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