Journal: Cell death and differentiation

Article Title: CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma.

doi: 10.1038/s41418-023-01192-3

Figure Lengend Snippet: Fig. 1 RNF8 binds to MAD2 to promote MCC formation. A Volcano plot showing the proteins which were enriched in BirA*-RNF8 compared to BirA*-GFP proximal proteomes. Western blot analysis of MAD2 and p31comet levels in the streptavidin pull down lysates (B) or myc immunoprecipitates (IP) (C) of HEK293T transfected with BirA*-RNF8 or GFP. D Western blot analysis of RNF8 levels in the MAD2 immunoprecipitates of HEK293T, with or without 200 ng/ml NOC (16 h). The band of interest is indicated by *. E Western blot analysis of MAD2 and flag-CDC20 levels in the flag IP from HEK293T cells co-transfected with flag-CDC20 and empty vector (EV)/RNF8-myc. Cells with nocodazole (NOC) treatment (200 ng/mL, 16 h) serve as the positive control. The MAD2/flag-CDC20 ratio was normalized to EV control. F Phos- tag SDS-PAGE analysis of wild-type or S102A HA-p31comet phosphorylation, with or without RNF8-myc overexpression. G Western blot analysis of endogenous MAD2 levels in the myc IP from HEK293T cell lysates overexpressing RNF8-myc, along with or without flag-p31comet or p31comet(QF) overexpression. The band of interest is indicated by *. MAD2/myc ratio was normalized to empty vector control. H Western blot analysis of H3 pS10 levels in the cell lysates of HEK293T overexpressing flag-tagged p31comet, p31comet(QF), or EV, along with myc-tagged RNF8. GAPDH serves as loading control. H3 pS10/GAPDH ratio was normalized to empty vector control. I Western blot analysis of endogenous MAD2 levels in the p31comet immunoprecipitates from HEK293T cell lysates transfected with different amount of RNF8-myc overexpressing plasmids. MAD2/p31comet ratio was normalized to empty vector control.

Article Snippet: GSCs were transduced with lentiviruses overexpressing GFP, RNF8, *FHA or *RING and fresh media containing 40 μM Z-VAD-FMK (MedChemExpress) was replenished after 4-hour post-transduction.

Techniques: Western Blot, Transfection, Plasmid Preparation, Positive Control, Control, SDS Page, Phospho-proteomics, Over Expression

Journal: Cell death and differentiation

Article Title: CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma.

doi: 10.1038/s41418-023-01192-3

Figure Lengend Snippet: Fig. 2 RNF8 associates with c-MAD2 stably via its RING domain without ubiquitinating MAD2. A Structure of human RNF8 protein. B Western blot analysis of MAD2 levels in the myc IP from HEK293T cell lysates overexpressing myc-tagged RNF8, *FHA, *RING or EV. C Western blot analysis of HA-MAD2 levels in the streptavidin pull down lysates of HEK293T transfected with EV, HA-MAD2, -MAD2Δ or –MAD2L13A, along with BirA*-RNF8. D Western blot analysis of HA-MAD2ΔC levels in the streptavidin pull down lysates from HEK293T cell lysates overexpressing BirA*-RNF8 or -*RING, along with HA-MAD2ΔC overexpression. E Western blot analysis of flag-MAD2 levels in the myc IP from HEK293T cell lysates overexpressing EV, flag-MAD2, or –MAD2RQ, along with myc-tagged RNF8. F In silico docking of c-MAD2 (gray) and RNF8’s RING domain (yellow) with zinc (gray sphere) using the program HADDOCK. Interacting residues are highlighted in sticks and the interactions are shown as dotted black lines. G Western blot analysis of MAD2 levels in the myc IP from HEK293T cell lysates overexpressing myc-tagged RNF8, 3A, or 4A mutants. H Western blot analysis of HA levels in flag IP from HEK293T cell lysates overexpressing flag-tagged MAD2 and HA-tagged Ub, with RNF8 overexpression.

Article Snippet: GSCs were transduced with lentiviruses overexpressing GFP, RNF8, *FHA or *RING and fresh media containing 40 μM Z-VAD-FMK (MedChemExpress) was replenished after 4-hour post-transduction.

Techniques: Stable Transfection, Western Blot, Transfection, Over Expression, In Silico

Journal: Cell death and differentiation

Article Title: CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma.

doi: 10.1038/s41418-023-01192-3

Figure Lengend Snippet: Fig. 3 RNF8 overexpression impairs GSC mitotic progression that is dependent on its FHA and RING domains. A Western blot analysis of RNF8 and SAC markers, including H3 pS10 and cyclin B1, in GSCs transduced with different RNF8 constructs. RNF8 serves as the positive control whereas GAPDH and β-actin serve as loading control respectively. B Tumorsphere formation of two GSC lines overexpressing GFP, RNF8, *FHA, or *RING (n = 6) (mean±SD). *P < 0.05; **P < 0.01. C, D Colony formation of two GSC lines overexpressing GFP, RNF8, *FHA, or *RING (n = 4) (mean ± SD). *P < 0.05; ***P < 0.001. D Representative images of (C). E Cell cycle analysis of TS543 overexpressing GFP, RNF8, *FHA or *RING, with 40 µM Z-VAD-FMK treatment (72 h) (n = 3) (mean±SD). ***P < 0.001. F Western blot analysis of MAD2 levels in the RNF8 IP from RNF8 overexpressing GSC TS543 lysates. G Western blot analysis of H3 pS10 and cyclin B1 levels of GSC TS543 with GFP or RNF8 overexpression, with or without MAD2 depletion. RNF8 and MAD2 serve as the positive controls, while GAPDH serves as the loading control. H, I Karyotyping analysis of GSC TS543 overexpressing different RNF8 constructs (n = 3). Near triploidy: 60–80 chromosomes; near tetraploidy: 81–110 chromosomes. Minimum of 100 spreads were analyzed per condition. I Representative karyotypes from GSCs overexpressing different RNF8 constructs (H). Scale bar 20 μm.

