Journal: Chromosoma

Article Title: COORDINATION OF THE RECRUITMENT OF THE FANCD2 AND PALB2 FANCONI ANEMIA PROTEINS BY A UBIQUITIN SIGNALING NETWORK

doi: 10.1007/s00412-016-0602-9

Figure Lengend Snippet: a Representative immunoblot showing knockdown of MDC1. b–c Representative images (b) and quantification (c) of FANCD2 focus formation in HeLa cells transfected with a control siRNA or two distinct siRNAs targeting MDC1 and treated with 0.5 µM MMC for 16 hr. FANCD2 foci (red) are also shown in merged images in b with DAPI signal in blue to indicate the position of nuclei. d–e Representative images of MMC-induced PALB2 foci (d) in cells transfected with either siLacZ or MDC1 siRNAs and quantification (e) of the percentage of HeLa cells with five or more PALB2 foci. PALB2 foci (red) are also shown in merged images in d with DAPI signal in blue. f Immunoblot showing FANCD2 monoubiquitination status in cells treated with a siRNA directed against RNF8 or MDC1. The intensity of the upper monoubiquitinated band divided by the intensity of the lower unubiquitinated band is shown for each lane. g Quantification of the percentage of HeLa cells with five or more ubiquitin foci following transfection with siRNAs and treatment with MMC. Values in c,e,g represent the mean of three independent counts of at least 150 cells each ± standard deviation; * indicates p<0.005.

Article Snippet: Antibodies Primary antibodies utilized for immunofluorescence microscopy and immunoblotting were as follows: FK2 (EMD Millipore, 04-263), FANCD2 (E35) ( Garcia-Higuera et al. 2001 ), PALB2 ( Zhang et al. 2009a ), γH2AX (EMDMillipore, JBW301), RNF8 (Santa Cruz, sc271462), MDC1 (Novus Biologicals, NB100-395), FAAP20 ( Ali et al. 2012 ), RAP80 (Bethyl Laboratory, A300-763A), and anti-HA (Covance, 16B12).

Techniques: Western Blot, Knockdown, Transfection, Control, Ubiquitin Proteomics, Standard Deviation

Journal: Cell Death and Differentiation

Article Title: JMJD6 modulates DNA damage response through downregulating H4K16ac independently of its enzymatic activity

doi: 10.1038/s41418-019-0397-3

Figure Lengend Snippet: JMJD6 is recruited to DSBs, and limits the spreading of histone ubiquitination around DSBs independently of its enzymatic activity. a EGFP-JMJD6 is recruited to DNA damage sites. EGFP-JMJD6 expression constructs were transfected into U2OS cells, and the localization of EGFP-JMJD6 was observed under a fluorescence microscope following laser microirradiation. Scale bar, 20 μm. b The endogenous JMJD6 is recruited to laser irradiated regions. Cell treated with microirradiation were subjected to immunofluorescent staining using anti-JMJD6 together with anti-γH2A.X. Scale bar, 20 μm. c JMJD6 overexpression does not affect the initial γH2A.X foci formation, but prevents the vanishment of those foci. U2OS cells transfected with FLAG-JMJD6 expression constructs were treated with 10 Gy of IR, and immunofluorescence assays were performed using anti-FLAG together with anti-γH2A.X at 1 and 8 h after irradiation, respectively. Scale bar, 20 μm. d JMJD6 overexpression does not alter MDC1 foci formation, but prevents the vanishment of those foci. Cells were transfected with FLAG-JMJD6 expression constructs, exposed to 10 Gy of IR, and immunostained for FLAG and MDC1 at the indicated time. Scale bar, 20 μm. e JMJD6 overexpression inhibits the spreading of histone ubiquitination in response to IR. U2OS cells transfected with FLAG-JMJD6 or FLAG-mutant expression constructs were treated with 10 Gy of IR, and 1 h later, immunofluorescence assays were performed using anti-FLAG together with FK2 antibodies. Scale bar, 20 μm. f The spreading of histone ubiquitination in response to IR is increased upon depletion of endogenous JMJD6. U2OS cells transfected with JMJD6 siRNAs or control siRNAs were treated with 10 Gy of IR, and 1 h later, immunofluorescence assays were performed using FK2 antibodies. Scale bar, 20 μm. The knockdown effect induced by JMJD6 specific siRNAs was examined by western blot analysis using anti-JMJD6 and anti-GAPDH. g JMJD6 overexpression inhibits the spreading of RNF168 around DSBs. U2OS cells transfected with FLAG-JMJD6 or FLAG-mutant expression constructs were treated with 10 Gy of IR, and immunofluorescence assays were performed using anti-FLAG together with anti-RNF168. Scale bar, 20 μm. h JMJD6 knockdown increases the spreading of RNF168 around DSBs. Scale bar, 20 μm. The knockdown effect induced by JMJD6 specific siRNAs was examined by western blot analysis. For Fig. 1c–h, at least 50 nuclei of FLAG-JMJD6 expressing cells or control cells (cells without FLAG-JMJD6 expressing) from triplicate experiments were used to quantify the number of foci, and the p -value was determined by Student’s t- test. **** p < 0.0001, ** p < 0.01, NS = not significant

