the pci vector backbone  (Promega)

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: A, HCT116 (p53 Wt and p53 Null ) lines were treated with the indicated dose of P5091 (0 to 80 µM) for 24 and 48 hrs, and cell viability was checked by MTT assay. Inset graph representing change in mean IC-50 value of both cell lines at different time points. B, Protein levels of USP7, PARP, Cleaved PARP, Caspase3, Cleaved Caspase3, p53, MDM2 and p21 were determined by immunoblotting (IB) where HCT116 (p53 Wt and p53 Null ) cell lines were treated with USP7 inhibitor P5091 (20µM) for 24 hrs. GAPDH was kept as loading control. C, Following treatment of HCT116 (p53 Null ) cells with P5091 in different doses (1, 5, 10µM) for 24 hrs, the increase in caspase 3/7 activity was determined using a fluorescence plate reader. Data represent mean ± SD of three independent biological replicates. D, Number of colonies formed by HCT116 (p53 Wt ), HepG2 (p53 Null ), and Huh7 (p53 Mut ) cells after treatment with P5091 (20 µM) for 24 hrs; colonies were counted after 15 days. E & F, Identification of USP7 and XIAP interacting proteins by Mass Spectrometry. Silver stained SDS-PAGE gels containing elute from respective pull-down as indicated. MS analysis identifies specific peptides of XIAP and USP7 respectively from GST-USP7 and GST-XIAP pull-down lanes by using HEK cell lysates. G, Identification of USP7 target proteome in HCT116 cells by label-free comparative proteomics analysis upon USP7 inhibition with P5091 (15µM for 24 hrs), figure represents the workflow of label-free quantitation (LTQ) by nano-LC−MS/M.S. on a Q-exactive followed by SIEVE TM processing. H, Graphical representation of calculated protein ratios of P5091 treated samples with respect to the vehicle control (left panel). Venn diagram showing identified number of Up-regulated, Unchanged and Down regulated proteins in HCT116 p53 wt and HCT116 p53 null cell lines upon P5091 treatment with respect to control (right panel). I & J, Whole-cell lysates were prepared from MCF7, MD-AMB 231, MD-AMB 468, T47D, C6, U87, LN18, HCT116 (p53 Wt ), HCT116 (p53 Null ), SW480, SW620, HeLa, SiHa, LnCap, A549, RAW, HEK293T, and HEK293 cells. IB analysis was performed using respective antibodies for USP7, XIAP and β-Actin. Normalized values were plotted to show a strong positive correlation between USP7 and XIAP where p value < 0.0001, n=2. Error bars in all the indicated sub-figures represent mean ± SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P<0.05 is represented as *, otherwise non-significant (ns).

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: MTT Assay, Western Blot, Control, Activity Assay, Fluorescence, Mass Spectrometry, Staining, SDS Page, Inhibition, Quantitation Assay, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet:

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques:

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: A, HEK cells transfected with 2µG of EV (pGZ) (lane-1&3), pGZ-USP7 (lane-2), and consecutively inactive form of USP7 mutant pGZ-USP7 C223S (lane-4); 48 hrs post-transfection, USP7 and XIAP protein levels were checked by IB. Additionally, three different shRNAs targeting USP7 and scramble shRNA were transfected (lanes-5,6,7&8) and the levels of XIAP and USP7 were analyzed by IB. GAPDH was kept as loading control. B, qRT-PCR analysis showing relative mRNA fold change upon USP7 inhibition by P5091 (15µM) for 24 hrs. Relative mRNA fold change of XIAP upon USP7 knockdown (using siUSP7) and USP7 over-expression. Protein expression of XIAP from the same experiment was also represented by IB, where GAPDH was used as an internal loading control. C, Multiple cancer cell lines such as HCT116, C6, SH-SY5Y, SiHa, and SW620 were treated with 15µM of USP7 inhibitor (P5091-lane 2, 4, 6, 8, 10; P22077-lane 11) for 24 hrs and XIAP was analyzed by IB and compared with respective control lanes (lane 1, 3, 5, 7, 9). IB analysis of XIAP protein level in HCT116 cells transfected with GFP-USP7 (4 µG, lane 13), pGZ-USP7 C223S (4 µG, lane 14), or siUSP7 (lane 16). EV (lane 12), control Si (lane 15) were kept for control. D, Huh7 and HT29 cells were treated with USP7 inhibitor P22077 in a dose-dependent manner (5, 10, 15, 20 µM) for 24 hrs. XIAP was checked by IB. GAPDH was kept as an internal loading control. E, HCT116 cells were transfected with either Control Si (upper left panel) or siUSP7 (Upper right panel) and HEK cells were transfected with either EV (Lower left panel) or GFP-USP7 (Lower right panel) before treatment with cycloheximide (CHX: 50µG/ml). XIAP protein levels at indicated time points were analyzed by IB as shown in the figure (left panels). Graph showing the change in XIAP half-life was determined in USP7 knockdown and overexpression conditions. Error bars in all the indicated sub-figures represent mean (±) SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P<0.05 is represented as *, otherwise non-significant (ns).