Article Snippet: GSCs were transduced with lentiviruses overexpressing GFP, RNF8, *FHA or *RING and fresh media containing 40 μM Z-VAD-FMK (MedChemExpress) was replenished after 4-hour post-transduction.

Techniques: Over Expression, Western Blot, Transduction, Construct, Positive Control, Control, Cell Cycle Assay

Journal: Cell death and differentiation

Article Title: CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma.

doi: 10.1038/s41418-023-01192-3

Figure Lengend Snippet: Fig. 4 CAMK2D associates with RNF8’s FHA domain weakly/transiently via p-Thr287 to mediate RNF8-induced mitotic checkpoint. A Volcano plot showing the proteins which are significantly depleted in *FHA vs RNF8 proximal proteomes (i.e., minus log2fold change (FC)). B Western blot analysis of endogenous CAMK2D levels in the streptavidin pull down lysates of HEK293T overexpressing BirA*-RNF8 or *FHA. CAMK2D/myc ratio was normalized to BirA*-RNF8 group. Western blot analysis of flag-CAMK2D levels in the streptavidin pull down lysates (C), or flag IP (D) of HEK293T co-transfected with flag-CAMK2D and BirA*-RNF8/*FHA. The band of interest is indicated by *. E Western blot analysis of flag levels in streptavidin pulldown lysates from HEK293T cell lysates overexpressing flag-CAMK2D or -CAMK2DT287A mutant, along with BirA*-RNF8. F Phos-tag SDS-PAGE analysis of flag-CAMK2D and -CAMK2DT287A mutant, along with co-expression of EV or myc-tagged RNF8/ *FHA. The p-T287/flag ratio was normalized to empty vector control. G Western blot analysis of H3 pS10 levels in GFP or RNF8 overexpressing GSC TS543, with or without CAMK2D KD. RNF8 and CAMK2D serve as positive control whereas GAPDH serve as the loading control. H Western blot analysis of H3 pS10 levels in wildtype CAMK2D or CAMK2DED overexpressing GSC TS543, with or without RNF8 KD. Flag and RNF8 serve as the positive controls, while GAPDH serves as the loading control.

Article Snippet: GSCs were transduced with lentiviruses overexpressing GFP, RNF8, *FHA or *RING and fresh media containing 40 μM Z-VAD-FMK (MedChemExpress) was replenished after 4-hour post-transduction.

Techniques: Western Blot, Transfection, Mutagenesis, SDS Page, Expressing, Plasmid Preparation, Control, Positive Control

Journal: Cell death and differentiation

Article Title: CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma.

doi: 10.1038/s41418-023-01192-3

Figure Lengend Snippet: Fig. 5 CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex. A Schematic diagram to illustrate the workflow for sucrose density gradient ultracentrifugation to assess RNF8 protein distribution in various fractions. B Western blot analysis of myc-RNF8, flag-CAMK2D, and MAD2 levels, with or without CAMK2D overexpression, in various fractions after sucrose density gradient ultracentrifugation of 293T cell lysates. C Western blot analysis of myc-RNF8 levels, with or without CAMK2D overexpression, in pooled fractions 6 to 8 from (B). The myc/ input ratio was normalized to the EV control. D Western blot analysis of myc-RNF8/*FHA, flag-CAMK2D and MAD2 levels, with CAMK2D overexpression, in various fractions after sucrose density gradient ultracentrifugation of 293T cell lysates. E Western blot analysis of myc-RNF8/ *FHA and MAD2 levels, with CAMK2D overexpression, in pooled fractions 6–8 from (D). The myc/input ratio was normalized to the RNF8 control. F Western blot analysis of flag-RNF8 and HA-MAD2 levels in the streptavidin pull down lysates of HEK293T overexpressing BirA*- CAMK2D, along with or without flag-RNF8 and HA-MAD2 overexpression. G Western blot analysis of flag-CAMK2D levels in the streptavidin pulldown lysates of BirA*-RNF8 or GFP-overexpressing HEK293T, with or without 200 ng/ml NOC (16 h).

Article Snippet: GSCs were transduced with lentiviruses overexpressing GFP, RNF8, *FHA or *RING and fresh media containing 40 μM Z-VAD-FMK (MedChemExpress) was replenished after 4-hour post-transduction.

Techniques: Western Blot, Over Expression, Control

Journal: Cell death and differentiation

Article Title: CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma.

doi: 10.1038/s41418-023-01192-3

Figure Lengend Snippet: Fig. 6 GBM with high HER2-EGFR signaling tends to avoid high RNF8 expression as RNF8 overexpression impedes GBM tumorigenicity. A Correlative analysis of RNF8 mRNA levels with glioma grades in two independent glioma patient cohorts. Wilcoxon-Mann-Whitney exact test. **P < 0.01. B, C Immunohistochemistry (IHC) scores of RNF8 in different grades of gliomas using glioma patient tissue microarray. C Representative images of the IHC scores of RNF8 in (B). D Correlative analysis of RNF8 mRNA levels with glioma patient survival in multiple glioma patient cohorts. OS: overall survival. Wald test. E Top 5 proteins that are upregulated in RNF8low vs RNF8high gliomas in the TCGA RPPA dataset using GlioVis (http://gliovis.bioinfo.cnio.es/). F RPPA analysis of HER2-pY1248 and EGFR-pY1173 in RNF8low versus RNF8high gliomas using TCGA cohort. ***P < 0.001, t-test. G, H In vivo bioluminescence-based imaging of post-orthotropic injection of TS543 overexpressing GFP or RNF8. H Representative images of the tumor-bearing NSG mice in (G) (n = 5) (mean±SD). *P < 0.05. I Kaplan Meier curves of mice implanted with GFP or RNF8 overexpressing TS543. Log rank test.

