Journal: Chromosoma

Article Title: COORDINATION OF THE RECRUITMENT OF THE FANCD2 AND PALB2 FANCONI ANEMIA PROTEINS BY A UBIQUITIN SIGNALING NETWORK

doi: 10.1007/s00412-016-0602-9

Figure Lengend Snippet: a Representative immunoblot showing knockdown of MDC1. b–c Representative images (b) and quantification (c) of FANCD2 focus formation in HeLa cells transfected with a control siRNA or two distinct siRNAs targeting MDC1 and treated with 0.5 µM MMC for 16 hr. FANCD2 foci (red) are also shown in merged images in b with DAPI signal in blue to indicate the position of nuclei. d–e Representative images of MMC-induced PALB2 foci (d) in cells transfected with either siLacZ or MDC1 siRNAs and quantification (e) of the percentage of HeLa cells with five or more PALB2 foci. PALB2 foci (red) are also shown in merged images in d with DAPI signal in blue. f Immunoblot showing FANCD2 monoubiquitination status in cells treated with a siRNA directed against RNF8 or MDC1. The intensity of the upper monoubiquitinated band divided by the intensity of the lower unubiquitinated band is shown for each lane. g Quantification of the percentage of HeLa cells with five or more ubiquitin foci following transfection with siRNAs and treatment with MMC. Values in c,e,g represent the mean of three independent counts of at least 150 cells each ± standard deviation; * indicates p<0.005.

Article Snippet: Antibodies Primary antibodies utilized for immunofluorescence microscopy and immunoblotting were as follows: FK2 (EMD Millipore, 04-263), FANCD2 (E35) ( Garcia-Higuera et al. 2001 ), PALB2 ( Zhang et al. 2009a ), γH2AX (EMDMillipore, JBW301), RNF8 (Santa Cruz, sc271462), MDC1 (Novus Biologicals, NB100-395), FAAP20 ( Ali et al. 2012 ), RAP80 (Bethyl Laboratory, A300-763A), and anti-HA (Covance, 16B12).

Techniques: Western Blot, Knockdown, Transfection, Control, Ubiquitin Proteomics, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: The BRCA1 RING and BRCT Domains Cooperate in Targeting BRCA1 to Ionizing Radiation-induced Nuclear Foci

doi: 10.1074/jbc.m408879200

Figure Lengend Snippet: FIG. 1. Endogenous BRCA1 and ec- topic YFP-BRCA1 form nuclear foci with BARD1 and MDC1. A, endogenous BRCA1 nuclear foci (stained with Ab-4 and Texas Red) co-localize with BARD1 and MDC1 (stained with fluorescein iso- thiocyanate) in T47D cells before and af- ter 15-Gy IR treatment and 4 h of recov- ery. B, YFP-tagged wild-type BRCA1 was transfected into MCF-7 cells and assessed 48 h later for nuclear foci, which also co- localized with BARD1 and MDC1 (stained with Texas Red). Note the redistribution of BRCA1 from few and larger spots in untreated cells to small and more dis- persed foci in IR-treated cells.

Article Snippet: For BARD1 and MDC1 staining, rabbit BARD1 antibody 59L (gift from Professor Richard Baer) and rabbit MDC1 BL578 antibody (Bethyl Laboratories) were used, respectively.

Techniques: Staining, Transfection

Journal: Journal of Biological Chemistry

Article Title: The BRCA1 RING and BRCT Domains Cooperate in Targeting BRCA1 to Ionizing Radiation-induced Nuclear Foci

doi: 10.1074/jbc.m408879200

Figure Lengend Snippet: FIG. 6. Co-localization of BRCA1 BRCT domain fragments and MDC1 in foci. Four YFP-BRCA1 constructs were transiently ex- pressed in MCF-7 cells, which were later irradiated (15 Gy, 4 h of recovery) and co-stained for MDC1 (Texas Red). All ectopic BRCA1 proteins displayed at least partial co-localization with MDC1 foci except the 5382insC mutant, which does not form foci.

Article Snippet: For BARD1 and MDC1 staining, rabbit BARD1 antibody 59L (gift from Professor Richard Baer) and rabbit MDC1 BL578 antibody (Bethyl Laboratories) were used, respectively.

