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: Journal of Clinical Investigation

Article Title: Ubiquitin ligase RNF8 suppresses Notch signaling to regulate mammary development and tumorigenesis

doi: 10.1172/jci120401

Figure Lengend Snippet: Figure 6. RNF8 mediates negative regulation of Notch signaling in human breast cancer. (A) RT-qPCR analysis of the fold change in the expression of Notch targets in the indicated RNF8-knockdown (shRNF8) human breast cancer cell lines compared with their controls (shCtr). Dot plots show mean ± SEM of 3 independent experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001, 1-way ANOVA followed by Tukey’s test. (B) Immunoblot analysis of the indi- cated proteins in MDA-MB-231 cells transfected with FLAG empty vector (–) or RNF8-FLAG. (C) Immunoblot analysis of cytoplasmic (CE) and nuclear (NE) extracts from indicated cells. (D) Supervised clustering (from low to high RNF8 expression) of the profiles of genes with a significant negative or positive correlation (Pearson’s correlation coefficients [PCCs]) with RNF8 in the TCGA breast cancer (BRCA) data set. The breast cancer subtypes based on PAM50 are indicated. Data in B and C are representative of at least 3 experiments.

Article Snippet: Antibodies used for immunoblotting were against N1ICD (Val1744; Cell Signaling Technology, 4147), HES1 (sc-25392, clone H-140; Santa Cruz Biotechnology), CCND1 (sc-717, clone C-20; Santa Cruz Biotechnology), TRP53 (sc-6243, clone FL393; Santa Cruz Biotechnology), P21 (sc-471, clone M19; Santa Cruz Biotechnology), BAX (sc-493, clone N-20; Santa Cruz Biotechnology), FLAG (F1804, clone M2; Sigma-Aldrich), HA (11583816001, clone 12CA5; Sigma-Aldrich), ubiquitin (NB300-130, clone Ubi-1; Novus Biologicals), RNF8 (sc-271462, clone B-2, Santa Cruz Biotechnology; and NB100-1176, Novus Biologicals), lamin B (sc-3739; Santa Cruz Biotechnology), α-tubulin (sc-53646; Santa Cruz Biotechnology), RBPJ (5442; Cell Signaling Technology), FBW7 (40-1500; Invitrogen), and β-actin (sc-47778, clone C4; Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Western Blot, Transfection, Plasmid Preparation

Journal: Journal of Clinical Investigation

Article Title: Ubiquitin ligase RNF8 suppresses Notch signaling to regulate mammary development and tumorigenesis

doi: 10.1172/jci120401

Figure Lengend Snippet: Figure 7. RNF8 polyubiquitylates N1ICD to promote its turnover. (A) N1ICD interacts with RNF8. Immunoblot (IB) analysis of N1ICD and RNF8-FLAG fol- lowing anti-FLAG immunoprecipitation (IP) from RNF8-deficient mammary tumor cells and their RNF8-restored isogenic controls. WCL, whole cell lysate. (B) Ubiquitylation of N1ICD depends on RNF8 in vivo. RNF8-deficient mammary tumor cells complemented with empty FLAG vector (–) or RNF8-FLAG (+) were either left untreated or treated with the proteasome inhibitor MG132 and subjected to IP with anti-N1ICD. Immunoblot analysis was performed with the indicated antibodies. (C) Ubiquitylation of endogenous N1ICD by RNF8. Nuclear (NE) and cytoplasmic (CE) extracts prepared from MDA-MB-231 cells deleted of RNF8 (sgRNF8) and their controls (sgCh10) were subjected to IP with anti-N1ICD or IgG, followed by immunoblot as indicated. (D) In vitro ubiq- uitylation of recombinant N1ICD in the presence of recombinant RNF8, UBE1 (E1), Ubc13 (E2), and Ub proteins. (E) Cycloheximide (CHX) chase analysis of N1ICD protein half-lives in RNF8-deficient mammary tumor cells and their RNF8-restored isogenic controls. Cells were incubated in the presence of CHX for the indicated times, and immunoblots were performed using the indicated antibodies. N1ICD levels were quantified by densitometry using ImageJ and normalized with β-actin level. N1ICD fold decrease is indicated. (F) Requirement of RNF8 catalytic activity for N1ICD ubiquitylation. HEK293T cells, trans- fected with empty FLAG vector (–), RNF8WT-FLAG, RNF8C406S-FLAG (catalytically inactive), and Ub-HA as indicated, were subjected to IP with anti-N1ICD followed by immunoblot for HA and N1ICD. WCL was analyzed by immunoblot as indicated. (G) FHA domain of RNF8, responsible for its recruitment to DSB sites, is dispensable for N1ICD turnover. Immunoblots showing expression of N1ICD and RNF8-FLAG in RNF8-deficient mammary tumor cells recon- stituted with FLAG-RNF8WT, FLAG-RNF8C406S, FLAG-RNF8R42A (FHA domain mutant), or empty FLAG vector (–) as indicated. Data in A–G are representative of at least 3 experiments.

