Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a Immunoblotting of Drosha, DGCR8, and β-actin in the LM2-DRR (expressing the pLCN DSB Repair Reporter) cell line transduced with DGCR8 shRNA. b Knockdown of DGCR8 decreased HR and NHEJ efficiency in LM2-DRR cells. Two days after co-transfection of I-SceI endonuclease and an exogenous donor for HR (pCAGGS DRR mCherry Donor EF1a BFP) into the DGCR8-knockdown LM2-DRR cells, the percentages of GFP-positive and mCherry-positive cells, gated on BFP-positive cells, were determined by flow cytometry. Repair by HR or NHEJ leads to mCherry or GFP expression. Data were normalized to the control cells. n = 3 biological replicates. c MYC-DGCR8-overexpressing LM2 cells were treated with IR (8 Gy) and cultured for 1 h, followed by pulldown with MYC beads and immunoblotting with the indicated antibodies. d Control and DGCR8-knockdown LM2 cells were treated with IR (8 Gy) and cultured for 1 h, followed by immunoprecipitation with an antibody against RNF168 or RNF8 and immunoblotting with the indicated antibodies. e Chromatin was extracted from LM2 cells that were treated with IR (8 Gy) and cultured for 1 h. The chromatin fractions, with or without MNase treatment, were immunoprecipitated with a DGCR8-specific antibody and immunoblotted with the indicated antibodies. f Quantification of MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in DGCR8-knockdown LM2 cells. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. g Control and DGCR8-knockdown LM2 cells with stable overexpression of FLAG-H2A and RNF8 or RNF168 were transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. LE long exposure, SE short exposure. Statistical significance in b and f was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. n.s . not statistically significant. Source data are provided as a file.

Article Snippet: The DGCR8 (#10921), Drosha (#10921), Dicer (#10921), Exportin-5 (#10921), HA-ubiquitin (WT: #17608; K48R: #17604; K48: #17605; K63: #17606), RNF8 (#99396), FLAG-H2A (#63560), p53 (#81754), pLCN DSB Repair Reporter (DRR) (#98895), and pCAGGS DRR mCherry Donor EF1a BFP (#98896) constructs were from Addgene.

Techniques: Western Blot, Expressing, Transduction, shRNA, Knockdown, Cotransfection, Flow Cytometry, Control, Cell Culture, Immunoprecipitation, Incubation, Over Expression, Transfection, Ubiquitin Proteomics, Lysis, Sonication, Two Tailed Test

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a Immunoblotting of USP36, USP51, and β-actin in parental and radioresistant LM2 cells with and without IR treatment (8 Gy followed by 24-h incubation). b Immunoblotting of DGCR8, USP36, USP51, and β-actin in USP36-knockdown and USP51-knockdown LM2 cells with or without IR treatment (8 Gy followed by 24-h incubation). c Co-IP of endogenous DGCR8 with endogenous USP51. LM2 and LM2-R cells were treated with 8-Gy IR. After 8 h, cells were lysed, immunoprecipitated with a DGCR8-specific antibody, and immunoblotted with antibodies against USP51 and DGCR8. SE short exposure, LE long exposure. d HEK293T cells with stable overexpression of MYC-DGCR8 were co-transfected with SFB-USP51 (wild-type or the C372S mutant) and HA-tagged ubiquitin or the lysine-specific mutant (K48 or K63), and then treated with IR (8 Gy). After 8 h, cells were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. e Knockdown of USP51 decreased HR and NHEJ efficiency in LM2-DRR cells. Two days after co-transfection of I-SceI endonuclease and an exogenous donor for HR (pCAGGS DRR mCherry Donor EF1a BFP) into the USP51-knockdown LM2-DRR cells, the percentages of GFP-positive and mCherry-positive cells, gated on BFP-positive cells, were determined by flow cytometry. Repair by HR or NHEJ leads to mCherry or GFP expression. Data were normalized to the control cells. n = 3 biological replicates. f Quantification of γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in USP51-knockdown LM2 cells. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. g Control and USP51-knockdown LM2 cells with stable overexpression of FLAG-H2A and RNF8 or RNF168 were transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. Statistical significance in e and f was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. Source data are provided as a file.

Article Snippet: The DGCR8 (#10921), Drosha (#10921), Dicer (#10921), Exportin-5 (#10921), HA-ubiquitin (WT: #17608; K48R: #17604; K48: #17605; K63: #17606), RNF8 (#99396), FLAG-H2A (#63560), p53 (#81754), pLCN DSB Repair Reporter (DRR) (#98895), and pCAGGS DRR mCherry Donor EF1a BFP (#98896) constructs were from Addgene.