Techniques: Construct, Irradiation, Staining, Mutagenesis

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/elife.61407

Figure Lengend Snippet: Figure 1. Global proteome analysis of cavin3 KO HeLa cells by label-free quantitative proteomics. (A) Z-score for HeLa WT and cavin3 KO cells (replicates Rep. 1–3) showing upregulated proteins (red) and downregulated proteins (blue). (B) Volcano plot showing proteins (red dots) identified by Gene Ontology Biological Process (GOBP) involved in DNA repair. (C) Volcano plot showing DNA repair proteins upregulated in cavin3 KO cells. (D) Volcano plot showing proteins of the BRCA1 A-complex, BRCA1, BRCC3, MDC1, and UBE4A downregulated in cavin3 HeLa KO cells and upregulation of 53BP1 with a heatmap analysis of the expression of each of these proteins in replicate (Rep. 1–3) HeLa WT and cavin3 KO cells. The online version of this article includes the following source data and figure supplement(s) for figure 1:

Article Snippet: DOI: https://doi.org/10.7554/eLife.61407 18 of 35 gamma catenin antibody (Cell Signaling, Cat# 2309, WB 1:3000), rabbit anti-CAV1 antibody (BD Biosciences, Cat# 610060, WB 1:5000), mouse anti-cavin1 antibody (Abmart, China, 1:100 PLA), and rabbit anti-cavin1 antibody were raised as described previously and used for immunofluorescence (Bastiani et al., 2009), rabbit anti-cavin1 antibody (Sigma-Aldrich, Cat# AV36965, RRID:AB 1855947, WB 1:2000), mouse anti-cavin3 antibody (Novus, Cat# H00112464-MO4, PLA 1:200), rabbit anti-cavin3 antibody (Proteintech, Millennium Sciences, Pty, Ltd, Cat# 16250-1-AP, RRID:AB_ 2171897, WB 1:2000, IF 1:300, PLA 1:200), rabbit anti-CHD3 antibody (GeneTex, Sapphire Bioscience, Cat# GTX131779, RRID:AB_2886520, WB 1:500), rabbit anti-DDX21 antibody (Novus, Cat# NBP1-88310, RRID:AB_11027665, WB 1:2000), rabbit anti-EGFR Clone LA22 antibody (Millipore, Cat# 05-104, RRID:AB_11210086, WB 1:4000), mouse-anti-FANCD2 antibody (GeneTex, Cat# GTX116037, RRID:AB2036898, WB 1:500), mouse anti-Flotillin Clone 18 antibody (BD Biosciences, Cat# 610821, RRID:AB_398140, PLA 1:100), mouse anti-GFP antibody (Roche, Cat# 11814460001, RRID:AB_390913, WB 1:4000, PLA 1:300), rabbit anti-Histone H2A.X-Chip Grade (Abcam, Cat# ab20669, RRID:AB_445689, WB 1:1000), rabbit phospho-Histone H2A.X (Ser 139) (20E3) antibody (Cell Signaling Technology, Cat# 9718, RRID:AB_2118009, IF 1:500), rabbit phospho-Histone H2A.X CHIP Grade antibody (Abcam, Cat# ab2893, RRID:AB_303388, WB: 1:3000), rabbit anti-HLTF antibody (Proteintech, Cat# 14286-1-AP, WB 1:2000), rabbit anti-MDC1 antibody (Novus, Cat# 10056657SS, RRID:AB_838567, WB 1:100), sheep anti-Merit40 antibody (R&D Systems, Cat# AF6604SP, RRID:AB_10717577, WB 1:500), rabbit anti-PARP1 antibody (GeneTex, Cat# GTX112864, RRID:AB_11173565, WB 1:1000), mouse anti-PCNA antibody (Millipore, Cat# NA03T, RRID:AB_ 2160357, PLA 1:100), rabbit anti-PGK1 antibody (GeneTex, Cat# GTX107614, RRID:AB_2037666, WB 1:3000), rabbit anti-PKM antibody (GeneTex, Cat# GTX107977, RRID:AB_1951264, WB 1:3000), mouse anti-Rad51 antibody (Novus, Cat# 100-184, RRID:AB_350083, WB 1:1000), rabbit anti-RAP80 D1T6Q antibody (Cell Signaling Technology, Cat# 14466, RRID:AB_2798487, WB 1:1000, IF 1:100), rabbit anti-RNF168 antibody (GeneTex, Cat# GTX118147, RRID:AB_11169617, WB 1:1000), and mouse anti-Tubulin DM1A antibody (Abcam, Cat# ab7291, RRID:AB_2241126, WB 1:4000).

