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    ATCC raw c6 u87 hela siha hepg2 huh7
    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 ), <t>HepG2</t> (p53 Null ), and <t>Huh7</t> (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, <t>U87,</t> LN18, HCT116 (p53 Wt ), HCT116 (p53 Null ), SW480, SW620, <t>HeLa,</t> 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).
    Raw C6 U87 Hela Siha Hepg2 Huh7, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 31385 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis"

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

    Journal: bioRxiv

    doi: 10.1101/2021.08.12.456108

    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).
    Figure Legend 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).

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

    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).
    Figure Legend 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).

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

    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).
    Figure Legend 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).

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



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    raw c6 u87 hela siha hepg2 huh7  (ATCC)
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    ATCC raw c6 u87 hela siha hepg2 huh7
    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 ), <t>HepG2</t> (p53 Null ), and <t>Huh7</t> (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, <t>U87,</t> LN18, HCT116 (p53 Wt ), HCT116 (p53 Null ), SW480, SW620, <t>HeLa,</t> 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).
    Raw C6 U87 Hela Siha Hepg2 Huh7, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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).

    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: Human colorectal cancer (CRC) cell lines [HCT 116 (p53 Wt and p53 Null ), SW-480, HT-29, SW-620]; human embryonic kidney cell line [HEK-293 and HEK-293T] and other cell lines (RAW, C6, U87, HeLa, SiHa, HepG2, Huh7) were used (ATCC.

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

    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).

    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: Human colorectal cancer (CRC) cell lines [HCT 116 (p53 Wt and p53 Null ), SW-480, HT-29, SW-620]; human embryonic kidney cell line [HEK-293 and HEK-293T] and other cell lines (RAW, C6, U87, HeLa, SiHa, HepG2, Huh7) were used (ATCC.

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

    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).

    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: Human colorectal cancer (CRC) cell lines [HCT 116 (p53 Wt and p53 Null ), SW-480, HT-29, SW-620]; human embryonic kidney cell line [HEK-293 and HEK-293T] and other cell lines (RAW, C6, U87, HeLa, SiHa, HepG2, Huh7) were used (ATCC.

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