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human aml cell lines k562  (DSMZ)


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    DSMZ human aml cell lines k562
    Treatment with daunorubicin increases autophagy in <t>K562</t> cells. ( A ) Schematic representation of the autophagy process with its components and related molecules. ( B ) K562 cells were treated with different indicated doses of daunorubicin (DNR) or vehicle (DMSO) for 24 h. ( C , D ) K562 cells were treated with 2 µM of daunorubicin for 24 h and 10 µM of chloroquine (CQ) for the last 4 h. ( B , C ) Level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. The untreated conditions were set to 1. The normalized means are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the untreated conditions, and in grey: CQ conditions compared with DNR and CQ conditions). ( D ) Double-labeling immunofluorescence and representative images of LC3 (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown.
    Human Aml Cell Lines K562, supplied by DSMZ, used in various techniques. Bioz Stars score: 96/100, based on 749 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/aml+cell+lines+k562/pmc08152749-189-1-10?v=DSMZ
    Average 96 stars, based on 749 article reviews
    human aml cell lines k562 - by Bioz Stars, 2026-07
    96/100 stars

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    1) Product Images from "MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin"

    Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22105153

    Treatment with daunorubicin increases autophagy in K562 cells. ( A ) Schematic representation of the autophagy process with its components and related molecules. ( B ) K562 cells were treated with different indicated doses of daunorubicin (DNR) or vehicle (DMSO) for 24 h. ( C , D ) K562 cells were treated with 2 µM of daunorubicin for 24 h and 10 µM of chloroquine (CQ) for the last 4 h. ( B , C ) Level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. The untreated conditions were set to 1. The normalized means are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the untreated conditions, and in grey: CQ conditions compared with DNR and CQ conditions). ( D ) Double-labeling immunofluorescence and representative images of LC3 (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown.
    Figure Legend Snippet: Treatment with daunorubicin increases autophagy in K562 cells. ( A ) Schematic representation of the autophagy process with its components and related molecules. ( B ) K562 cells were treated with different indicated doses of daunorubicin (DNR) or vehicle (DMSO) for 24 h. ( C , D ) K562 cells were treated with 2 µM of daunorubicin for 24 h and 10 µM of chloroquine (CQ) for the last 4 h. ( B , C ) Level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. The untreated conditions were set to 1. The normalized means are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the untreated conditions, and in grey: CQ conditions compared with DNR and CQ conditions). ( D ) Double-labeling immunofluorescence and representative images of LC3 (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown.

    Techniques Used: Western Blot, Expressing, Control, Software, Labeling, Immunofluorescence, Staining

    Treatment with 3-MA partially prevents the effects of DNR on autophagy and cell growth. K562 were treated with 2 µM of daunorubicin and 3 mM of 3-methyladenine (3-MA). ( A ) Proteins were extracted after 24 h, and the level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. The untreated conditions were set to 1. The normalized means are shown with SEM. (*, p < 0.05, **, p < 0.01, in black: all the conditions compared with the untreated conditions, and in grey: DNR conditions compared with DNR and 3-MA conditions). ( B ) Viable cells were counted in the presence of Trypan Blue after 1, 2, 3, and 4 days of culture. The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (***, p < 0.001).
    Figure Legend Snippet: Treatment with 3-MA partially prevents the effects of DNR on autophagy and cell growth. K562 were treated with 2 µM of daunorubicin and 3 mM of 3-methyladenine (3-MA). ( A ) Proteins were extracted after 24 h, and the level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. The untreated conditions were set to 1. The normalized means are shown with SEM. (*, p < 0.05, **, p < 0.01, in black: all the conditions compared with the untreated conditions, and in grey: DNR conditions compared with DNR and 3-MA conditions). ( B ) Viable cells were counted in the presence of Trypan Blue after 1, 2, 3, and 4 days of culture. The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (***, p < 0.001).