Article Snippet: The antibodies used were γH2A.X antibodies (clone JBW301, 05-636) and RNF168 antibodies (ABE367) from Merck KGaA (Darmstadt, Germany); MDC1 antibodies (NB100-397) from Novus Biologicals (Littleton, USA); FK2 antibodies (PW8810) from Enzo Life Sciences (Farmingdale, New York, USA); anti-JMJD6 (277011) from Synaptic Systems (Goettingen, Germany); anti-53BP1 (4937) from Cell Signaling Technology (Danvers, MA, USA); anti-JMJD6 (ab65770) from Abcam Inc. (Cambridge, MA, USA); anti-BRCA1 (sc-6954) and anti-JMJD6 (sc-28348) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-FLAG (M2, F3165) and anti-β-actin (A1978) from Sigma-Alderich (St Louis, MO, USA).

Techniques: Ubiquitin Proteomics, Activity Assay, Expressing, Construct, Transfection, Fluorescence, Microscopy, Irradiation, Staining, Over Expression, Immunofluorescence, Mutagenesis, Control, Knockdown, Western Blot

Journal: Cancers

Article Title: Low-Dose Pesticides Alter Primary Human Bone Marrow Mesenchymal Stem/Stromal Cells through ALDH2 Inhibition

doi: 10.3390/cancers13225699

Figure Lengend Snippet: Pesticide exposure for 21 days induces DNA damage in normal BM-MSCs. ( A ) DNA damage with double strand breaks, quantified by ℗-S139-γH2AX immunoreactivity by flow cytometry, increases in BM-MSCs after pesticide exposure for 21 days (left panel, n = 6), as shown in a representative experiment (middle panel). Comet assays reveal increased nucleus DNA migration after exposure of BM-MSCs to pesticides for 21 days, as shown in a representative experiment (right panel). ( B ) ℗-S139 γH2AX, 53BP1, MDC1 triple immunostaining in BM-MSCs indicates a higher number of double positive 53BP1/℗-S139 γH2AX signals in the nucleus of exposed cells (FOCI) and a loss of MDC1 staining. Data are expressed as percent of the control ± SEM; *, p < 0.05; **, p < 0.01; Friedman followed by post hoc Dunn’s multiple comparisons test. Scalebar: 50 µm ( A ) and 10 µm ( B ).

Article Snippet: After 21 days, cells were fixed with 4% paraformaldehyde, permeabilized (PBS/Triton X-100 0.1%, 10 min, room temperature), blocked with gelatin from cold water fish skin 5%, Triton X-100 0.1% (Sigma-Aldrich) in PBS, and were incubated overnight at 4 °C with anti-phospho-S139 γH2AX monoclonal mouse antibody (Abcam) at 1/1000, anti-MDC1 polyclonal rabbit antibody (Novus Biologicals, Centennial, CO, USA) at 1/500 and anti-53BP1 polyclonal goat antibody (R&D systems) at 1/1000, followed by a secondary anti-mouse antibody labeled with TRITC (Jackson Immunoresearch, Ely, UK), Alexa Fluor ® 647 conjugated anti-goat (Jackson Immunoresearch) and FITC-conjugated anti-rabbit (Jackson Immunoresearch) at 1/2000 each at room temperature for 2 h. Fluorescence images were acquired with a Leica TCS SP8 (Leica, Wetzlar, Germany) confocal microscope and analyzed using ImageJ software.

Techniques: Flow Cytometry, Migration, Pesticides, Triple Immunostaining, Staining, Control