Article Snippet: GSCs were transduced with lentiviruses overexpressing GFP, RNF8, *FHA or *RING and fresh media containing 40 μM Z-VAD-FMK (MedChemExpress) was replenished after 4-hour post-transduction.

Techniques: Expressing, Over Expression, MANN-WHITNEY, Immunohistochemistry, Microarray, In Vivo, Imaging, Injection

Journal: Cell death and differentiation

Article Title: CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma.

doi: 10.1038/s41418-023-01192-3

Figure Lengend Snippet: Fig. 7 CMA using the RNF8 overexpression gene signature identifies PLK1i that synergizes with HSP90i to reduce GSC proliferation and stemness in vitro. A CMA using a RNF8 overexpression query signature identifies PLK1 inhibitor. B Top 5 compounds identified with CMA by using the RNF8 overexpression query signature. C Correlative analysis of PLK1 and RNF8 mRNA levels in glioma patients. D Schematic diagram showing the drug treatment regimen to be used in (G–I). E Western blot analysis of H3 pS10 and securin levels in TS543 lysates upon treatment with DMSO or BI 2536/Volasertib at the indicated concentrations for two days. GAPDH and β-actin serve as loading controls. F Western blot analysis of Akt levels in TS543 lysates upon treated with DMSO or 17-AAG at the indicated concentrations for 1 day. GAPDH serves as loading control. G Cell viability assay of TS543 upon treatment with BI 2536/Volasertib, 17-AAG, or combination of BI 2536/ Volasertib and 17-AAG at the indicated concentrations (n = 6) (mean ± SD). ***P < 0.001. H Cell viability assay of TS576 and non-cancerous mouse astrocytes upon treatment with Volasertib, 17-AAG, or Volasertib/17-AAG combination at the indicated concentrations (n = 6) (mean ± SD). ***P < 0.001. I Western blot analysis of cleaved-caspase 3 (CC3) and select GSC stemness marker levels in TS543 lysates upon treatment with BI 2536/ Volasertib, 17-AAG, or combination of BI 2536/ Volasertib and 17-AAG. β-actin serves as loading control.

Article Snippet: GSCs were transduced with lentiviruses overexpressing GFP, RNF8, *FHA or *RING and fresh media containing 40 μM Z-VAD-FMK (MedChemExpress) was replenished after 4-hour post-transduction.

Techniques: Over Expression, In Vitro, Western Blot, Control, Viability Assay, Marker

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: UBE2T-regulated H2AX monoubiquitination induces hepatocellular carcinoma radioresistance by facilitating CHK1 activation

doi: 10.1186/s13046-020-01734-4

Figure Lengend Snippet: UBE2T induces HCC cell radioresistance in coordination with RNF8. a 293 T cells were transfected with FLAG-UBE2T and treated with IR (4 Gy). IP was performed by using FLAG antibody, and the IP product was analyzed by immunoblotting. b Immunoblotting analysis of FLAG-IP derived from the irradiated 293 T cells transfected with empty vector or FLAG-RNF8. c Representative images of immunofluorescence staining for UBE2T (green) and RNF8 (red) in MHCC-97H cells treated with IR (4 Gy). d Immunoblotting analysis of the cytosolic and chromatin fractions of IR (4 Gy) treated MHCC-97H cells transfected with siRNA-RNF8 or siRNA-control. e Representative images and quantification of UBE2T overexpressing MHCC-97H cells stained for RNF8 (red) foci before and after IR (4 Gy). f Representative images and quantification of UBE2T silencing MHCC-97H cells stained for RNF8 (red) foci before and after IR (4 Gy). g UBE2T overexpressing cells or control cells were transfected with siRNA-RNF8 or siRNA-control, and harvested at the indicated timepoints after IR (4 Gy). h Cells with the same treatment as that in panel g were collected at the indicated timepoints after IR (4 Gy) to test cell cycle distribution. i Cells with the same treatment as that in panel g were collected at the indicated timepoints after IR to test γH2AX level. j Colony formation assays were conducted in UBE2T stably overexpressing MHCC-97H cells transduced with lentivirus coding control shRNA or shRNA targeting RNF8. Data represent the mean ± SD. In ( e ) and ( f ), * P < 0.05, by 2-tailed paired Student’s t test. In ( h ), ns, not significant, * P < 0.05, by one-way ANOVA

Article Snippet: The sources of antibodies against the following proteins were as follows: H2AX (D17A3; 7631), γH2AX (Ser139, 20E3; 9718), p-ATR (2853), p-ATM (13050), and p-CHK1 (Ser345, 133D3; 2348) from Cell Signaling Technology; UBE2T (10105–2-AP), CHK1 (25887–1-AP), RNF8 (14112–1-AP), TRIM21 (12108–1-AP), BRCA1 (22362–1-AP), RAD51 (14961–1-AP), BMI1 (10832–1-AP), RING2 (16031–1-AP), ATR (19787–1-AP), ATM (27156–1-AP), HSP70 (10995–1-AP) and LaminB1 (12987–1-AP) from Proteintech Group; H2AX (ab229914) from Abcam; FLAG (F1804) from Sigma-Aldrich; GAPDH (RM2002), HA (RM1004), myc (RM1003) and β-actin (RM2001) from Rui Antibody Biotech.