    Techniques Used: Western Blot, Expressing, Control, Software

    miR-15a-5p inhibits autophagy induced by daunorubicin. K562 cells were transiently transfected with miR-15a-5p mimic (15a) or scrambled mimic (Sc) as control for 48 h, and cells were treated with 2 µM of daunorubicin (DNR) for the last 24 h and with 10 µM of chloroquine (CQ) for the last 16 h. ( A ) RNA was extracted and miR-15a-5p expression was measured by RT-qPCR. Normalization was completed with the endogenous control RNU44 . ( B ) Proteins were extracted, and the level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. ( C ) The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. ( D ) Double-labeling immunofluorescence and representative images of LC3 (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown. LC3B and LAMP-2 immunofluorescence intensities of three independent experiments were quantified using the ImageJ software and divided by the nuclear immunofluorescence. ( A , C , D ) The untreated scrambled condition was set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the untreated scrambled condition, and in grey: miR-15a conditions compared with scrambled conditions).
    Figure Legend Snippet: miR-15a-5p inhibits autophagy induced by daunorubicin. K562 cells were transiently transfected with miR-15a-5p mimic (15a) or scrambled mimic (Sc) as control for 48 h, and cells were treated with 2 µM of daunorubicin (DNR) for the last 24 h and with 10 µM of chloroquine (CQ) for the last 16 h. ( A ) RNA was extracted and miR-15a-5p expression was measured by RT-qPCR. Normalization was completed with the endogenous control RNU44 . ( B ) Proteins were extracted, and the level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. ( C ) The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. ( D ) Double-labeling immunofluorescence and representative images of LC3 (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown. LC3B and LAMP-2 immunofluorescence intensities of three independent experiments were quantified using the ImageJ software and divided by the nuclear immunofluorescence. ( A , C , D ) The untreated scrambled condition was set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the untreated scrambled condition, and in grey: miR-15a conditions compared with scrambled conditions).

    Techniques Used: Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Software, Labeling, Immunofluorescence, Staining

    miR-15a-5p decreases the downregulation of cell growth by daunorubicin. K562 cells were transiently transfected with miR-15a-5p mimic (miR-15a) or scrambled mimic (Sc) as control, and cells were treated with 0.1 µM of daunorubicin (DNR), 0.5 µM of DNR, or vehicle for 72 h ( A ) Viable cells were counted in the presence of Trypan Blue after 1, 2, and 3 days of culture. The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (*, p < 0.05, ***, p < 0.001). ( B ) Cell viability assay (CellTiter-Glo ® Luminescent assay) was performed at 72 h of treatment. The untreated scrambled condition was set to 1. The normalized means are shown with SEM (**, p < 0.01).
    Figure Legend Snippet: miR-15a-5p decreases the downregulation of cell growth by daunorubicin. K562 cells were transiently transfected with miR-15a-5p mimic (miR-15a) or scrambled mimic (Sc) as control, and cells were treated with 0.1 µM of daunorubicin (DNR), 0.5 µM of DNR, or vehicle for 72 h ( A ) Viable cells were counted in the presence of Trypan Blue after 1, 2, and 3 days of culture. The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (*, p < 0.05, ***, p < 0.001). ( B ) Cell viability assay (CellTiter-Glo ® Luminescent assay) was performed at 72 h of treatment. The untreated scrambled condition was set to 1. The normalized means are shown with SEM (**, p < 0.01).

    Techniques Used: Transfection, Control, Viability Assay, Luminescence Assay

    Inhibition of mir-15a-5p induces autophagy. ( A – C ) K562 cells were transiently transfected with miR-15a-5p inhibitor (15ai) or scrambled inhibitor (Sci) as control for 48 h, and cells were treated with 2 µM of daunorubicin (DNR) for the last 24 h. ( A ) RNA was extracted and miR-15a-5p expression was measured by RT-qPCR and normalized with the expression of RNU44 . ( B ) Protein were extracted, and level of autophagy was analyzed by Western blot with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. ( C ) The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. ( D ) Double-labeling immunofluorescence and representative images of LC3B (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown. LC3B and LAMP-2 immunofluorescence intensities of three independent experiments were quantified using the ImageJ software and divided by the nuclear immunofluorescence. ( D , E ) K562 cells were transiently transfected with miR-15a-5p inhibitor (miR-15ai) or scrambled inhibitor (Sci) as control, and cells were treated with 0.1 µM of daunorubicin (DNR), 0.5 µM of DNR, or vehicle for 72 h. ( D ) Viable cells were counted in the presence of Trypan Blue after 1, 2, and 3 days of culture. ( E ) Cell viability assay (CellTiter-Glo ® Luminescent assay) was performed at 72 h of treatment. ( A – C , E ) The untreated scrambled conditions were set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, all the conditions compared with the untreated scrambled condition). ( D ) The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (**, p < 0.01).
    Figure Legend Snippet: Inhibition of mir-15a-5p induces autophagy. ( A – C ) K562 cells were transiently transfected with miR-15a-5p inhibitor (15ai) or scrambled inhibitor (Sci) as control for 48 h, and cells were treated with 2 µM of daunorubicin (DNR) for the last 24 h. ( A ) RNA was extracted and miR-15a-5p expression was measured by RT-qPCR and normalized with the expression of RNU44 . ( B ) Protein were extracted, and level of autophagy was analyzed by Western blot with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. ( C ) The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. ( D ) Double-labeling immunofluorescence and representative images of LC3B (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown. LC3B and LAMP-2 immunofluorescence intensities of three independent experiments were quantified using the ImageJ software and divided by the nuclear immunofluorescence. ( D , E ) K562 cells were transiently transfected with miR-15a-5p inhibitor (miR-15ai) or scrambled inhibitor (Sci) as control, and cells were treated with 0.1 µM of daunorubicin (DNR), 0.5 µM of DNR, or vehicle for 72 h. ( D ) Viable cells were counted in the presence of Trypan Blue after 1, 2, and 3 days of culture. ( E ) Cell viability assay (CellTiter-Glo ® Luminescent assay) was performed at 72 h of treatment. ( A – C , E ) The untreated scrambled conditions were set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, all the conditions compared with the untreated scrambled condition). ( D ) The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (**, p < 0.01).