Techniques: Quantitative Proteomics, Expressing

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/elife.61407

Figure Lengend Snippet: Figure 8. Cavin3-deficient HeLa cells exhibit abolishment of DNA repair. (A) Representative western blot analysis of WT and cavin3 KO cells UV time course for cavin3, BRCA1, CAV1, Rad51, and Tubulin. (B) Protein components of the BRCA1 A-complex. Blue-colored circles: proteins downregulated in the label-free quantitative (LFQ) proteomics; yellow-colored circles: proteins not detected in the LFQ proteomics of cavin3 KO cells. (C) Representative western blot analysis of cavin3, BRCA1, pH2AX, UIM1C/Rap80, BARD1, Rad51, MDC1, RNF168, BRCC36, Merit40, BRCA2, CAV, PKM, PGK1, and Actin in WT and cavin3 KO HeLa cells untreated (-) or UV treated (UV) followed by a 4 hr chase. Quantitation of protein levels from three independent Figure 8 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.61407 18 of 35 gamma catenin antibody (Cell Signaling, Cat# 2309, WB 1:3000), rabbit anti-CAV1 antibody (BD Biosciences, Cat# 610060, WB 1:5000), mouse anti-cavin1 antibody (Abmart, China, 1:100 PLA), and rabbit anti-cavin1 antibody were raised as described previously and used for immunofluorescence (Bastiani et al., 2009), rabbit anti-cavin1 antibody (Sigma-Aldrich, Cat# AV36965, RRID:AB 1855947, WB 1:2000), mouse anti-cavin3 antibody (Novus, Cat# H00112464-MO4, PLA 1:200), rabbit anti-cavin3 antibody (Proteintech, Millennium Sciences, Pty, Ltd, Cat# 16250-1-AP, RRID:AB_ 2171897, WB 1:2000, IF 1:300, PLA 1:200), rabbit anti-CHD3 antibody (GeneTex, Sapphire Bioscience, Cat# GTX131779, RRID:AB_2886520, WB 1:500), rabbit anti-DDX21 antibody (Novus, Cat# NBP1-88310, RRID:AB_11027665, WB 1:2000), rabbit anti-EGFR Clone LA22 antibody (Millipore, Cat# 05-104, RRID:AB_11210086, WB 1:4000), mouse-anti-FANCD2 antibody (GeneTex, Cat# GTX116037, RRID:AB2036898, WB 1:500), mouse anti-Flotillin Clone 18 antibody (BD Biosciences, Cat# 610821, RRID:AB_398140, PLA 1:100), mouse anti-GFP antibody (Roche, Cat# 11814460001, RRID:AB_390913, WB 1:4000, PLA 1:300), rabbit anti-Histone H2A.X-Chip Grade (Abcam, Cat# ab20669, RRID:AB_445689, WB 1:1000), rabbit phospho-Histone H2A.X (Ser 139) (20E3) antibody (Cell Signaling Technology, Cat# 9718, RRID:AB_2118009, IF 1:500), rabbit phospho-Histone H2A.X CHIP Grade antibody (Abcam, Cat# ab2893, RRID:AB_303388, WB: 1:3000), rabbit anti-HLTF antibody (Proteintech, Cat# 14286-1-AP, WB 1:2000), rabbit anti-MDC1 antibody (Novus, Cat# 10056657SS, RRID:AB_838567, WB 1:100), sheep anti-Merit40 antibody (R&D Systems, Cat# AF6604SP, RRID:AB_10717577, WB 1:500), rabbit anti-PARP1 antibody (GeneTex, Cat# GTX112864, RRID:AB_11173565, WB 1:1000), mouse anti-PCNA antibody (Millipore, Cat# NA03T, RRID:AB_ 2160357, PLA 1:100), rabbit anti-PGK1 antibody (GeneTex, Cat# GTX107614, RRID:AB_2037666, WB 1:3000), rabbit anti-PKM antibody (GeneTex, Cat# GTX107977, RRID:AB_1951264, WB 1:3000), mouse anti-Rad51 antibody (Novus, Cat# 100-184, RRID:AB_350083, WB 1:1000), rabbit anti-RAP80 D1T6Q antibody (Cell Signaling Technology, Cat# 14466, RRID:AB_2798487, WB 1:1000, IF 1:100), rabbit anti-RNF168 antibody (GeneTex, Cat# GTX118147, RRID:AB_11169617, WB 1:1000), and mouse anti-Tubulin DM1A antibody (Abcam, Cat# ab7291, RRID:AB_2241126, WB 1:4000).

