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: 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

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: 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