Fig 1: Inhibition of DSB repair by low stiffness is dependent on MAP4K4/6/7.(A) Western blots showing the expression levels of MST1, MST2, MAP4K4, MAP4K6, and MAP4K7 in control (MM0), MM2KO, MM3KO, and MM5KO HEK293 cells. Mw, 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.
Fig 2: Low stiffness inhibits DSB repair at the level of RNF8 in the DSB repair pathway.(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.
Fig 3: Phosphorylation of ubiquitin blocks RNF8/RNF168-mediated ubiquitin conjugation in vitro and in cells.(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.
Fig 4: Phosphorylation of ubiquitin mediated DNA repair blockage in cells at low stiffness.(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.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for RNF8 Antibody - Azide and BSA Free
Available conjugates: Available conjugates: UnconjugatedSizes Available: 0.1 mg