Techniques: Transfection, Western Blot, Derivative Assay, Irradiation, Plasmid Preparation, Immunofluorescence, Staining, Control, Stable Transfection, Transduction, shRNA

Journal: Oncogenesis

Article Title: RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks

doi: 10.1038/s41389-021-00315-0

Figure Lengend Snippet: A Endogenous RecQL4 or RNF8 were detected in anti-Flag immunoprecipitated fractions from Flag-RNF8/Flag-RecQL4 transfected U2OS cells by western blotting analysis. Flag-GFP/Empty vector-transfected cells were used as negative controls. B Interaction between endogenous RecQL4 and RNF8. Immunoprecipitated fraction from U2OS lysate was prepared using anti-RNF8 antibody (14112-1-AP, Proteintech), followed by western blotting analysis with anti-RecQL4 (25470002, SDIX) or RNF8 antibodies (sc-271462, Santa cruz). RecQL4 protein was detected in the RNF8 immunoprecipitated complex. C Direct interaction between RecQL4 and RNF8 was demonstrated by an in vitro pull-down assay. Purified recombinant GST-RNF8 was immobilized on Glutathione resin and incubated with purified Flag-RecQL4 in the IP buffer, and the bound proteins were examined by western blotting. D Colocalization of GFP-tagged 53BP1 and mCherry-tagged RNF8/RecQL4 at DSB track induced by UV micro-point laser. E Co-localization of mCherry-RNF8 and GFP-RecQL4 at DSB track induced by UV micro-point laser. F Co-localization of endogenous RNF8 and RecQL4 after X-ray irradiation. U2OS cells were exposed to 10 Gy of X-ray irradiation (25 mA, 160 kV; dose rate 0.995 Gy/min, X-RAD RS2000, Rad Source, USA), and fixed with 4% paraformaldehyde at 3.5 h post treatment. Indirect immunostaining was performed using primary anti-RecQL4 and RNF8 and fluorescence-dye conjugated secondary antibodies. After counterstaining with DAPI, images were captured using a fluorescence microscope (Leica DM5000 Microsystems).

Article Snippet: Immunoprecipitated fraction from U2OS lysate was prepared using anti-RNF8 antibody (14112-1-AP, Proteintech), followed by western blotting analysis with anti-RecQL4 (25470002, SDIX) or RNF8 antibodies (sc-271462, Santa cruz).

Techniques: Immunoprecipitation, Transfection, Western Blot, Plasmid Preparation, In Vitro, Pull Down Assay, Purification, Recombinant, Incubation, Irradiation, Immunostaining, Fluorescence, Microscopy

Journal: Oncogenesis

Article Title: RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks

doi: 10.1038/s41389-021-00315-0

Figure Lengend Snippet: A RecQL4 ubiquitination level was decreased upon RNF8 depletion. U2OS cells were transfected with Flag-RecQL4 plasmid, followed by control/RNF8 shRNA and HA-Ub plasmids. Immunoprecipitation was performed on the cell lysates using anti-Flag (M2) beads, and RecQL4 ubiquitination level was determined by western blotting analysis with anti-HA antibody. B RNF8 promotes RecQL4 ubiquitination in vivo. U2OS cells were transfected with Flag-RecQL4, HA-Ub, and RNF8 plasmids. Empty vector was used as control. C , D RecQL4 CT mutant (3M: K876R-K2048R-K1101R) showed a markedly decreased ubiquitination level relative to wild-type RecQL4 CT in both in vivo and in vitro ubiquitination assays.

Article Snippet: Immunoprecipitated fraction from U2OS lysate was prepared using anti-RNF8 antibody (14112-1-AP, Proteintech), followed by western blotting analysis with anti-RecQL4 (25470002, SDIX) or RNF8 antibodies (sc-271462, Santa cruz).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Control, shRNA, Immunoprecipitation, Western Blot, In Vivo, Mutagenesis, In Vitro

Journal: Oncogenesis

Article Title: RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks

doi: 10.1038/s41389-021-00315-0

Figure Lengend Snippet: A Knockdown of RNF8 significantly inhibits the dissociation of RecQL4 from DSBs. U2OS cells with or without RNF8 depletion were treated with 365-nm micro-point laser. The recruitment of GFP-RecQL4 at DSBs was recorded and the fluorescence density was quantified. B The prolonged and enhanced recruitment of RecQL4-3M at DSBs induced by UV micro-point laser. U2OS cells were transfected with GFP-tagged WT RecQL4 or its 3M mutant, and subjected to UV micro-point laser treatment. The recruitment of GFP-RecQL4 or its mutant at DSBs were recorded and the fluorescence density at DSBs was quantified. At least 15 cells were analyzed for each treatment. The data represent mean ± SEM from three independent experiments. ** p < 0.01.

Article Snippet: Immunoprecipitated fraction from U2OS lysate was prepared using anti-RNF8 antibody (14112-1-AP, Proteintech), followed by western blotting analysis with anti-RecQL4 (25470002, SDIX) or RNF8 antibodies (sc-271462, Santa cruz).

Techniques: Knockdown, Fluorescence, Transfection, Mutagenesis

Journal: Oncogenesis

Article Title: RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks

doi: 10.1038/s41389-021-00315-0

Figure Lengend Snippet: A , B RecQL4 depletion significantly decreased the HR- and NHEJ-mediated DSB repair in U2OS cells quantified by DR-GFP and EJ5-GFP reporter system, respectively. The defective DSB repair was significantly restored by re-introduction of wild-type RecQL4 but not its mutant. The percentage of GFP-positive cells was quantified by Flow cytometry. The data represent mean ± SEM from three independent experiments. (* p < 0.05, Student’s t test). C Analysis of the time-dependent recruitment of various DSB repair proteins in U2OS cells after micro-point laser treatment. Cells were transfected with GFP-tagged RecQL4, RNF8, Ku80, 53BP1, or mCherry-tagged MDC1 plasmids, followed by the treatment of micro-point laser. The images were captured using time-lapse microscopy. At least 15 cells for each transfection were recorded and analyzed for the earliest time point of protein aggregate formation at DSB track. D RecQL4 ubiquitination status affects the recruitment of its directly associated downstream protein-CtIP. RecQL4 was first silenced in U2OS cells followed by transfection with GFP-tagged CtIP and mCherry-tagged wild type RecQL4 or its mutant (3M). The mCherry-positive cells were treated with micro-point laser and the images were captured using microscopy. Both recruitment time and fluorescence density were recorded and at least 15 cells were analyzed. The data represent mean ± SEM from three independent experiments. ** p < 0.01. E RecQL4 3M mutant interferes with its capacity in processing ssDNA formation at DSB ends estimated by an end resection assay. RecQL4 was first silenced by shRNA infection in U2OS cells which were then transfected with either a control, RecQL4 WT, or 3M mutant for 24 h, followed by the treatment with 1 μM CPT (C9911, Sigma) for 1 h. Cells were fixed for RPA2 (ab2175, Abcam) immunostaining. A total of 200 cells were analyzed and RPA2-foci (>15) positive cells were scored for each individual experiment. Data represent mean ± SEM of three independent experiments (** p < 0.01, the Single Factor Anova test). Fluorescence images were captured using a LEICA TCS SP8 confocal microscope system. Scale bar, 22 μm.