Techniques: Western Blot, Incubation, Knockdown, Co-Immunoprecipitation Assay, Immunoprecipitation, Over Expression, Transfection, Mutagenesis, Ubiquitin Proteomics, Cotransfection, Flow Cytometry, Expressing, Control, Cell Culture, Lysis, Sonication, Two Tailed Test

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a , b MYC-GFP-, WT DGCR8-, S677A-DGCR8-, and S677D-DGCR8-overexpressing LM2 cells with or without IR treatment ( a , 8 Gy followed by 1-h incubation; b , 8 Gy followed by 8-h incubation) were subjected to pulldown with MYC beads and immunoblotting with the indicated antibodies. c HEK293T cells with stable overexpression of MYC-tagged WT DGCR8, S677A-DGCR8, or S677D-DGCR8 were co-transfected with SFB-USP51 (WT or the C372S mutant) and HA-tagged ubiquitin, and then treated with IR (8 Gy). After 8 h, cells were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. d Quantification of γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in DRCR8-knockdown LM2 cells with ectopic expression of WT DGCR8, S677A-DGCR8, or S677D-DGCR8. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. e DRCR8-knockdown LM2 cells with ectopic expression of WT DGCR8 or the S677A mutant were transduced with FLAG-H2A and RNF8 or RNF168. The cells were then transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. LE long exposure, SE short exposure. Source data are provided as a file.

Article Snippet: The DGCR8 (#10921), Drosha (#10921), Dicer (#10921), Exportin-5 (#10921), HA-ubiquitin (WT: #17608; K48R: #17604; K48: #17605; K63: #17606), RNF8 (#99396), FLAG-H2A (#63560), p53 (#81754), pLCN DSB Repair Reporter (DRR) (#98895), and pCAGGS DRR mCherry Donor EF1a BFP (#98896) constructs were from Addgene.

Techniques: Incubation, Western Blot, Over Expression, Transfection, Mutagenesis, Ubiquitin Proteomics, Immunoprecipitation, Knockdown, Expressing, Two Tailed Test, Transduction, Cell Culture, Lysis, Sonication

Journal: Human Genetics and Genomics Advances

Article Title: Prediction of breast cancer risk based on flow variant analysis of circulating peripheral blood mononuclear cells

doi: 10.1016/j.xhgg.2022.100085

Figure Lengend Snippet: Replication of CR-B FVAs (A) Replication of BRCA1 nuclear localization, BRCA2 nuclear localization, and p53 in matched LCLs and PBMCs derived from the same individuals (N = 20). Correlation cofficients are shown. (B) Replication of BRCA1 nuclear localization, BRCA2 nuclear localization, and p53 phosphorylation of days 2–4 following sample collection, compared to day 1. Correlation cofficients are shown.

Article Snippet: Expression plasmids (1 μg) for BRCA1 (pDEST-FRT/T0-GFP-BRCA1, cat. no. 71116, GFP tag), BRCA2 (pMH-SFB-BRCA2, cat. no. 99395, SFP tag), PALB2 (pDEST-FRT/T0-GFP-PALB2, cat. no. 71113, GFP tag), and ATM (pcDNA3.1(+)FLAG-His-ATM WT, cat. no. 31985, Addgene, Watertown, MA) were transfected into WT LCLs or those with P/LP or B/LB variants.

Techniques: Derivative Assay, Phospho-proteomics

Journal: Human Genetics and Genomics Advances

Article Title: Prediction of breast cancer risk based on flow variant analysis of circulating peripheral blood mononuclear cells

doi: 10.1016/j.xhgg.2022.100085

Figure Lengend Snippet: Sensitivity, specificity, and accuracy of FVAs and RCS for Coriell, Montefiore, and Northwell cohorts and all cohorts

Article Snippet: Expression plasmids (1 μg) for BRCA1 (pDEST-FRT/T0-GFP-BRCA1, cat. no. 71116, GFP tag), BRCA2 (pMH-SFB-BRCA2, cat. no. 99395, SFP tag), PALB2 (pDEST-FRT/T0-GFP-PALB2, cat. no. 71113, GFP tag), and ATM (pcDNA3.1(+)FLAG-His-ATM WT, cat. no. 31985, Addgene, Watertown, MA) were transfected into WT LCLs or those with P/LP or B/LB variants.