    Techniques Used: Inhibition, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Software, Labeling, Immunofluorescence, Staining, Viability Assay, Luminescence Assay

    miR-15a-5p directly regulates the expression of autophagy target genes. ( A , B ) K562 cells were transiently transfected with miR-15a-5p mimic (15a) or scrambled mimic (Sc) as control and treated with 2 µM of daunorubicin (DNR) for 24 h before protein and RNA extraction. ( A ) Protein expression of ATG9A, ATG14, and GABARAPL1 was analyzed by Western blotting. Expression of β-actin was also detected as a loading control. ( B ) The expression of ATG9A, ATG14, GABARAPL1, and SMPD1 was measured by RT-qPCR and normalized with the expression of a housekeeping gene RPLP0. ( C ) HEK 293T cells were co-transfected with wild-type (WT) or mutated (Mut∆) pMIR-luciferase vector, with either miR-15a-5p mimic or the scrambled oligonucleotide as control, and the pEF-β-galactosidase vector as internal control. After 24 h of transfection, cells were lysed, and the luminescence and β-gal activities were measured. ( B , C ) The normalized means of three independent experiments are shown with SEM. The scrambled condition was set to 1. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the scrambled condition, and in grey: miR-15a conditions compared with scrambled conditions, n.s.: non-significant).
    Figure Legend Snippet: miR-15a-5p directly regulates the expression of autophagy target genes. ( A , B ) K562 cells were transiently transfected with miR-15a-5p mimic (15a) or scrambled mimic (Sc) as control and treated with 2 µM of daunorubicin (DNR) for 24 h before protein and RNA extraction. ( A ) Protein expression of ATG9A, ATG14, and GABARAPL1 was analyzed by Western blotting. Expression of β-actin was also detected as a loading control. ( B ) The expression of ATG9A, ATG14, GABARAPL1, and SMPD1 was measured by RT-qPCR and normalized with the expression of a housekeeping gene RPLP0. ( C ) HEK 293T cells were co-transfected with wild-type (WT) or mutated (Mut∆) pMIR-luciferase vector, with either miR-15a-5p mimic or the scrambled oligonucleotide as control, and the pEF-β-galactosidase vector as internal control. After 24 h of transfection, cells were lysed, and the luminescence and β-gal activities were measured. ( B , C ) The normalized means of three independent experiments are shown with SEM. The scrambled condition was set to 1. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the scrambled condition, and in grey: miR-15a conditions compared with scrambled conditions, n.s.: non-significant).