Article Snippet: Immunoprecipitated fraction from U2OS lysate was prepared using anti-RNF8 antibody (14112-1-AP, Proteintech), followed by western blotting analysis with anti-RecQL4 (25470002, SDIX) or RNF8 antibodies (sc-271462, Santa cruz).

Techniques: Mutagenesis, Flow Cytometry, Transfection, Time-lapse Microscopy, Ubiquitin Proteomics, Microscopy, Fluorescence, Resection Assay, shRNA, Infection, Control, Immunostaining

Journal: Oncogenesis

Article Title: RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks

doi: 10.1038/s41389-021-00315-0

Figure Lengend Snippet: A RecQL4 interacts with WRAP53β. Endogenous WRAP53β was present in Flag immunoprecipitated fractions using the lysate of HEK293 cells transfected with Flag-RecQL4. Direct interaction between RecQL4 and WRAP53β was further demonstrated by an in vitro pull-down assay using the purified recombinant GST-WRAP53β to pull down Flag-RecQL4. B A markedly enhanced interaction between RecQL4 and RNF8 was observed upon the increased input of WRAP53β protein assessed by an in vitro Flag pull-down assay, followed by the western blotting analysis. C Dynamic recruitment of GFP-RNF8 to DSBs induced by UV micro-point laser in WRAP53β silenced U2OS cells relative to scrambled siRNA-transfected cells. D Dynamic recruitment of GFP-RecQL4 to DSBs in scrambled siRNA or WRAP53β siRNA-transfected U2OS cells.

Article Snippet: Immunoprecipitated fraction from U2OS lysate was prepared using anti-RNF8 antibody (14112-1-AP, Proteintech), followed by western blotting analysis with anti-RecQL4 (25470002, SDIX) or RNF8 antibodies (sc-271462, Santa cruz).

Techniques: Immunoprecipitation, Transfection, In Vitro, Pull Down Assay, Purification, Recombinant, Western Blot

Journal: Cell reports

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

doi: 10.1016/j.celrep.2021.109506

Figure Lengend Snippet: (A) Predicted let-7 miRNA binding sites in Rnf8 mRNA. (B–F) Western blot analysis and densitometric quantification of RNF8 protein (B, D, and F) and qRT-PCR of Rnf8 mRNA (C and E) in let7b/c2 +/+ and let7b/c2 ΔHep (B and C); EGFP and let-7 sponge AAV-transduced (D and E); EGFP and pre-let-7c-1 AAV-transduced (F) livers treated with HFD feeding. (G) 3′ UTR reporter assays in HepG2 cells transfected with Rnf8 wild-type or mutant 3′ UTR reporter constructs and a let-7c mimic expression vector. (H and I) Western blot analysis (H) and densitometric quantification (I) of RXRα expression in Rxra - and Rnf8 -transfected Hepa-1 cells. (J and K) Fold change of Rnf8 (J) and Rxra (K) mRNA by qRT-PCR analysis in Rxra - and Rnf8 -transfected Hepa-1 cells. (L) Western blot analysis and the densitometric quantification of RXRα in Rxra - and Rnf8- transfected Hepa-1 cells treated with the proteasome inhibitor MG-132. (M) Ubiquitination assays for Rxra - and Rnf8 -transfected and MG-132-treated Hepa-1 cells. RXRα was immunoprecipitated and polyubiquitin detected by anti-ubiquitin antibody. RXRα expression was confirmed in whole-cell lysate as input. (N) Scheme of 3-step inhibition for PPARα/RXRα pathway that the current study demonstrates.

Article Snippet: pSG5-mouse Rxra ( ) and pCMV6-mouse Rnf8 (Origene) expression plasmids were co-transfected into Hepa-1c1c7 cells (ATCC) using Lipofectamine 3000 (Thermo Fisher Scientific). pRK5-HA-Ubiquitin-WT (Addgene) were co-transfected for polyubiquitin type analysis.

Techniques: Binding Assay, Western Blot, Quantitative RT-PCR, Transfection, Mutagenesis, Construct, Expressing, Plasmid Preparation, Ubiquitin Proteomics, Immunoprecipitation, Inhibition

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: pSG5-mouse Rxra ( ) and pCMV6-mouse Rnf8 (Origene) expression plasmids were co-transfected into Hepa-1c1c7 cells (ATCC) using Lipofectamine 3000 (Thermo Fisher Scientific). pRK5-HA-Ubiquitin-WT (Addgene) were co-transfected for polyubiquitin type analysis.