Techniques:

Journal: Human Genetics and Genomics Advances

Article Title: Prediction of breast cancer risk based on flow variant analysis of circulating peripheral blood mononuclear cells

doi: 10.1016/j.xhgg.2022.100085

Figure Lengend Snippet: Expression plasmid rescue of genetic variants in LCLs (A–D) Gene rescue for (A) BRCA1 , (B) BRCA2 , (C) ATM , and (D) PALB2 variants by RCS by boxplots. P-values for pairwise comparisons are shown.

Article Snippet: Expression plasmids (1 μg) for BRCA1 (pDEST-FRT/T0-GFP-BRCA1, cat. no. 71116, GFP tag), BRCA2 (pMH-SFB-BRCA2, cat. no. 99395, SFP tag), PALB2 (pDEST-FRT/T0-GFP-PALB2, cat. no. 71113, GFP tag), and ATM (pcDNA3.1(+)FLAG-His-ATM WT, cat. no. 31985, Addgene, Watertown, MA) were transfected into WT LCLs or those with P/LP or B/LB variants.

Techniques: Expressing, Plasmid Preparation

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway.

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet: Figure 4. Interactome of the USP7 TRAF domain in cortical neurons (A) Domain structure of mouse USP7 protein. R105, D165, and W165 are three amino acids necessary for substrate binding.18,23 USP, ubiquitin-specific protease domain; Ubl, ubiquitin-like domain. (B) Silver staining of the FLAG-immunoprecipitated USP7 TRAF domain (arrowhead) with and without RDW mutations (R105A+D165A+W166A). (C) Protein-protein association map of the top 50 USP7 TRAF interactors (full STRING network, confidence > 0.4). Proteins in complexes are colored according to (D). (D) GO analysis (cellular component) of the top 50 USP7 TRAF interactors. nBAF complex, neuronal SWI/SNF (BAF) complex. (E) Heatmap showing adjusted p values for enrichment of the USP7 TRAF interactors in disease genes. ID, intellectual disability; ADHD, attention deficit hyperactivity disorder; SCZ, schizophrenia; Ns, not significant. The p values were calculated by one-sided hypergeometric test with Benjamini-Hochberg correction. See also Figure S5.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptideFLAG (SFB)-taggedUSP7 construct61 (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Binding Assay, Ubiquitin Proteomics, Silver Staining, Immunoprecipitation

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway.

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet: Figure 5. Acute USP7 loss transforms the proteome of cortical neurons and depletes candidate substrate at the protein level (A) Flowchart to characterize dynamics of the neuronal proteome in response to acute Usp7 knockout with 16-plex TMT-MS. (B) Immunoblot of USP7 depletion and Cre expression in primary cortical neurons over time. 14-3-3 is the loading control.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptideFLAG (SFB)-taggedUSP7 construct61 (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Knock-Out, Western Blot, Expressing, Control

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway.

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet: Figure 6. USP7 targets and deubiquitinates Ppil4 to promote dendritic spinogenesis (A) Representative GFP and PSD95 micrographs of dendritic spines (arrowheads) of DIV18 primary cortical neurons with knockdown of Ppil4 or luciferase (Luci). Micrographs of dendritic spines with knockdown of other candidate substrates are shown in Figure S8. Scale bar, 5 mm. (B–G) Density of all (B–D) and PSD95+ (E–G) spines on apical, basal, and all dendrites of cortical neurons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Dunnett’s multiple-comparisons test (compared to Luci). (H) Streptavidin pull-down of HEK293T lysate with overexpression of streptavidin-binding peptide-FLAG (SFB)-tagged USP7 followed by immunoblot analyses. (I) IP of endogenous Ppil4 in HEK293T lysates followed by immunoblot analyses. (J) Reciprocal IP of endogenous USP7 and Ppil4 in mouse brain at age P4, followed by immunoblot analyses. Input lysate with long exposure for clear visualization is shown on the left. (K) TUBE pull-down of MG132-treated neuronal lysate with USP7 loss driven by lentiviral Cre followed by immunoblotting of Ppil4. Densitometric quantification is shown at the bottom. IB, immunoblot; a.u., arbitrary unit.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptideFLAG (SFB)-taggedUSP7 construct61 (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Knockdown, Luciferase, Over Expression, Binding Assay, Western Blot