Journal: Human Molecular Genetics

Article Title: Amyotrophic lateral sclerosis-associated TDP-43 mutation Q331K prevents nuclear translocation of XRCC4-DNA ligase 4 complex and is linked to genome damage-mediated neuronal apoptosis

doi: 10.1093/hmg/ddz062

Figure Lengend Snippet: Targeted sequencing of exon-6 of the TARDBP gene in spinal cord specimens from ALS patients. (A) Chromatogram showing detection of the Q331K mutation in genomic DNA isolated from spinal cord tissue from an ALS patient. (B) Immunoblots probed with TDP-43 antibody and γH2AX antibody show reduced monomeric TDP-43 protein and increased γH2AX in an ALS specimen carrying the Q331K mutation compared to an age-matched control sample. GAPDH served as the loading control. (C) Analysis of DNA integrity by LA-PCR. Representative agarose gel images indicate reduced DNA amplification of OCT3/4, NONOG, Polβ and HPRT gene segments in TDP-43-Q331K spinal cord compared to control. The 200 bp short amplicon served as control.( D) Quantitation of PCR products by Pico Green-based DNA quantitation from triplicate experiments. (E) IHC of spinal cord from an ALS patient carrying a Q331K mutation for TDP-43, γH2AX and TUNEL staining. Representative images acquired at 20× magnification. (F) Quantitation of mean γH2AX signal and mean TUNEL-positive cells per field in ALS spinal cord sample and age-matched controls. **P<0.05; ****P<0.0001.

Article Snippet: The TDP-43 coding DNA sequence (CDS) along with the N-terminal 1X FLAG tag sequence was PCR-amplified from FLAG-TDP-43 pcDNA 3.1 vector using high-fidelity Deep Vent DNA polymerase (# M0258, NEB, Ipswich, MA).

Techniques: Sequencing, Mutagenesis, Isolation, Western Blot, Agarose Gel Electrophoresis, Amplification, Quantitation Assay, TUNEL Assay, Staining

Journal: Human Molecular Genetics

Article Title: Amyotrophic lateral sclerosis-associated TDP-43 mutation Q331K prevents nuclear translocation of XRCC4-DNA ligase 4 complex and is linked to genome damage-mediated neuronal apoptosis

doi: 10.1093/hmg/ddz062

Figure Lengend Snippet: Q331K expression induces ROS stress and accumulation DNA strand breaks in neurons. (A) Cellular Reactive Oxygen Species Detection Assay by IF microscopy. Upper panels represent WT-expressing cells; lower panels represent Q331K-expressing cells. The two panels on the left represent uninduced cells. Panels to the right represent cells after Dox induction. Deep RED dye staining indicates the presence of ROS (Scale bar, 10 μm). (B) Quantitation of cellular ROS using a microplate fluorescence reader. (C) LA-PCR analysis of genomic DNA isolated from TDP-43-Q331K neurons shows reduced DNA integrity. Representative agarose gel images of amplified DNA products. (D) Quantification of PCR products by Pico Green-based DNA quantitation from triplicate experiments. (E) Alkaline comet analysis of differentiated SH-SY5Y cells expressing WT or Q331K (lower panel). Quantitation of mean tail moment before and after Dox induction in 25–50 cells reveals an ~5-fold increase in DNA damage in Q331K cells (Scale bar, 10 μm). *P<0.01; **P<0.05; ***P<0.0005.

Article Snippet: The TDP-43 coding DNA sequence (CDS) along with the N-terminal 1X FLAG tag sequence was PCR-amplified from FLAG-TDP-43 pcDNA 3.1 vector using high-fidelity Deep Vent DNA polymerase (# M0258, NEB, Ipswich, MA).