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

Journal: Biochemical and biophysical research communications

Article Title: Targeting RNF8 effectively reverses cisplatin and doxorubicin resistance in endometrial cancer.

doi: 10.1016/j.bbrc.2021.01.046

Figure Lengend Snippet: Fig. 1. Expression of RNF8 is increased in cisplatin and doxorubicin resistant EC cells. A Cell viability of ISH-WT, ISH-Cis-R, B AN3CA-WT and AN3CA-Cis-R cells. Cisplatin con- centrations are 0 mM, 5 mM, 10 mM, 20 mM, and 40 mM. Data are represented as mean ± standard deviations (SD) of three independent experiments. C Cell viability of ISH-WT, ISH- Dox-R, D AN3CA-WT and AN3CA-Dox-R cells. Doxorubicin concentrations are 0 mM, 1 mM, 2 mM, 4 mM, and 8 mM. Data are represented as mean ± SD of three independent ex- periments. E RNF8 mRNA expression in ISH-WT, ISH-Cis-R, F AN3CA-WT, AN3CA-Cis-R, G ISH-WT, ISH-Dox-R, H AN3CA-WT and AN3CA-Dox-R cells. ***: P < 0.001. I Western blotting analysis of RNF8 protein expression in ISH-WT, ISH-Cis-R, AN3CA-WT and AN3CA-Cis-R, ISH-WT, ISH-Dox-R, AN3CA-WT and AN3CA-Dox-R cells. J RNF8 mRNA expression in EC patient samples. Platinum sensitive group: n ¼ 32. Platinum resistant group: n ¼ 32. ***: p < 0.001.

Article Snippet: A pool of 3 plasmids encoding Cas9 coding gene and RNF8 guide RNA (cat. no. sc-401909; Santa Crus Biotechnology, Inc.) or CRISPR/ Cas9-Ctr Plasmid (cat. no. sc-418922; Santa Crus Biotechnology, Inc.) was transfected into AN3CA Cis-R and AN3CA Dox-R cells using Lipofectamine® 2000.

Techniques: Expressing, Western Blot

Journal: Biochemical and biophysical research communications

Article Title: Targeting RNF8 effectively reverses cisplatin and doxorubicin resistance in endometrial cancer.

doi: 10.1016/j.bbrc.2021.01.046

Figure Lengend Snippet: Fig. 2. Depletion of RNF8 sensitize cisplatin and doxorubicin resistant EC cells. A Western blotting analysis of RNF8 protein expression in AN3CA-Cis-R-Ctr, RNF8 knockout AN3CA- Cis-R clone 1 (AN3CA-Cis-R-KO1), RNF8 knockout AN3CA-Cis-R clone 2 (AN3CA-Cis-R-KO2), RNF8 ectopic expressed AN3CA-Cis-R-KO1 (AN3CA-Cis-R-KO1þRNF8), RNF8 ectopic expressed AN3CA-Cis-R-KO2 (AN3CA-Cis-R-KO2þRNF8), B AN3CA-Dox-R-Ctr, RNF8 knockout AN3CA-Dox-R clone 1 (AN3CA-Dox-R-KO1), RNF8 knockout AN3CA-Dox-R clone 2 (AN3CA-Dox-R-KO2), RNF8 ectopic expressed AN3CA-Dox-R-KO1 (AN3CA-Dox-R-KO1þRNF8), RNF8 ectopic expressed AN3CA-Dox-R-KO2 (AN3CA-Dox-R-KO2þRNF8) cells. C Cell viability of AN3CA-Cis-R-Ctr, AN3CA-Cis-R-KO1, AN3CA-Cis-R-KO2, AN3CA-Cis-R-KO1þRNF8 and AN3CA-Cis-R-KO2þRNF8 cells to cisplatin. Data are represented as mean ± SD of three independent experiments. D Cell viability of AN3CA-Dox-R-Ctr, AN3CA-Dox-R-KO1, AN3CA-Dox-R-KO2, AN3CA-Dox-R-KO1þRNF8, AN3CA-Dox-R-KO2þRNF8 cells to doxo- rubicin. Data are represented as mean ± SD of three independent experiments.

Article Snippet: A pool of 3 plasmids encoding Cas9 coding gene and RNF8 guide RNA (cat. no. sc-401909; Santa Crus Biotechnology, Inc.) or CRISPR/ Cas9-Ctr Plasmid (cat. no. sc-418922; Santa Crus Biotechnology, Inc.) was transfected into AN3CA Cis-R and AN3CA Dox-R cells using Lipofectamine® 2000.

Techniques: Western Blot, Expressing, Knock-Out

Journal: Biochemical and biophysical research communications

Article Title: Targeting RNF8 effectively reverses cisplatin and doxorubicin resistance in endometrial cancer.

doi: 10.1016/j.bbrc.2021.01.046

Figure Lengend Snippet: Fig. 3. RNF8 deficiency reduces NHEJ efficiency in cisplatin and doxorubicin resistant EC cells. A Relative NHEJ events in AN3CA-Cis-R, AN3CA-Cis-R-KO1, AN3CA-Cis-R-KO2, AN3CA- Cis-R-KO1þRNF8 and AN3CA-Cis-R-KO2þRNF8 cells. Data are represented as mean ± SD of three independent experiments. **: P < 0.01. B Relative NHEJ events in AN3CA-Dox-R, AN3CA-Dox-R-KO1, AN3CA-Dox-R-KO2, AN3CA-Dox-R-KO1þRNF8, AN3CA-Dox-R-KO2þRNF8 cells. Data are represented as mean ± SD of three independent experiments. ***: P < 0.001. C Localization of Ku80 at DNA damage sites upon DNA damage in AN3CA-Cis-R-Ctr, AN3CA-Cis-R-KO1 and AN3CA-Cis-R-KO1þRNF8 cells. Cells were subjected to

Article Snippet: A pool of 3 plasmids encoding Cas9 coding gene and RNF8 guide RNA (cat. no. sc-401909; Santa Crus Biotechnology, Inc.) or CRISPR/ Cas9-Ctr Plasmid (cat. no. sc-418922; Santa Crus Biotechnology, Inc.) was transfected into AN3CA Cis-R and AN3CA Dox-R cells using Lipofectamine® 2000.

