usp1 uaf1 complex  (R&D Systems)

Journal: Nature

Article Title: RNA transcripts stimulate homologous recombination by forming DR-loops

doi: 10.1038/s41586-021-03538-8

Figure Lengend Snippet: a, b, HR efficiency was measured by qPCR in transcriptionally on and off states (+Dox and −Dox, respectively) after knockdown of CtIP, BRCA1 or RAD51 using siRNA targeting CtIP (also known as RBB8), BRCA1 or RAD51, respectively (a). Ratios of HR efficiencies between the off and on states (−Dox/+Dox) were determined (b). Data are mean ± s.e.m. (n = 3 independent experiments). c, d, HR efficiency was measured by qPCR in transcriptionally on and off states (+Dox and −Dox, respectively) after knockdown of RAD51AP1 (c, d), CtIP (c) and UAF1 (d). Data are mean ± s.e.m. (n = 3 independent experiments). e, HR efficiency was measured by qPCR in control and RAD51AP1-knockdown cells with or without Dox and RNAg-t-GFP as indicated. Relative HR efficiencies were normalized to that of control cells in the transcriptionally on state (+Dox). Data are mean ± s.e.m. (n = 3 independent experiments). f, Sister chromatid exchange (SCE) was quantified in control and RAD51AP1-knockdown cells treated with 3 nM CPT, 200 nM etoposide or no drug. Data are mean ± s.d. (n = 25 cells analysed in 1 experiment for each condition). *P < 0.05, ***P < 0.001 (two-sided Student’s t test; P = 0. 014 for no drug; P = 0.0003 for CPT; P < 0.0001 for etoposide).

Article Snippet: Recombinant UAF1 protein was purchased from Bio-Techne Corporation (E-566).

Techniques:

Journal: Nature

Article Title: RNA transcripts stimulate homologous recombination by forming DR-loops

doi: 10.1038/s41586-021-03538-8

Figure Lengend Snippet: a, Cells were transfected with control or two independent RAD51AP1 siRNAs. Levels of endogenous RAD51AP1 and β-actin (loading control) were analysed by western blot. A representative western blot of three similar experiments is shown. b, HR efficiency was measured by FACS using U2OS-Tet-DR-GFP reporter cells after knockdown of RAD51AP1. Data are mean ± s.e.m (n = 3 independent experiments). c, Cells were transfected with control or UAF1 siRNA. Levels of UAF1 mRNA were analysed by RT–qPCR. Data are mean (n = 2 independent experiments). d, Cells were transfected with control, RAD51AP1 or UAF1 siRNA. Levels of endogenous RAD51AP1 and β-actin (loading control) were analysed by western blot. A representative western blot of three similar experiments is shown. e, HR efficiency was measured by FACS using U2OS-Tet-DR-GFP reporter cells after knockdown of RAD51AP1 or UAF1. Data are mean (n = 2 independent experiments). f, Cells were transfected with control or two independent RAD52 siRNAs. Levels of endogenous RAD52 and KU80 (loading control) were analysed by western blot. A representative western blot of three similar experiments is shown. g, HR efficiency was measured by FACS using U2OS-Tet-DR-GFP reporter cells after knockdown of RAD52. Data are mean ± s.e.m (n = 3 independent experiments). h, HR efficiency was measured by qPCR in transcriptionally on and off states (+/−Dox) after knockdown of RAD52. The HR efficiency of control siRNA transfected cells in the transcriptionally on state (+Dox) serves as a reference. Data are mean ± s.e.m (n = 3 independent experiments).

Article Snippet: Recombinant UAF1 protein was purchased from Bio-Techne Corporation (E-566).