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: Transfection, Mutagenesis, shRNA, Control, Quantitative RT-PCR, Inhibition, Knockdown, Over Expression, Expressing

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: A , Confocal images of HEK cells showing stains for USP7 (green), XIAP (red), and USP7-XIAP merged (yellow). Cells were counterstained with DAPI (blue). Images were captured at 60X optical magnification. B, Purified Myc-His USP7 incubated separately with purified GST and GST-XIAP in the presence of GST-Bead for 4 hrs. Proteins were separated by SDS-PAGE and analyzed by Coomassie blue staining to visualize the specific bands for GST (lane 1), GST-XIAP and Myc-His USP7 (lane 2). Figure shows the representative data of three biological replicates. C, HEK cell lysate was incubated separately with purified GST and GST-XIAP in the presence of GST-bead for 4 hrs, followed by IB using anti-USP7 and GST antibody. D, HEK cell lysate was immunoprecipitated with XIAP antibody followed by IB using antibodies against USP7 and XIAP to identify their interaction at the endogenous condition. E, HEK cells were transiently transfected with FLAG-USP7 and GFP-XIAP. Equal amounts of cell lysates were used for immunoprecipitation with Flag antibody and Normal Rabbit Serum, followed by IB using antibodies against Flag and GFP to identify their interaction under overexpressed conditions. F, HEK cells were treated with either DMSO or MG132 (25µM) for 8 hrs. Equal amounts of lysates were used for IP using USP7 antibody followed by IB using indicated antibodies to show an increased interaction of USP7 and XIAP upon blocking the proteasomal system. Veriblot secondary antibody was used to prevent nonspecific detection of the heavy and light chains. G, Workflow of domain wise docking of USP7 and XIAP proteins using three independent Docking software to identify the probable domains responsible for interaction. H, Venn diagram showing domain pair of USP7 and XIAP by using three docking softwares and identified CAT-BIR2 and UBL-BIR3 as the probable interacting domains.

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: Purification, Incubation, SDS Page, Staining, Immunoprecipitation, Transfection, Blocking Assay, Software

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: A, Workflow representing generation of full-length USP7 and XIAP followed by docking with Swarmdock and simulation of docking complex to identify the stable orientation of USP7 and XIAP interaction. B, Upper panels showing full-length USP7 and XIAP proteins generated by stitching the available USP7 and XIAP domains in PDB. Lower panels showing USP7-XIAP complex, where interface region highlighted in yellow. C, Schematic representation of USP7 deletion mutants and their interactions with full-length XIAP protein. D, HEK cells were co-transfected with indicated plasmid constructs; prepared the cell lysates followed by pull-down with Ni-NTA beads for 1 hr at room temperature. The pulled-down proteins and input were analyzed by IB with the indicated antibodies. Ponceau S staining indicates an equal loading in input lanes. HEK cells were transfected with the indicated plasmid constructs before the preparation of cell lysates. E, Equal amounts of lysate from plates transiently transfected as indicated, were incubated separately with purified proteins GST (right panel) or GST-XIAP fusion protein (left panel) including glutathione beads followed by IB analysis using indicated antibodies. The experiment performed thrice for biological replicates. F, Schematic representation of XIAP deletion mutants and their interaction with full-length USP7 protein. G, HEK cells were transiently transfected with Flag-USP7. The lysate was prepared and incubated separately with purified different GST tagged XIAP deletion mutants and GST protein (Control) overnight at 4°C. After pull-down with GST-bead for 2 hrs at RT, pulled-down proteins were analyzed by IB.

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: Generated, Transfection, Plasmid Preparation, Construct, Staining, Incubation, Purification, Control