    Techniques Used: Expressing, Transfection, Control, RNA Extraction, Western Blot, Quantitative RT-PCR, Luciferase, Plasmid Preparation

    miR-15a-5p regulates autophagy by targeting autophagy genes. K562 cells were transiently co-transfected with miR-15a-5p inhibitor or scrambled inhibitor as control and with either a combination of four siRNA-targets ( ATG9A, ATG14, GABARAPL1, and SMPD1 ) or a siRNA-control for 24 h before protein and RNA extractions. ( A ) Level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Protein expression of ATG9A, ATG14, and GABARAPL1 was also analyzed, as well as the expression of β-actin as a loading control. ( B ) In all the conditions, the expression of miR-15a-5p, ATG9A, ATG14, GABARAPL1, and SMPD1 was measured by RT-qPCR and normalized with the expression of RNU44 for miR-15a-5p or with RPLP0 for the autophagy target genes. The scrambled inhibitor and siRNA condition was set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, all the conditions compared with the scrambled condition).
    Figure Legend Snippet: miR-15a-5p regulates autophagy by targeting autophagy genes. K562 cells were transiently co-transfected with miR-15a-5p inhibitor or scrambled inhibitor as control and with either a combination of four siRNA-targets ( ATG9A, ATG14, GABARAPL1, and SMPD1 ) or a siRNA-control for 24 h before protein and RNA extractions. ( A ) Level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Protein expression of ATG9A, ATG14, and GABARAPL1 was also analyzed, as well as the expression of β-actin as a loading control. ( B ) In all the conditions, the expression of miR-15a-5p, ATG9A, ATG14, GABARAPL1, and SMPD1 was measured by RT-qPCR and normalized with the expression of RNU44 for miR-15a-5p or with RPLP0 for the autophagy target genes. The scrambled inhibitor and siRNA condition was set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, all the conditions compared with the scrambled condition).

    Techniques Used: Transfection, Control, Western Blot, Expressing, Quantitative RT-PCR



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    Image Search Results


    Treatment with daunorubicin increases autophagy in K562 cells. ( A ) Schematic representation of the autophagy process with its components and related molecules. ( B ) K562 cells were treated with different indicated doses of daunorubicin (DNR) or vehicle (DMSO) for 24 h. ( C , D ) K562 cells were treated with 2 µM of daunorubicin for 24 h and 10 µM of chloroquine (CQ) for the last 4 h. ( B , C ) Level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. The untreated conditions were set to 1. The normalized means are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the untreated conditions, and in grey: CQ conditions compared with DNR and CQ conditions). ( D ) Double-labeling immunofluorescence and representative images of LC3 (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown.

    Journal: International Journal of Molecular Sciences

    Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

    doi: 10.3390/ijms22105153

    Figure Lengend Snippet: Treatment with daunorubicin increases autophagy in K562 cells. ( A ) Schematic representation of the autophagy process with its components and related molecules. ( B ) K562 cells were treated with different indicated doses of daunorubicin (DNR) or vehicle (DMSO) for 24 h. ( C , D ) K562 cells were treated with 2 µM of daunorubicin for 24 h and 10 µM of chloroquine (CQ) for the last 4 h. ( B , C ) Level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. The untreated conditions were set to 1. The normalized means are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the untreated conditions, and in grey: CQ conditions compared with DNR and CQ conditions). ( D ) Double-labeling immunofluorescence and representative images of LC3 (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown.

    Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

    Techniques: Western Blot, Expressing, Control, Software, Labeling, Immunofluorescence, Staining

    Treatment with 3-MA partially prevents the effects of DNR on autophagy and cell growth. K562 were treated with 2 µM of daunorubicin and 3 mM of 3-methyladenine (3-MA). ( A ) Proteins were extracted after 24 h, and the level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. The untreated conditions were set to 1. The normalized means are shown with SEM. (*, p < 0.05, **, p < 0.01, in black: all the conditions compared with the untreated conditions, and in grey: DNR conditions compared with DNR and 3-MA conditions). ( B ) Viable cells were counted in the presence of Trypan Blue after 1, 2, 3, and 4 days of culture. The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (***, p < 0.001).

    Journal: International Journal of Molecular Sciences

    Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

    doi: 10.3390/ijms22105153

    Figure Lengend Snippet: Treatment with 3-MA partially prevents the effects of DNR on autophagy and cell growth. K562 were treated with 2 µM of daunorubicin and 3 mM of 3-methyladenine (3-MA). ( A ) Proteins were extracted after 24 h, and the level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. The untreated conditions were set to 1. The normalized means are shown with SEM. (*, p < 0.05, **, p < 0.01, in black: all the conditions compared with the untreated conditions, and in grey: DNR conditions compared with DNR and 3-MA conditions). ( B ) Viable cells were counted in the presence of Trypan Blue after 1, 2, 3, and 4 days of culture. The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (***, p < 0.001).

    Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

    Techniques: Western Blot, Expressing, Control, Software

    miR-15a-5p inhibits autophagy induced by daunorubicin. K562 cells were transiently transfected with miR-15a-5p mimic (15a) or scrambled mimic (Sc) as control for 48 h, and cells were treated with 2 µM of daunorubicin (DNR) for the last 24 h and with 10 µM of chloroquine (CQ) for the last 16 h. ( A ) RNA was extracted and miR-15a-5p expression was measured by RT-qPCR. Normalization was completed with the endogenous control RNU44 . ( B ) Proteins were extracted, and the level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. ( C ) The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. ( D ) Double-labeling immunofluorescence and representative images of LC3 (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown. LC3B and LAMP-2 immunofluorescence intensities of three independent experiments were quantified using the ImageJ software and divided by the nuclear immunofluorescence. ( A , C , D ) The untreated scrambled condition was set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the untreated scrambled condition, and in grey: miR-15a conditions compared with scrambled conditions).

    Journal: International Journal of Molecular Sciences

    Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

    doi: 10.3390/ijms22105153

    Figure Lengend Snippet: miR-15a-5p inhibits autophagy induced by daunorubicin. K562 cells were transiently transfected with miR-15a-5p mimic (15a) or scrambled mimic (Sc) as control for 48 h, and cells were treated with 2 µM of daunorubicin (DNR) for the last 24 h and with 10 µM of chloroquine (CQ) for the last 16 h. ( A ) RNA was extracted and miR-15a-5p expression was measured by RT-qPCR. Normalization was completed with the endogenous control RNU44 . ( B ) Proteins were extracted, and the level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. ( C ) The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. ( D ) Double-labeling immunofluorescence and representative images of LC3 (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown. LC3B and LAMP-2 immunofluorescence intensities of three independent experiments were quantified using the ImageJ software and divided by the nuclear immunofluorescence. ( A , C , D ) The untreated scrambled condition was set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the untreated scrambled condition, and in grey: miR-15a conditions compared with scrambled conditions).

    Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

    Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Software, Labeling, Immunofluorescence, Staining

    miR-15a-5p decreases the downregulation of cell growth by daunorubicin. K562 cells were transiently transfected with miR-15a-5p mimic (miR-15a) or scrambled mimic (Sc) as control, and cells were treated with 0.1 µM of daunorubicin (DNR), 0.5 µM of DNR, or vehicle for 72 h ( A ) Viable cells were counted in the presence of Trypan Blue after 1, 2, and 3 days of culture. The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (*, p < 0.05, ***, p < 0.001). ( B ) Cell viability assay (CellTiter-Glo ® Luminescent assay) was performed at 72 h of treatment. The untreated scrambled condition was set to 1. The normalized means are shown with SEM (**, p < 0.01).

    Journal: International Journal of Molecular Sciences

    Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

    doi: 10.3390/ijms22105153

    Figure Lengend Snippet: miR-15a-5p decreases the downregulation of cell growth by daunorubicin. K562 cells were transiently transfected with miR-15a-5p mimic (miR-15a) or scrambled mimic (Sc) as control, and cells were treated with 0.1 µM of daunorubicin (DNR), 0.5 µM of DNR, or vehicle for 72 h ( A ) Viable cells were counted in the presence of Trypan Blue after 1, 2, and 3 days of culture. The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (*, p < 0.05, ***, p < 0.001). ( B ) Cell viability assay (CellTiter-Glo ® Luminescent assay) was performed at 72 h of treatment. The untreated scrambled condition was set to 1. The normalized means are shown with SEM (**, p < 0.01).

    Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

    Techniques: Transfection, Control, Viability Assay, Luminescence Assay

    Inhibition of mir-15a-5p induces autophagy. ( A – C ) K562 cells were transiently transfected with miR-15a-5p inhibitor (15ai) or scrambled inhibitor (Sci) as control for 48 h, and cells were treated with 2 µM of daunorubicin (DNR) for the last 24 h. ( A ) RNA was extracted and miR-15a-5p expression was measured by RT-qPCR and normalized with the expression of RNU44 . ( B ) Protein were extracted, and level of autophagy was analyzed by Western blot with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. ( C ) The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. ( D ) Double-labeling immunofluorescence and representative images of LC3B (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown. LC3B and LAMP-2 immunofluorescence intensities of three independent experiments were quantified using the ImageJ software and divided by the nuclear immunofluorescence. ( D , E ) K562 cells were transiently transfected with miR-15a-5p inhibitor (miR-15ai) or scrambled inhibitor (Sci) as control, and cells were treated with 0.1 µM of daunorubicin (DNR), 0.5 µM of DNR, or vehicle for 72 h. ( D ) Viable cells were counted in the presence of Trypan Blue after 1, 2, and 3 days of culture. ( E ) Cell viability assay (CellTiter-Glo ® Luminescent assay) was performed at 72 h of treatment. ( A – C , E ) The untreated scrambled conditions were set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, all the conditions compared with the untreated scrambled condition). ( D ) The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (**, p < 0.01).