Techniques: Western Blot, Quantitation Assay

Journal: Cancers

Article Title: Low-Dose Pesticides Alter Primary Human Bone Marrow Mesenchymal Stem/Stromal Cells through ALDH2 Inhibition

doi: 10.3390/cancers13225699

Figure Lengend Snippet: Pesticide exposure for 21 days induces DNA damage in normal BM-MSCs. ( A ) DNA damage with double strand breaks, quantified by ℗-S139-γH2AX immunoreactivity by flow cytometry, increases in BM-MSCs after pesticide exposure for 21 days (left panel, n = 6), as shown in a representative experiment (middle panel). Comet assays reveal increased nucleus DNA migration after exposure of BM-MSCs to pesticides for 21 days, as shown in a representative experiment (right panel). ( B ) ℗-S139 γH2AX, 53BP1, MDC1 triple immunostaining in BM-MSCs indicates a higher number of double positive 53BP1/℗-S139 γH2AX signals in the nucleus of exposed cells (FOCI) and a loss of MDC1 staining. Data are expressed as percent of the control ± SEM; *, p < 0.05; **, p < 0.01; Friedman followed by post hoc Dunn’s multiple comparisons test. Scalebar: 50 µm ( A ) and 10 µm ( B ).

Article Snippet: After 21 days, cells were fixed with 4% paraformaldehyde, permeabilized (PBS/Triton X-100 0.1%, 10 min, room temperature), blocked with gelatin from cold water fish skin 5%, Triton X-100 0.1% (Sigma-Aldrich) in PBS, and were incubated overnight at 4 °C with anti-phospho-S139 γH2AX monoclonal mouse antibody (Abcam) at 1/1000, anti-MDC1 polyclonal rabbit antibody (Novus Biologicals, Centennial, CO, USA) at 1/500 and anti-53BP1 polyclonal goat antibody (R&D systems) at 1/1000, followed by a secondary anti-mouse antibody labeled with TRITC (Jackson Immunoresearch, Ely, UK), Alexa Fluor ® 647 conjugated anti-goat (Jackson Immunoresearch) and FITC-conjugated anti-rabbit (Jackson Immunoresearch) at 1/2000 each at room temperature for 2 h. Fluorescence images were acquired with a Leica TCS SP8 (Leica, Wetzlar, Germany) confocal microscope and analyzed using ImageJ software.

Techniques: Flow Cytometry, Migration, Pesticides, Triple Immunostaining, Staining, Control

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 following antibodies were used: antibodies against DGCR8 (1:2000, Abcam, #ab191875), Dicer (1:1000, Cell Signaling Technology, #5362S), Drosha (1:1000, Cell Signaling Technology, #3364S), Exportin-5 (1:1000, Cell Signaling Technology, #12565), γH2AX (1:1000, Cell Signaling Technology, #9718S), H2AX (1:1000, Cell Signaling Technology, #2595S), H2A (1:1000, Cell Signaling Technology, #2578S), p-CHK1 (1:1000, Cell Signaling Technology, #12302S), CHK1 (1:1000, Cell Signaling Technology, #2360S), p-CHK2 (1:1000, Cell Signaling Technology, #2661S), CHK2 (1:1000, Cell Signaling Technology, #6334S), p-ATM (1:1000, Cell Signaling Technology, #5883S), ATM (1:1000, Cell Signaling Technology, #2873S), p-ATR (1:1000, Cell Signaling Technology, #2853S), ATR (1:1000, Cell Signaling Technology, #2790S), p-S/TQ (1:1000, Cell Signaling Technology, 2851S), MBP (1:1000, Cell Signaling Technology, 2396S), GST (1:1000, Cell Signaling Technology, #2622S), MDC1 (1:1000, R&D Systems, #MAB6497), RNF8 (1:1000, Millipore, #09-813), RNF168 (1:1000, Millipore, #ABE367), USP36 (1:1000, a gift from Dr Masayuki Komada at Tokyo Institute of Technology), USP51 (1:3000, a gift from Dr Sharon Dent at MD Anderson Cancer Center), β-actin (1:1000, Santa Cruz Biotechnology, #sc-47778), FLAG (1:5000, Sigma, #F3165, clone M2), HA (1:2000, Santa Cruz Biotechnology, #sc-7392), and MYC (1:2000, Santa Cruz Biotechnology, #sc-40, clone 9E10).