Techniques: Transfection, Western Blot, Quantitative RT-PCR

Journal: Nature

Article Title: RNA transcripts stimulate homologous recombination by forming DR-loops

doi: 10.1038/s41586-021-03538-8

Figure Lengend Snippet: a, U2OS cells carrying an array of tetracycline responsive elements (TREs) were transfected with a plasmid expressing the TetR-VP16-KillerRed (TA-KR) fusion protein. The TA-KR fusion protein binds the TRE array and induces DSBs upon light activation. The formation of RAD51AP1 or RAD51 foci at the TA-KR-marked locus was analysed by immunostaining. Scale bars, 10 μm. b, Immunostaining of RAD51AP1 in cells that were not irradiated or were irradiated with 2 Gy IR. Cells were analysed 2 h after IR. Scale bars, 10 μm. c, Correlation between the numbers of RAD51AP1 and RPA32 foci in IR-treated cells as determined by linear regression. The numbers of RAD51AP1 and RPA32 foci were quantified in individual cells (n = 260 cells analysed in one experiment). Individual cells were plotted according to the numbers of RAD51AP1 and RPA32 foci in them. d–g, Asynchronously growing U2OS cells were treated with or without DRB for 4 h, exposed to 2 Gy IR or mock-treated, and analysed in 2 h. d, Immunostaining of RAD51AP1 and γH2AX. Scale bars, 10 μm. e, Numbers of RAD51AP1 foci in individual cells were plotted as mean ± s.d. (n = 401 cells for no IR, n = 408 cells for +IR, and n = 408 cells for +IR +DRB, analysed in one experiment). f, Numbers of γH2AX foci in individual cells were plotted as mean ± s.d. (n = 313 cells for − DRB and n = 294 cells for +DRB, analysed in one experiment). g, Numbers of RAD51AP1 foci in PCNA+ cells were plotted as mean ± s.d. (n = 360 cells for −DRB and n = 303 cells for +DRB, analysed in one experiment). h, i, U2OS cells transfected with control, UAF1 (h) or CtIP (i) siRNA were irradiated with 2 Gy IR. Immunostaining of RAD51AP1 was done 2 h after IR. Numbers of RAD51AP1 foci in individual cells were plotted as mean ±s.d. ***P < 0.001 (two-sided Student’s t test; P < 0.0001 in h, i. h, n = 483 cells for siCTRL, n = 527 cells for siUAF1 analysed in one experiment. i, n = 200 cells for siCTRL, n = 182 cells for siCtIP analysed in one experiment.

Article Snippet: Recombinant UAF1 protein was purchased from Bio-Techne Corporation (E-566).

Techniques: Transfection, Plasmid Preparation, Expressing, Activation Assay, Immunostaining, Irradiation

Journal: Nature

Article Title: RNA transcripts stimulate homologous recombination by forming DR-loops

doi: 10.1038/s41586-021-03538-8

Figure Lengend Snippet: a, In vitro D-loop formation with RAD51 or the RAD51AP1–UAF1 complex. RAD51 was incubated with labelled 60-nt ssDNA and then with RAD51AP1 or the RAD51AP1–UAF1 complex. A dsDNA plasmid containing a sequence homologous to the ssDNA was then added to the reactions. Formation of D-loops was analysed by native gel electrophoresis. Representative results from 2 similar experiments are shown. b, In vitro R-loop formation with the RAD51AP1–UAF1 complex. Preformed RAD51AP1–UAF1 complexes were incubated with labelled 63-nt ssRNA and then with a dsDNA plasmid containing a sequence homologous to the ssRNA. Formation of R-loops was analysed by native gel electrophoresis. The efficiency of R-loop formation was determined by quantifying the shifted and unshifted bands in a light exposure of the gel. Data are mean ± s.d. (n = 3 independent experiments).

Article Snippet: Recombinant UAF1 protein was purchased from Bio-Techne Corporation (E-566).

Techniques: In Vitro, Incubation, Plasmid Preparation, Sequencing, Nucleic Acid Electrophoresis

Journal: Theranostics

Article Title: CRISPR/Cas9-based genome-wide screening for deubiquitinase subfamily identifies USP1 regulating MAST1-driven cisplatin-resistance in cancer cells.