    Journal: International Journal of Molecular Sciences

    Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

    doi: 10.3390/ijms22105153

    Figure Lengend Snippet: Inhibition of mir-15a-5p induces autophagy. ( A – C ) K562 cells were transiently transfected with miR-15a-5p inhibitor (15ai) or scrambled inhibitor (Sci) as control for 48 h, and cells were treated with 2 µM of daunorubicin (DNR) for the last 24 h. ( A ) RNA was extracted and miR-15a-5p expression was measured by RT-qPCR and normalized with the expression of RNU44 . ( B ) Protein were extracted, and level of autophagy was analyzed by Western blot with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. ( C ) The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. ( D ) Double-labeling immunofluorescence and representative images of LC3B (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown. LC3B and LAMP-2 immunofluorescence intensities of three independent experiments were quantified using the ImageJ software and divided by the nuclear immunofluorescence. ( D , E ) K562 cells were transiently transfected with miR-15a-5p inhibitor (miR-15ai) or scrambled inhibitor (Sci) as control, and cells were treated with 0.1 µM of daunorubicin (DNR), 0.5 µM of DNR, or vehicle for 72 h. ( D ) Viable cells were counted in the presence of Trypan Blue after 1, 2, and 3 days of culture. ( E ) Cell viability assay (CellTiter-Glo ® Luminescent assay) was performed at 72 h of treatment. ( A – C , E ) The untreated scrambled conditions were set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, all the conditions compared with the untreated scrambled condition). ( D ) The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (**, p < 0.01).

    Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

    Techniques: Inhibition, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Software, Labeling, Immunofluorescence, Staining, Viability Assay, Luminescence Assay

    miR-15a-5p directly regulates the expression of autophagy target genes. ( A , B ) K562 cells were transiently transfected with miR-15a-5p mimic (15a) or scrambled mimic (Sc) as control and treated with 2 µM of daunorubicin (DNR) for 24 h before protein and RNA extraction. ( A ) Protein expression of ATG9A, ATG14, and GABARAPL1 was analyzed by Western blotting. Expression of β-actin was also detected as a loading control. ( B ) The expression of ATG9A, ATG14, GABARAPL1, and SMPD1 was measured by RT-qPCR and normalized with the expression of a housekeeping gene RPLP0. ( C ) HEK 293T cells were co-transfected with wild-type (WT) or mutated (Mut∆) pMIR-luciferase vector, with either miR-15a-5p mimic or the scrambled oligonucleotide as control, and the pEF-β-galactosidase vector as internal control. After 24 h of transfection, cells were lysed, and the luminescence and β-gal activities were measured. ( B , C ) The normalized means of three independent experiments are shown with SEM. The scrambled condition was set to 1. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the scrambled condition, and in grey: miR-15a conditions compared with scrambled conditions, n.s.: non-significant).

    Journal: International Journal of Molecular Sciences

    Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

    doi: 10.3390/ijms22105153

    Figure Lengend Snippet: miR-15a-5p directly regulates the expression of autophagy target genes. ( A , B ) K562 cells were transiently transfected with miR-15a-5p mimic (15a) or scrambled mimic (Sc) as control and treated with 2 µM of daunorubicin (DNR) for 24 h before protein and RNA extraction. ( A ) Protein expression of ATG9A, ATG14, and GABARAPL1 was analyzed by Western blotting. Expression of β-actin was also detected as a loading control. ( B ) The expression of ATG9A, ATG14, GABARAPL1, and SMPD1 was measured by RT-qPCR and normalized with the expression of a housekeeping gene RPLP0. ( C ) HEK 293T cells were co-transfected with wild-type (WT) or mutated (Mut∆) pMIR-luciferase vector, with either miR-15a-5p mimic or the scrambled oligonucleotide as control, and the pEF-β-galactosidase vector as internal control. After 24 h of transfection, cells were lysed, and the luminescence and β-gal activities were measured. ( B , C ) The normalized means of three independent experiments are shown with SEM. The scrambled condition was set to 1. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the scrambled condition, and in grey: miR-15a conditions compared with scrambled conditions, n.s.: non-significant).

    Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

    Techniques: Expressing, Transfection, Control, RNA Extraction, Western Blot, Quantitative RT-PCR, Luciferase, Plasmid Preparation

    miR-15a-5p regulates autophagy by targeting autophagy genes. K562 cells were transiently co-transfected with miR-15a-5p inhibitor or scrambled inhibitor as control and with either a combination of four siRNA-targets ( ATG9A, ATG14, GABARAPL1, and SMPD1 ) or a siRNA-control for 24 h before protein and RNA extractions. ( A ) Level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Protein expression of ATG9A, ATG14, and GABARAPL1 was also analyzed, as well as the expression of β-actin as a loading control. ( B ) In all the conditions, the expression of miR-15a-5p, ATG9A, ATG14, GABARAPL1, and SMPD1 was measured by RT-qPCR and normalized with the expression of RNU44 for miR-15a-5p or with RPLP0 for the autophagy target genes. The scrambled inhibitor and siRNA condition was set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, all the conditions compared with the scrambled condition).

    Journal: International Journal of Molecular Sciences

    Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

    doi: 10.3390/ijms22105153

    Figure Lengend Snippet: miR-15a-5p regulates autophagy by targeting autophagy genes. K562 cells were transiently co-transfected with miR-15a-5p inhibitor or scrambled inhibitor as control and with either a combination of four siRNA-targets ( ATG9A, ATG14, GABARAPL1, and SMPD1 ) or a siRNA-control for 24 h before protein and RNA extractions. ( A ) Level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Protein expression of ATG9A, ATG14, and GABARAPL1 was also analyzed, as well as the expression of β-actin as a loading control. ( B ) In all the conditions, the expression of miR-15a-5p, ATG9A, ATG14, GABARAPL1, and SMPD1 was measured by RT-qPCR and normalized with the expression of RNU44 for miR-15a-5p or with RPLP0 for the autophagy target genes. The scrambled inhibitor and siRNA condition was set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, all the conditions compared with the scrambled condition).

    Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

    Techniques: Transfection, Control, Western Blot, Expressing, Quantitative RT-PCR

    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 <xref ref-type=Figure S1 and , , and . " width="100%" height="100%">

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

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

    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 .

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

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

    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 .

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

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

    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 <xref ref-type=Figure S2 . " width="100%" height="100%">

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

    Techniques: Inhibition

    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 ( <xref ref-type=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 . " width="100%" height="100%">

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

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

    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 <xref ref-type=Figure S4 . " width="100%" height="100%">

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

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

    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 ( <xref ref-type=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 . " width="100%" height="100%">

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

    Techniques: Inhibition, RNA Sequencing, ChIP-sequencing

    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 <xref ref-type=Figure S6 . " width="100%" height="100%">

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

    Techniques: Inhibition, Control, 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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

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

    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 <xref ref-type=Figure S1 and , , and . " width="100%" height="100%">

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

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

    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 .

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

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

    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 .

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

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

    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 <xref ref-type=Figure S2 . " width="100%" height="100%">

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

    Techniques: Inhibition

    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 ( <xref ref-type=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 . " width="100%" height="100%">

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

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

    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 <xref ref-type=Figure S4 . " width="100%" height="100%">

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

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

    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 ( <xref ref-type=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 . " width="100%" height="100%">

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

    Techniques: Inhibition, RNA Sequencing, ChIP-sequencing

    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 <xref ref-type=Figure S6 . " width="100%" height="100%">

    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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

    Techniques: Inhibition, Control, 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: The AML cell lines K562, HL60, MV4-11 (ATCC: CCL-243,CCL-240, CRL-9591), MOLM-13, NB4, OCI-AML2, OCI-AML3 (DSMZ: ACC-554, ACC-207, ACC-99, ACC-582), and THP1 containing a doxycycline-inducible CRISPR/Cas9 USP7 knockout model ( ) were cultured in RPMI 1640 (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FCS, HyClone Laboratories, Logan, Utah, US) and 1% penicillin/streptomycin (p/s, PAA Laboratories).

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