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 following antibodies were used: antibodies against DGCR8 (1:2000, Abcam, #ab191875), Dicer (1:1000, Cell Signaling Technology, #5362S), Drosha (1:1000, Cell Signaling Technology, #3364S), Exportin-5 (1:1000, Cell Signaling Technology, #12565), γH2AX (1:1000, Cell Signaling Technology, #9718S), H2AX (1:1000, Cell Signaling Technology, #2595S), H2A (1:1000, Cell Signaling Technology, #2578S), p-CHK1 (1:1000, Cell Signaling Technology, #12302S), CHK1 (1:1000, Cell Signaling Technology, #2360S), p-CHK2 (1:1000, Cell Signaling Technology, #2661S), CHK2 (1:1000, Cell Signaling Technology, #6334S), p-ATM (1:1000, Cell Signaling Technology, #5883S), ATM (1:1000, Cell Signaling Technology, #2873S), p-ATR (1:1000, Cell Signaling Technology, #2853S), ATR (1:1000, Cell Signaling Technology, #2790S), p-S/TQ (1:1000, Cell Signaling Technology, 2851S), MBP (1:1000, Cell Signaling Technology, 2396S), GST (1:1000, Cell Signaling Technology, #2622S), MDC1 (1:1000, R&D Systems, #MAB6497), RNF8 (1:1000, Millipore, #09-813), RNF168 (1:1000, Millipore, #ABE367), USP36 (1:1000, a gift from Dr Masayuki Komada at Tokyo Institute of Technology), USP51 (1:3000, a gift from Dr Sharon Dent at MD Anderson Cancer Center), β-actin (1:1000, Santa Cruz Biotechnology, #sc-47778), FLAG (1:5000, Sigma, #F3165, clone M2), HA (1:2000, Santa Cruz Biotechnology, #sc-7392), and MYC (1:2000, Santa Cruz Biotechnology, #sc-40, clone 9E10).

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 following antibodies were used: antibodies against DGCR8 (1:2000, Abcam, #ab191875), Dicer (1:1000, Cell Signaling Technology, #5362S), Drosha (1:1000, Cell Signaling Technology, #3364S), Exportin-5 (1:1000, Cell Signaling Technology, #12565), γH2AX (1:1000, Cell Signaling Technology, #9718S), H2AX (1:1000, Cell Signaling Technology, #2595S), H2A (1:1000, Cell Signaling Technology, #2578S), p-CHK1 (1:1000, Cell Signaling Technology, #12302S), CHK1 (1:1000, Cell Signaling Technology, #2360S), p-CHK2 (1:1000, Cell Signaling Technology, #2661S), CHK2 (1:1000, Cell Signaling Technology, #6334S), p-ATM (1:1000, Cell Signaling Technology, #5883S), ATM (1:1000, Cell Signaling Technology, #2873S), p-ATR (1:1000, Cell Signaling Technology, #2853S), ATR (1:1000, Cell Signaling Technology, #2790S), p-S/TQ (1:1000, Cell Signaling Technology, 2851S), MBP (1:1000, Cell Signaling Technology, 2396S), GST (1:1000, Cell Signaling Technology, #2622S), MDC1 (1:1000, R&D Systems, #MAB6497), RNF8 (1:1000, Millipore, #09-813), RNF168 (1:1000, Millipore, #ABE367), USP36 (1:1000, a gift from Dr Masayuki Komada at Tokyo Institute of Technology), USP51 (1:3000, a gift from Dr Sharon Dent at MD Anderson Cancer Center), β-actin (1:1000, Santa Cruz Biotechnology, #sc-47778), FLAG (1:5000, Sigma, #F3165, clone M2), HA (1:2000, Santa Cruz Biotechnology, #sc-7392), and MYC (1:2000, Santa Cruz Biotechnology, #sc-40, clone 9E10).

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