doi: 10.7150/thno.72826

Figure Lengend Snippet: Figure 3. USP1 interacts with and regulates the MAST1 protein. (A) Schematic representation of the sgRNAs targeting exon 5 of the USP1 gene. Red arrowheads indicate the positions of sgRNAs that target the top strand. sgRNA sequences are in red; PAM sequences are in bold blue font. (B) Validation of sgRNA efficiency targeting USP1 by transient transfection of sgRNA1 and sgRNA2 into HeLa cells and immunoblotting with USP1 antibody. (C) HeLa cells were transfected with sgRNA1 and shRNA1 targeting USP1, and the endogenous protein levels of USP1 and MAST1 were checked by Western blotting. (D) HeLa cells were transfected with increasing concentrations of Flag-USP1 to check the endogenous MAST1 protein level. (E) HeLa cells were transfected with increasing concentrations of Flag-USP1CS to assess the endogenous MAST1 protein level. (F) The reconstitution effect of Flag-USP1 on endogenous MAST1 protein in USP1-depleted HeLa cells. The protein band intensities (Fig 3C-F) were estimated using ImageJ software with reference to the GAPDH control band for each individual sgRNA (MAST1/GAPDH) and presented below the blot. (G) Interactions between endogenous and (H)

Article Snippet: The cells were lysed using IP lysis buffer and then incubated with TUBE2 beads at 4 °C for 3 h. The beads were washed three times with IP lysis buffer and two times with ubiquitination buffer (50 mM Tris‐HCl (pH 8.0), 10 mM MgCl2, 0.2 mM CaCl2, and 1 mM DTT) along with protease inhibitor cocktail, followed by incubation with 1.5 μg of recombinant USP1 protein (rUSP1) (catalog No. E-568-050; R&D Systems) at 37 °C for 1 h. The rUSP1treated samples were eluted with 30 μL of 2X SDS sample loading buffer and boiled for 5 min before subjecting to Western blot analysis.

Techniques: Biomarker Discovery, Transfection, Western Blot, Software, Control

Journal: Theranostics

Article Title: CRISPR/Cas9-based genome-wide screening for deubiquitinase subfamily identifies USP1 regulating MAST1-driven cisplatin-resistance in cancer cells.

doi: 10.7150/thno.72826

Figure Lengend Snippet: Figure 5. USP1 extends MAST1 protein half-life by its deubiquitinating activity. (A) The ubiquitination and deubiquitination of endogenous MAST1 were analyzed by transfecting HeLa cells with Flag-USP1, Flag-USP1CS, or sgRNA targeting USP1 followed by immunoprecipitation with an anti-MAST1 antibody and immunoblotting with an anti-ubiquitin antibody. The cells were treated with MG132 for 6 h prior to harvest. (B) The K48- and K63-linked polyubiquitination of MAST1 was analyzed by transfecting HEK293 cells with Myc-MAST1, HA-ubiquitin, HA-K48-ubiquitin, and HA-K63-ubiquitin, followed by immunoprecipitation with an anti-Myc antibody and immunoblotting with anti-HA and anti-Myc antibodies. (C) The deubiquitination of K48-linked ubiquitination of MAST1 by USP1 was analyzed by transfecting HEK293 cells with Myc-MAST1 and HA-K48-ubiquitin along with Flag-USP1 or Flag-USP1CS, followed by immunoprecipitation with an anti-Myc antibody and immunoblotting with anti-HA and anti-Myc antibodies.

Article Snippet: The cells were lysed using IP lysis buffer and then incubated with TUBE2 beads at 4 °C for 3 h. The beads were washed three times with IP lysis buffer and two times with ubiquitination buffer (50 mM Tris‐HCl (pH 8.0), 10 mM MgCl2, 0.2 mM CaCl2, and 1 mM DTT) along with protease inhibitor cocktail, followed by incubation with 1.5 μg of recombinant USP1 protein (rUSP1) (catalog No. E-568-050; R&D Systems) at 37 °C for 1 h. The rUSP1treated samples were eluted with 30 μL of 2X SDS sample loading buffer and boiled for 5 min before subjecting to Western blot analysis.

