Journal: Epigenetics & Chromatin

Article Title: HP1β-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs

doi: 10.1186/1756-8935-7-39

Figure Lengend Snippet: Recruitment of GFP-UBF1, HP1β, γH2AX, and 53BP1 to UVA-induced DNA lesions. (A) Cells transiently expressing GFP-UBF1 were microirradiated using a 355-nm UVA laser. Recombinant GFP-UBF1 (green) in (Aa) immortalized mouse embryonic fibroblasts and (Ab) primary mouse embryonic fibroblasts was monitored after local microirradiation by a 355-nm UVA laser. Magnification of the irradiated nucleus is shown. (B) Endogenous UBF1/2 (red) was analyzed by immunofluorescence after local UVA microirradiation of regions of interest (yellow) in 3T3 cells stably expressing GFP-HP1β (green). Irradiated cells were fixed in 4% formaldehyde and stained with appropriate antibodies against (C) HP1β (red), (D) γH2AX (red), and (E) 53BP1 (red). Regions of interest (yellow) were irradiated by a 355-nm UVA laser. Cell nuclei were visualized by DAPI (blue), and UBF1 was tagged by GFP (green). For each event, 10 nuclei were analyzed in three independent experiments. IF, immunofluorescence; LCI, live-cell image; Nu, nucleolus.

Article Snippet: RNA interference (RNAi) for HP1β was performed using Lipofectamine RNAiMax transfection reagent (#13778-075, Invitrogen), siRNA (HP1β) (#sc-35588, Santa Cruz Biotechnology, Inc., Germany), control siRNA (#sc-36869, Santa Cruz Biotechnology, Inc.), and Opti-Mem reduced serum medium (#31985047, Invitrogen).

Techniques: Expressing, Recombinant, Irradiation, Immunofluorescence, Stable Transfection, Staining

Journal: Epigenetics & Chromatin

Article Title: HP1β-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs

doi: 10.1186/1756-8935-7-39

Figure Lengend Snippet: Protein interactions at DNA lesions. (A) FRET results for reference interacting partners p53 (green) and 53BP1 (red). (B) FRET analysis of UBF1/2 dimerization. FRET analysis of (C) UBF1/2 (red) and γH2AX (green), (D) GFP-UBF1 (green) and 53BP1 (red), and (E) HP1β (green) and UBF1/2 (red). Studies were performed in control and γ-irradiated cells using a Leica TSC SP5 confocal microscope. For each event, 10 nuclei were analyzed in three independent experiments. IF, immunofluorescence; non-irrad., non-irradiated cells.

Article Snippet: RNA interference (RNAi) for HP1β was performed using Lipofectamine RNAiMax transfection reagent (#13778-075, Invitrogen), siRNA (HP1β) (#sc-35588, Santa Cruz Biotechnology, Inc., Germany), control siRNA (#sc-36869, Santa Cruz Biotechnology, Inc.), and Opti-Mem reduced serum medium (#31985047, Invitrogen).

Techniques: Control, Irradiation, Microscopy, Immunofluorescence

Journal: Epigenetics & Chromatin

Article Title: HP1β-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs

doi: 10.1186/1756-8935-7-39

Figure Lengend Snippet: siRNA silencing of HP1β reduced UBF1/2 accumulation after UVA irradiation. (A) Recruitment of endogenous UBF1/2 (red) to locally induced, γH2AX-positive (blue) DNA lesions (yellow frames) in 3T3 cells stably expressing GFP-HP1β (green) in control sample relevant to siRNA experiments. (B) Effect of HP1β siRNA on UBF1/2 (red) accumulation after UVA irradiation by a 355-nm UVA laser. Induced DNA lesions were positive for γH2AX (blue). (C) Percentage of irradiated cells with accumulated UBF1/2. Thirty cell nuclei were evaluated for each event. Asterisks show statistically significant differences from control values ( P ≤ 0.05) using Student’s t tests. (D) Efficiency of HP1β siRNA was determined by (a) Western blots showing the level of HP1β and UBF1/2. Samples were loaded according to identical total protein levels. Results of (b) qRT-PCR experiments show up-regulation of HP1β in UVA-irradiated cells. Results were obtained from two biological replicates. The transcripts were normalized to two housekeeping reference genes ( GAPDH and β-actin ). Irradiation was performed by UVA lamp (E) Analysis of CPD accumulation at DNA lesions in (a) control cells and (b) cells exposed to UBF siRNA. Image acquisition was performed 7 min, 15 min, 30 min, 1 h, 2 h, and 3 h after local microirradiation by a 355-nm UVA laser. For panel E, 40 nuclei were examined. C, control; Exp 1, first independent experiment; Exp 2, second independent experiment; IF, immunofluorescence; LCI, live-cell image.