Techniques:

Journal: Biochemical and biophysical research communications

Article Title: Targeting RNF8 effectively reverses cisplatin and doxorubicin resistance in endometrial cancer.

doi: 10.1016/j.bbrc.2021.01.046

Figure Lengend Snippet: Fig. 4. Inhibition of RNF8 increases sensitivity to cisplatin in vivo. A Photograph of tumors dissected from mice at day 21 in AN3CA-Cis-R-Ctr and AN3CA-Cis-R-KO1 xenograft. Female BALB/c nude mice were treated intraperitoneally with vehicle (DMSO) or cisplatin (8 mg/kg/3 days). Group size: 6 mice/group. B Tumor growth, C Tumor size and D body weight of AN3CA-Cis-R-Ctr and AN3CA-Cis-R-KO1 xenograft in response to cisplatin. *: p < 0.05, ****: p < 0.0001.

Article Snippet: A pool of 3 plasmids encoding Cas9 coding gene and RNF8 guide RNA (cat. no. sc-401909; Santa Crus Biotechnology, Inc.) or CRISPR/ Cas9-Ctr Plasmid (cat. no. sc-418922; Santa Crus Biotechnology, Inc.) was transfected into AN3CA Cis-R and AN3CA Dox-R cells using Lipofectamine® 2000.

Techniques: Inhibition, In Vivo

Journal: Science Advances

Article Title: Extracellular matrix stiffness determines DNA repair efficiency and cellular sensitivity to genotoxic agents

doi: 10.1126/sciadv.abb2630

Figure Lengend Snippet: ( A to G ) HEK293 cells were grown for 24 hours on fibronectin-coated hydrogels of different stiffness. Cells were fixed 1 hour after irradiation (1 Gy) and stained with anti–γ-H2AX (A), MDC1 (B), RNF8 (C), FK2 (D), 53BP1 (F), and BRCA1(G) antibodies. For RNF168 (E), HEK293 cells expressing mCherry-RNF168 were plated on fibronectin-coated hydrogels of different stiffness. Cells were fixed 1 hour after irradiation (1 Gy), and RNF168 foci were visualized with mCherry. Quantification is described in Methods. Data are presented as means ± SD, n = 3 biologically independent samples (** P < 0.01). ( H ) A model showing the affected DNA repair steps by low stiffness.

Article Snippet: Antibodies were obtained from the following sources: Ub (Santa Cruz Biotechnology, sc-8017), actin (Sigma-Aldrich, A2228), γ-H2AX (Millipore, 05-636), FLAG (Sigma-Aldrich, F1804), HA-tag [HA; Cell Signaling Technology (CST), 5017], Myc (Santa Cruz Biotechnology, 9E10), 53BP1 (Novus Biologicals, NB100-304), BRCA1 (Santa Cruz Biotechnology, sc-6954), FK2 (Millipore, 04-263), RNF8 (Novus Biologicals, H00009025-D01P, Anti-Ubiquitin Antibody), MAP4K4 (Bethyl, A301-503A), MAP4K6 (Novus, NBP1-22990), MAP4K7 (Genetex, GTX13141), YAP/TAZ (Santa Cruz Biotechnology, sc-101199), V5 (Abcam, ab9116), pLATS1 T1079 (CST, 8654), LATS1 (CST, 3477), LATS2 (CST,5888), MST1 (CST, 3682), MST2 (CST, 3952), and Rap2 (Biosciences, 610215).

Techniques: Irradiation, Staining, Expressing

Journal: Science Advances

Article Title: Extracellular matrix stiffness determines DNA repair efficiency and cellular sensitivity to genotoxic agents

doi: 10.1126/sciadv.abb2630

Figure Lengend Snippet: ( A ) Western blots showing the expression levels of MST1, MST2, MAP4K4, MAP4K6, and MAP4K7 in control (MM0), MM2KO, MM3KO, and MM5KO HEK293 cells. M w , weight average molecular weight. ( B to D ) MM0, MM2KO, MM3KO, and MM5KO cells were grown on soft (1 kPa) and stiff (30 kPa) fibronectin-coated hydrogels. Cells were fixed 1 hour after irradiation (1 Gy) and stained with anti–γ-H2AX and MDC1 (B), RNF8 and FK2 (C), and 53BP1 and BRCA1 (D) antibodies. Scale bars, 10 μM. ( E to H ) Quantification of (B) to (D) is described in Methods. ( K and L ) MM0, MM2KO, MM3KO, and MM5KO cells were grown on soft (1 kPa) and stiff (30 kPa) fibronectin-coated hydrogels. Effect of ECM stiffness on the efficiency of NHEJ (K) and HR (L) in indicated cells was analyzed by flow cytometry. ( M ) MAP4K4/6/7 kinases are required for regulation of stiffness-induced radiation sensitivity. Colony formation assays were performed to examine survival of WT (MM0) and MM3KO HEK293 cells on soft (1 kPa) and stiff (30 kPa) fibronectin-coated hydrogels when exposed to the indicated doses of radiation.

Article Snippet: Antibodies were obtained from the following sources: Ub (Santa Cruz Biotechnology, sc-8017), actin (Sigma-Aldrich, A2228), γ-H2AX (Millipore, 05-636), FLAG (Sigma-Aldrich, F1804), HA-tag [HA; Cell Signaling Technology (CST), 5017], Myc (Santa Cruz Biotechnology, 9E10), 53BP1 (Novus Biologicals, NB100-304), BRCA1 (Santa Cruz Biotechnology, sc-6954), FK2 (Millipore, 04-263), RNF8 (Novus Biologicals, H00009025-D01P, Anti-Ubiquitin Antibody), MAP4K4 (Bethyl, A301-503A), MAP4K6 (Novus, NBP1-22990), MAP4K7 (Genetex, GTX13141), YAP/TAZ (Santa Cruz Biotechnology, sc-101199), V5 (Abcam, ab9116), pLATS1 T1079 (CST, 8654), LATS1 (CST, 3477), LATS2 (CST,5888), MST1 (CST, 3682), MST2 (CST, 3952), and Rap2 (Biosciences, 610215).