Techniques: Activity Assay, Ubiquitin Proteomics, Immunoprecipitation, Western Blot

Journal: Theranostics

Article Title: CRISPR/Cas9-based genome-wide screening for deubiquitinase subfamily identifies USP1 regulating MAST1-driven cisplatin-resistance in cancer cells.

doi: 10.7150/thno.72826

Figure Lengend Snippet: Figure 6. Clinical correlation between USP1 and MAST1 expression in various cancer tissues. (A) Box plot showing the difference between USP1 expression in tumor and normal tissues using Correlation AnalyzeR. Significance was determined via the Wilcoxon rank sum test: ****P < 0.0001. (B) Box plot showing the difference between MAST1 expression in tumor and normal tissues using Correlation AnalyzeR. Significance was determined via the Wilcoxon rank sum test: *P < .05, ****P < 0.0001. VST stands for variance-stabilizing transform. (C) A heat map showing mRNA expression levels of USP1 and MAST1 derived from the CCLE database. Representative samples are arranged from high to low mRNA levels of MAST1, and corresponding USP1 values are sorted. (D) A scatterplot showing the expression correlation between USP1 and MAST1 mRNA levels. Pearson correlations (r) quantifying the relationship between USP1 and MAST1 are given. (E) Endogenous protein expression patterns of USP1 and MAST1 in different cancer and non-cancer cell lines were assessed by Western blotting. GAPDH was used as the loading control. (F–H) Representative immunohistochemical (IHC) staining images of endogenous USP1 and MAST1 in (F) human lung cancer (n = 32), (G) colon cancer (n = 32), and (H) breast cancer (n = 21) tissues. All IHC images were quantified with an H-score. Scale bar = 30 µm.

Article Snippet: The cells were lysed using IP lysis buffer and then incubated with TUBE2 beads at 4 °C for 3 h. The beads were washed three times with IP lysis buffer and two times with ubiquitination buffer (50 mM Tris‐HCl (pH 8.0), 10 mM MgCl2, 0.2 mM CaCl2, and 1 mM DTT) along with protease inhibitor cocktail, followed by incubation with 1.5 μg of recombinant USP1 protein (rUSP1) (catalog No. E-568-050; R&D Systems) at 37 °C for 1 h. The rUSP1treated samples were eluted with 30 μL of 2X SDS sample loading buffer and boiled for 5 min before subjecting to Western blot analysis.

Techniques: Expressing, Derivative Assay, Western Blot, Control, Immunohistochemical staining, Immunohistochemistry

Journal: Theranostics

Article Title: CRISPR/Cas9-based genome-wide screening for deubiquitinase subfamily identifies USP1 regulating MAST1-driven cisplatin-resistance in cancer cells.

doi: 10.7150/thno.72826

Figure Lengend Snippet: Figure 7. Depletion of USP1 promotes apoptosis, DNA damage, and tumor growth arrest. Mock control, USP1-KO1, and USP1-KO1 cells reconstituted with either USP1 or MAST1 were used to perform the following experiments. (A) Western blot analyses to validate the expression of USP1 and MAST1 using USP1- and MAST1-specific antibodies. GAPDH was used as the loading control. (B) The cells were treated with either vehicle or cisplatin (2 µg/mL) for 24 h and subjected to immunofluorescence analysis to estimate γH2AX foci formation. Green, γH2AX; blue, nucleus stained by DAPI. Scale bar = 100 µm. The right panel depicts the percentage of γH2AX-positive cells. (C) The cells were treated with cisplatin (2 µg/mL) for 24 h, and MEK1 activation and apoptosis-related factors were determined using Western blotting. GAPDH was used as the internal loading control. (D) The cells were treated with either vehicle or cisplatin (2 µg/mL) for 48 h and subjected to flow cytometry to measure the DNA content using PI staining and (E) annexin-V and 7-AAD staining. (F) The cells were treated with a sub-lethal dose of cisplatin (2 µg/mL) for 48 h, and cell viability was assayed using CCK-8 reagent. Data are presented as the mean and standard deviation of three independent experiments (n = 3). (G-I) Vehicle- or cisplatin-treated cells were

Article Snippet: The cells were lysed using IP lysis buffer and then incubated with TUBE2 beads at 4 °C for 3 h. The beads were washed three times with IP lysis buffer and two times with ubiquitination buffer (50 mM Tris‐HCl (pH 8.0), 10 mM MgCl2, 0.2 mM CaCl2, and 1 mM DTT) along with protease inhibitor cocktail, followed by incubation with 1.5 μg of recombinant USP1 protein (rUSP1) (catalog No. E-568-050; R&D Systems) at 37 °C for 1 h. The rUSP1treated samples were eluted with 30 μL of 2X SDS sample loading buffer and boiled for 5 min before subjecting to Western blot analysis.

