or P22077 by intraperitoneal injection for 14 days. GFP + LSK cells from the BM of the two groups ( n = 6 biologically independent samples per group) were evaluated by FACS. Data are mean ± s.d. p- values were analyzed by two-sided Student’s t -test. **** p < 0.0001. l Wild-type mice received BCR-ABL retroviral transplantation for 21 days, and the mice were trea t ed with IM for 40 days. Mice were divided into two groups ( n = 6 biologically independent samples per group). One group received P22077, and the other group received the vehicle and used as the control. After 12 days, the number of GFP + LSK cells in the BM of the mice was examined. Data are mean ± s.d. p- values were analyzed by two-sided Student’s t -test. **** p < 0·0001. Source data are provided as a Source Data file.

Article Snippet: Between 15 and 30 days, the mice were treated through tail vein injection of vehicle (10% DMSO + 10% BASF-ELP + 10% 1,2-Propanediol + 70% physiological saline) ( n = 6), P22077 (dissolved in the vehicle) (intraperitoneally, 30 mg/kg, n = 6), IM (intraperitoneally, 50 mg/kg, n = 6) or P22077 + IM (intraperitoneally, n = 6).

Techniques: CCK-8 Assay, Western Blot, Injection, Derivative Assay, Retroviral, Transplantation Assay, Control

Journal: Nature Reviews. Drug Discovery

Article Title: Deubiquitylating enzymes and drug discovery: emerging opportunities

doi: 10.1038/nrd.2017.152

Figure Lengend Snippet: DUB inhibitors in development PowerPoint slide

Article Snippet: P5091 (shown right), P22077 , , Progenra , Oncology, Immuno-oncology , Preclinical , .

Techniques: Analogues