Techniques: Western Blot, Expressing, Control, Molecular Weight, Irradiation, Staining, Flow Cytometry

Journal: Science Advances

Article Title: Extracellular matrix stiffness determines DNA repair efficiency and cellular sensitivity to genotoxic agents

doi: 10.1126/sciadv.abb2630

Figure Lengend Snippet: ( A ) Purified Ub variants were analyzed by electrospray ionization mass spectrometry. ( B ) Ub variants were treated with or without λ-PPase and blotted with indicated antibodies. ( C ) The assembly of ubiquitin chains was determined in the presence of Ube1, UbcH5c, and RNF8 and indicated Ub variants. Samples were taken at the indicated time and immunoblotted with anti-Ub antibody. ( D ) The assembly of ubiquitin chains was determined in the presence of Ube1, indicated E2s, RNF8, and indicated Ub variants. Polyubiquitin chains were detected by immunoblotting with an anti-Ub antibody.( E ) The assembly of ubiquitin chains was determined in the presence of Ube1, UbcH5c, RNF8, and indicated Ub variants. Samples were taken at the indicated time and immunoblotted with anti-Ub antibody. ( F ) HEK293 expressing WT, T66A, and T66E mutant ubiquitin were blotted with anti-Ub antibody. ( G ) Cells as in (F) were transfected with indicated plasmids. Cells were irradiated (10 Gy) and blotted with anti-HA antibody. ( H to J ) Cells as in (F) were grown on glass cover slips. Cells were fixed 1 hour after irradiation (1 Gy) and stained with indicated antibodies. ( K to P ) Quantification of (H) to (J). ** P < 0.01.

Article Snippet: Antibodies were obtained from the following sources: Ub (Santa Cruz Biotechnology, sc-8017), actin (Sigma-Aldrich, A2228), γ-H2AX (Millipore, 05-636), FLAG (Sigma-Aldrich, F1804), HA-tag [HA; Cell Signaling Technology (CST), 5017], Myc (Santa Cruz Biotechnology, 9E10), 53BP1 (Novus Biologicals, NB100-304), BRCA1 (Santa Cruz Biotechnology, sc-6954), FK2 (Millipore, 04-263), RNF8 (Novus Biologicals, H00009025-D01P, Anti-Ubiquitin Antibody), MAP4K4 (Bethyl, A301-503A), MAP4K6 (Novus, NBP1-22990), MAP4K7 (Genetex, GTX13141), YAP/TAZ (Santa Cruz Biotechnology, sc-101199), V5 (Abcam, ab9116), pLATS1 T1079 (CST, 8654), LATS1 (CST, 3477), LATS2 (CST,5888), MST1 (CST, 3682), MST2 (CST, 3952), and Rap2 (Biosciences, 610215).

Techniques: Purification, Mass Spectrometry, Ubiquitin Proteomics, Western Blot, Expressing, Mutagenesis, Transfection, Irradiation, Staining

Journal: Science Advances

Article Title: Extracellular matrix stiffness determines DNA repair efficiency and cellular sensitivity to genotoxic agents

doi: 10.1126/sciadv.abb2630

Figure Lengend Snippet: ( A to F ) Ubiquitin-replacement HEK293 cells expressing wild-type (WT), T66A, or T66E mutant ubiquitin were plated on soft (1 kPa) and stiff (30 kPa) fibronectin-coated hydrogels. Cells were fixed 1 hour after irradiation and stained with anti–γ-H2AX (A), MDC1 (B), RNF8 (C), FK2 (D), 53BP1 (E), and BRCA1 (F) antibodies. Data are presented as means ± SD, n = 3 biologically independent samples (** P < 0.01). ( G and H ) Phosphorylation of ubiquitin inhibits HR and NHEJ. Effect of ubiquitin phosphorylation on the efficiency of NHEJ (G) and HR (H) was analyzed by flow cytometry. Data are presented as means ± SD. n = 3 biologically independent samples (** P < 0.01). ( I to L ) Phosphorylation of ubiquitin regulates genotoxic sensitivity. Ubiquitin-replacement HEK293 cells expressing wild-type (WT), T66A, or T66E mutant ubiquitin were plated on soft (1 kPa) fibronectin-coated hydrogels. Cells were treated with indicated genotoxic agents. Colony formation assays were performed to examine survival of cells expressing wild-type (WT), T66A, or T66E mutant ubiquitin on soft (1 kPa) fibronectin-coated hydrogels. Data are presented as means ± SD. n = 3 biologically independent samples.

Article Snippet: Antibodies were obtained from the following sources: Ub (Santa Cruz Biotechnology, sc-8017), actin (Sigma-Aldrich, A2228), γ-H2AX (Millipore, 05-636), FLAG (Sigma-Aldrich, F1804), HA-tag [HA; Cell Signaling Technology (CST), 5017], Myc (Santa Cruz Biotechnology, 9E10), 53BP1 (Novus Biologicals, NB100-304), BRCA1 (Santa Cruz Biotechnology, sc-6954), FK2 (Millipore, 04-263), RNF8 (Novus Biologicals, H00009025-D01P, Anti-Ubiquitin Antibody), MAP4K4 (Bethyl, A301-503A), MAP4K6 (Novus, NBP1-22990), MAP4K7 (Genetex, GTX13141), YAP/TAZ (Santa Cruz Biotechnology, sc-101199), V5 (Abcam, ab9116), pLATS1 T1079 (CST, 8654), LATS1 (CST, 3477), LATS2 (CST,5888), MST1 (CST, 3682), MST2 (CST, 3952), and Rap2 (Biosciences, 610215).

Techniques: Ubiquitin Proteomics, Expressing, Mutagenesis, Irradiation, Staining, Phospho-proteomics, Flow Cytometry