Techniques: Control, Western Blot, Expressing, Immunofluorescence, Staining, Activation Assay, Flow Cytometry, CCK-8 Assay, Standard Deviation

Journal: Theranostics

Article Title: CRISPR/Cas9-based genome-wide screening for deubiquitinase subfamily identifies USP1 regulating MAST1-driven cisplatin-resistance in cancer cells.

doi: 10.7150/thno.72826

Figure Lengend Snippet: Figure 8. Combination of pimozide and lestaurtinib inhibits MAST1 protein and cisplatin-resistant tumor growth more than either single treatment. (A) The effect of USP1 inhibition on MAST1 protein level was determined by treating HeLa-cisR cells with increasing concentrations of pimozide for 24 h. The protein expression of MAST1 was determined by Western blotting. GAPDH was used as an internal loading control. (B) The effect of combination treatment of pimozide and lestaurtinib on MAST1-mediated MEK phosphorylation. HeLa-cisR cells were treated with pimozide (50 µM) and lestaurtinib (200 nM) in the presence of sub-lethal doses of cisplatin (5 µg/mL) for 24 h. The activity of MAST1 was assessed by a Western blot analysis of the phospho-MEK1 and phospho-ERK levels. GAPDH was used as an internal loading control. (C, D) The effect of combined treatment with pimozide and lestaurtinib on (C) cisplatin sensitivity (n = 3) (D) and cell viability in A549-cisR and HeLa-cisR cells (n = 4). Data are presented as the mean and standard deviation of at least three independent experiments. Two-way ANOVA followed by Tukey's post hoc test was used with the indicated P values. (E) Combination index (CI) plots for the synergistic effect of pimozide and lestaurtinib in A549-cisR and HeLa-cisR cells. (F–H) The effect of combination treatment with pimozide and lestaurtinib was validated using (F) colony formation assay, (G) wound-healing assay, and (H) Transwell cell-invasion assay. Data are presented as the mean and standard deviation of three independent experiments (n = 3). Two-way ANOVA followed by Tukey's post hoc test was used with the indicated P values. (I) Xenografts were generated by subcutaneously injecting A549-cisR cells into the right flanks of NSG mice (n = 4). Mice were treated with pimozide (10 mg/kg), lestaurtinib (20 mg/kg), and cisplatin (5 mg/kg) beginning 26 days after xenograft implantation, and tumor size was monitored. The right panel shows the tumors excised from the mice after the experiment. (J) Tumor volume and tumor weight were measured and are presented graphically. Data are presented as the mean and standard deviation of four independent experiments (n = 4). Two-way ANOVA followed by Tukey's post hoc test was used with the indicated P values. For brevity, statistical significance is shown only for comparisons between the groups of interest, except for the negative control group.

Article Snippet: The cells were lysed using IP lysis buffer and then incubated with TUBE2 beads at 4 °C for 3 h. The beads were washed three times with IP lysis buffer and two times with ubiquitination buffer (50 mM Tris‐HCl (pH 8.0), 10 mM MgCl2, 0.2 mM CaCl2, and 1 mM DTT) along with protease inhibitor cocktail, followed by incubation with 1.5 μg of recombinant USP1 protein (rUSP1) (catalog No. E-568-050; R&D Systems) at 37 °C for 1 h. The rUSP1treated samples were eluted with 30 μL of 2X SDS sample loading buffer and boiled for 5 min before subjecting to Western blot analysis.

Techniques: Inhibition, Expressing, Western Blot, Control, Phospho-proteomics, Activity Assay, Standard Deviation, Colony Assay, Wound Healing Assay, Invasion Assay, Generated, Negative Control