Article Snippet: RNA interference (RNAi) for HP1β was performed using Lipofectamine RNAiMax transfection reagent (#13778-075, Invitrogen), siRNA (HP1β) (#sc-35588, Santa Cruz Biotechnology, Inc., Germany), control siRNA (#sc-36869, Santa Cruz Biotechnology, Inc.), and Opti-Mem reduced serum medium (#31985047, Invitrogen).

Techniques: Irradiation, Stable Transfection, Expressing, Control, Western Blot, Quantitative RT-PCR, Immunofluorescence

Journal: Epigenetics & Chromatin

Article Title: HP1β-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs

doi: 10.1186/1756-8935-7-39

Figure Lengend Snippet: Functional analysis of HP1βΔCSD, HP1βΔhinge, and HP1βΔCD for UBF1 recruitment to UVA-induced DNA lesions. (A) Analysis of endogenous UBF1/2 (red) recruitment to UVA-induced DNA lesions (yellow regions of interest) in MEFs transiently expressing (a) GFP-HP1β-ΔCSD, (b) GFP-HP1β-Δhinge, or (c) GFP-HP1β-ΔCD (all green). (B) Quantification of fluorescence intensity of UBF1/2 (red) and HP1β (green) from panel A. Quantification was performed for (a) GFP-HP1β-ΔCSD, (b) GFP-HP1β-Δhinge, and (c) GFP-HP1β-ΔCD (all green). (C) Percentage of cells with recruitment of studied proteins to DNA lesions. Thirty cells were analyzed for each event in three independent experiments. (D) Analysis of endogenous HP1β (red) at UVA-induced DNA lesions (yellow regions of interest ) in mouse embryonic fibroblasts transiently expressing GFP-HP1β-ΔCSD (green). For panel D, 20 nuclei were analyzed. CD, chromodomain; CSD, chromo shadow domain; IF, immunofluorescence; LCI, live-cell image.

Article Snippet: RNA interference (RNAi) for HP1β was performed using Lipofectamine RNAiMax transfection reagent (#13778-075, Invitrogen), siRNA (HP1β) (#sc-35588, Santa Cruz Biotechnology, Inc., Germany), control siRNA (#sc-36869, Santa Cruz Biotechnology, Inc.), and Opti-Mem reduced serum medium (#31985047, Invitrogen).

Techniques: Functional Assay, Expressing, Fluorescence, Immunofluorescence

Journal: Epigenetics & Chromatin

Article Title: HP1β-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs

doi: 10.1186/1756-8935-7-39

Figure Lengend Snippet: Interaction of UBF1 with UVA-damaged chromatin. (A) Cells expressing full-length GFP-HP1β and GFP-HP1β-ΔCSD were immunoprecipitated with antibody against UBF1/2, and subsequent Western blot analysis of HP1β was performed. Quantification of immunoprecipitation fragments and original Western blots are shown. (B) FRET analysis of potential interactions between UBF1/2 (Alexa 594, red) and DNA (stained by TO-PRO-3, far-red, 642/661 nm). (Ba) Regions of interest (yellow) inspected by FRET; (Bb) quantification of UBF1/2 interaction with DNA in the nucleolus and nucleoplasm of irradiated and non-irradiated cells. FRET efficiency is shown in percentage ± standard error. For each event, 10 nuclei were analyzed in three independent experiments. (C) Immunoprecipitation verified the interaction between HP1β and UBF1/2; HP1β and H3K9me3 were used as reference interacting partners. Immunoprecipitation was performed in the presence or absence of EtBr; 20 μg of protein lysate was used as input. IF, immunofluorescence; irrad., irradiated; Nu, nucleolus; Nupl, nucleoplasm.

Article Snippet: RNA interference (RNAi) for HP1β was performed using Lipofectamine RNAiMax transfection reagent (#13778-075, Invitrogen), siRNA (HP1β) (#sc-35588, Santa Cruz Biotechnology, Inc., Germany), control siRNA (#sc-36869, Santa Cruz Biotechnology, Inc.), and Opti-Mem reduced serum medium (#31985047, Invitrogen).

Techniques: Expressing, Immunoprecipitation, Western Blot, Staining, Irradiation, Immunofluorescence