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: HEK cells were used in experiments A-E, H & L and mentioned in other cases. A, Cells co-transfected with HA-Ub and GFP-XIAP and 24 hrs post-transfection cells were further treated with MG-132 (25µM) and vehicle control for an additional 4 hrs. GFP-tagged proteins were pulled down from lysates using GFP antibody and Protein-A Sepharose bead; analyzed by IB using indicated antibodies. B, Cells were transfected with indicated constructs; 24 hrs post-transfection, cells were treated with P5091 (15µM) for another 24 hrs. Before harvesting, the cells were further treated with MG-132 for 4 hrs. Lysates were prepared and used for pull-down using GFP antibody followed by IB using indicated antibodies. C, Cells were transfected individually with EV, USP7, and USP7 C223S plasmids. Pull-down assay was performed using XIAP antibody, bead-bound proteins and total cell lysate were analyzed by IB to detect the change in poly-ubiquitination pattern. D, Cells were transfected with indicated plasmids; 24 hr post-transfection, treated with P5091 (15µM) and vehicle control for another 24 hours. Cells were harvested after MG-132 treatment for further 4 hrs, followed by pull-down assay was performed using GFP antibody and IB with indicated antibodies. E, Cells were transfected with either WT or different Ub mutants as depicted in the figure. Following lysate preparation, pulled down the proteins using GFP antibody followed by IB using the indicated antibodies. Input (3%) was run separately for Control. F, Cell lysates prepared from p53 WT and p53 null HCT116 cells subjected to IB with the indicated antibodies. G, HCT116 (p53 wt ) cells were treated with Nutlin3A (5µM) for 24 hrs, the protein level of XIAP and other p53 responsive genes were analyzed by IB. H, Cells were treated with Nutlin3A in a dose-dependent manner (5µM and 10µM) to analyze the level of indicated proteins by IB. I, HCT116 (p53 wt ) and HT29 (p53 mut ) cells were treated with P5091 in a dose-dependent manner (0, 10, and 20µM) for 24 hrs and check the levels of the indicated proteins by IB. J, Expression of indicated genes in HCT116 cells containing either p53 wt or p53 mut was analyzed by qRT-PCR. K, HCT116 cells were treated with Doxorubicin (10µM) for 3 hrs, washed and kept in fresh media without Dox for another 21hrs before harvesting them. Expression (mRNA level) of the indicated genes was analyzed by qRT-PCR. L, Cells were transfected with either EV (left panel) or GFP-USP7 (right panel) for 24 hrs followed by Dox treatment in a dose-dependent manner (0, 1, 2, 3µM) for another 24 hrs. Expression of the indicated proteins was analyzed by IB. Expression of XIAP was normalized against GAPDH and plotted here. The data are representative of three biological replicates. qRT-PCR data represents the mean ± SD of three independent biological replicates. Indicated P-values were calculated using Student’s t-test and P<0.0001 is represented as *, otherwise non-significant (ns).

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: Transfection, Control, Construct, Pull Down Assay, Ubiquitin Proteomics, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: A, HCT 116 (p53 Wt ) cells were transfected with indicated plasmids and treated with Doxorubicin for 24 hrs to induced apoptosis, prepared the lysates and analyzed by IB using the panel of indicated antibodies. B, Similarly, HEK cells were transfected with indicated plasmids and treated with Doxorubicin (1 µM) for 24hrs to induced apoptosis, prepared the lysates and analyzed by IB using the panel of indicated antibodies. C, Huh7 (p53 Mut ) and HepG2 cells (p53 Wt ) cells were transfected with Flag-USP7 and GFP-XIAP for the indicated time points, prepared the lysates and analyzed by IB. D, HCT116 (p53 Null ) cells were transfected with either Flag-USP7 or GFP-XIAP. After 48 hrs, cells were treated with P5091 (15µM) or Doxorubicin (1µM). Prepared the cell lysates and analyzed by IB using the indicated antibodies. XIAP, USP7, and Cleaved Caspase 3 were plotted after normalization against GAPDH. E, HCT116 (p53 Null - upper panel) and HCT116 (p53 Wt - lower panel) cells were treated with either XIAP inhibitor Embellin or USP7 inhibitor P5091 for the indicated doses. Prepared the lysates and analyzed by IB. XIAP expression was plotted after normalization against GAPDH. F, HEK and C6 cells were seeded in a 6-well plate and treated with P5091 (15µM and 20 µM for HEK and, 15 µM for C6) for 24 hrs. Treated cells were stained as per protocol to detect TUNNEL positivity. G, Cell cycle profile of HCT116 (p53 WT ) cells were analyzed as per protocol after treatment with an increasing dose of P5091 (5µM, 10µM and 15µM 24 hrs). H, Scratch assay was performed to evaluate the migration of HepG2 (p53 Wt ) and Huh7 (p53 Mut ) cells pre-treated with P5091 (20µM). I, percent of cell population in different apoptosis phases of HCT116 (p53 WT ) cells were analyzed as per protocol after treatment with indicated dose of P5091 for 24 hrs. Error bars in all the indicated sub-figures represent mean (±) SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P<0.0001 is represented as *, otherwise non-significant (ns).

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: Transfection, Expressing, Staining, Wound Healing Assay, Migration

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: USP7 deubiquitinates and stabilizes anti-apoptotic protein XIAP and promote cancer cell survival. Overexpression of either USP7 or XIAP confers chemotherapy resistance and associated with increased survival of cancer cells. USP7 inhibition by small molecule inhibitor acts as a potential therapeutic intervention to fight against both p53 Wt and p53 Mut/Null cancers through MDM2 and XIAP respectively.

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: Over Expression, Inhibition