Article Snippet: Antibodies used for immunoblotting were against N1ICD (Val1744; Cell Signaling Technology, 4147), HES1 (sc-25392, clone H-140; Santa Cruz Biotechnology), CCND1 (sc-717, clone C-20; Santa Cruz Biotechnology), TRP53 (sc-6243, clone FL393; Santa Cruz Biotechnology), P21 (sc-471, clone M19; Santa Cruz Biotechnology), BAX (sc-493, clone N-20; Santa Cruz Biotechnology), FLAG (F1804, clone M2; Sigma-Aldrich), HA (11583816001, clone 12CA5; Sigma-Aldrich), ubiquitin (NB300-130, clone Ubi-1; Novus Biologicals), RNF8 (sc-271462, clone B-2, Santa Cruz Biotechnology; and NB100-1176, Novus Biologicals), lamin B (sc-3739; Santa Cruz Biotechnology), α-tubulin (sc-53646; Santa Cruz Biotechnology), RBPJ (5442; Cell Signaling Technology), FBW7 (40-1500; Invitrogen), and β-actin (sc-47778, clone C4; Santa Cruz Biotechnology).

Techniques: Western Blot, Immunoprecipitation, In Vivo, Plasmid Preparation, In Vitro, Recombinant, Incubation, Activity Assay, Expressing, Mutagenesis

Journal: Journal of Clinical Investigation

Article Title: Ubiquitin ligase RNF8 suppresses Notch signaling to regulate mammary development and tumorigenesis

doi: 10.1172/jci120401

Figure Lengend Snippet: Figure 9. A schematic diagram depicting a model for RNF8-mediated coregulation of Notch signaling and DSB repair and the breast cancer– suppressive function of RNF8. RNF8 is recruited to DSBs, where it ubiquitylates histones at the flanking DNA damage sites. This ubiquitylation triggers recruitment of downstream DSB signaling and repair proteins (e.g., RNF168 and BRCA1) to DNA damage sites, allowing their repair. Loss of RNF8 expression or function impairs DSB repair, leading to genomic instability and increased breast cancer risk. Our data reveal an important role for RNF8 in mediating negative regulation of Notch signaling. RNF8 ubiquitylates the active form of NOTCH1 (N1ICD) to promote its turnover. Thus, impaired RNF8 expression or function results in constitutive activation of Notch signaling in mam- mary luminal progenitors, a process that promotes expansion of these progenitors and increases their risk for malignant transformation. We propose that RNF8 coregulation of Notch signaling and DSB repair is critical for its suppressor function in breast cancer. Small filled circles indicate γH2ax (black); phosphorylation (gray); and Ub (red).

Article Snippet: Antibodies used for immunoblotting were against N1ICD (Val1744; Cell Signaling Technology, 4147), HES1 (sc-25392, clone H-140; Santa Cruz Biotechnology), CCND1 (sc-717, clone C-20; Santa Cruz Biotechnology), TRP53 (sc-6243, clone FL393; Santa Cruz Biotechnology), P21 (sc-471, clone M19; Santa Cruz Biotechnology), BAX (sc-493, clone N-20; Santa Cruz Biotechnology), FLAG (F1804, clone M2; Sigma-Aldrich), HA (11583816001, clone 12CA5; Sigma-Aldrich), ubiquitin (NB300-130, clone Ubi-1; Novus Biologicals), RNF8 (sc-271462, clone B-2, Santa Cruz Biotechnology; and NB100-1176, Novus Biologicals), lamin B (sc-3739; Santa Cruz Biotechnology), α-tubulin (sc-53646; Santa Cruz Biotechnology), RBPJ (5442; Cell Signaling Technology), FBW7 (40-1500; Invitrogen), and β-actin (sc-47778, clone C4; Santa Cruz Biotechnology).

Techniques: Expressing, Activation Assay, Transformation Assay, Phospho-proteomics

Journal: Cell reports

Article Title: Feedback repression of PPARα signaling by Let-7 microRNA

doi: 10.1016/j.celrep.2021.109506

Figure Lengend Snippet:

Article Snippet: pCMV6-mouse Rnf8 , Origene , Cat# MR207821.

Techniques: Recombinant, Protease Inhibitor, TaqMan microRNA Assay, Chromatin Immunoprecipitation, Knock-Out, Negative Control, Software