Techniques: Expressing, Detection Assay, Microscopy, Staining, Quantitation Assay, Fluorescence, Isolation, Agarose Gel Electrophoresis, Amplification

Journal: Human Molecular Genetics

Article Title: Amyotrophic lateral sclerosis-associated TDP-43 mutation Q331K prevents nuclear translocation of XRCC4-DNA ligase 4 complex and is linked to genome damage-mediated neuronal apoptosis

doi: 10.1093/hmg/ddz062

Figure Lengend Snippet: The Q331K mutation affects the nuclear translocation of XRCC4-DNA ligase 4. (A) IF of DNA ligase 4 in WT or Q331K cells shows increased cytoplasmic presence in mutant cells (Scale bar, 10 μm). The 2.5-dimensional view of the localization is shown in the image below. (B) IF of XRCC4 in WT or Q331K cells shows their reduced nuclear presence in mutant cells (Scale bar, 10 μm). The 2.5-dimensional view of the localization is shown in the image below. (C) PLA of FLAG versus DNA ligase 4 and FLAG versus XRCC4 in cells expressing WT and mutant TDP-43 (Scale bar, 10 μm). The higher number of foci in the cytoplasm of Q331K-expressing cells compared to WT-expressing cells indicates the increased interaction of XRCC4-DNA ligase 4 after DNA-damage induction by IR (3 Gy). **P<0.05 ; ****P<0.0001.

Article Snippet: The TDP-43 coding DNA sequence (CDS) along with the N-terminal 1X FLAG tag sequence was PCR-amplified from FLAG-TDP-43 pcDNA 3.1 vector using high-fidelity Deep Vent DNA polymerase (# M0258, NEB, Ipswich, MA).

Techniques: Mutagenesis, Translocation Assay, Expressing

Journal: Human Molecular Genetics

Article Title: Amyotrophic lateral sclerosis-associated TDP-43 mutation Q331K prevents nuclear translocation of XRCC4-DNA ligase 4 complex and is linked to genome damage-mediated neuronal apoptosis

doi: 10.1093/hmg/ddz062

Figure Lengend Snippet: The Q331K mutation affects DNA-ligation activity. (A) In vitro DNA-ligation assay using extracts of neurons expressing WT or mutant TDP-43 before and after DNA damage induction by IR (3 Gy). Quantitation of DNA-ligation activity from three independent experiments expressed as fold change in the histogram. (B) IP of WT- and Q331K-expressing cells with anti-FLAG antibody or IgG (mouse) before and after DNA-damage induction by IR (3 Gy) shows reduce DNA ligase 4 interaction in Q331K mutant cells. (C) His-affinity pulldown assay using recombinant TDP-43-WT and TDP-43-Q331K shows reduced association of the Q331K protein with XRCC4-DNA ligase 4 complex in vitro. (D) A model showing TDP-43 WT facilitates XRCC4-Ligase4 complex nuclear translocation in healthy neuronal cells leading to efficient DNA repair. ALS-linked Q331K mutation nuclear clearance and cytoplasmic aggregation impairs XRCC4-Ligase4 complex nuclear translocation leading to persistent DNA damage accumulation. **P<0.05.

Article Snippet: The TDP-43 coding DNA sequence (CDS) along with the N-terminal 1X FLAG tag sequence was PCR-amplified from FLAG-TDP-43 pcDNA 3.1 vector using high-fidelity Deep Vent DNA polymerase (# M0258, NEB, Ipswich, MA).

Techniques: Mutagenesis, DNA Ligation, Activity Assay, In Vitro, Expressing, Quantitation Assay, Recombinant, Translocation Assay

Journal: Epigenetics

Article Title: SFRP1 CpG island methylation locus is associated with renal cell cancer susceptibility and disease recurrence.

doi: 10.4161/epi.19614

Figure Lengend Snippet: Figure 1. Quantitative SFRP1 CGI methylation and gene expression analysis in renal cell lines and normal primary cells. (A) Illustration of SFRP1 CGI structure and relative positions of methylation analysis. (B) Example for quantitative methylation analysis of 11 CpG sites (gray bars) and one control site (narrow gray bar) in the CGI region of the SFRP1 gene using pyrosequencing of bisulfite treated DNA. Here, an average methylation of 35% was determined while a nearly complete bisulfite conversion of 99.6% has been achieved. (C) Quantitative SFRP1 CGI methylation analysis and relative quantitation of SFRP1 mRNA expression in renal cell cancer cell lines and primary cells from normal kidney (RPTEC). (D) Duplicate measurements and Lin’s concordance correlation analysis. The solid line represents the regression line of measurements while the dashed line indicates the line of perfect concordance. Only minute deviations from ideal concordance were observed.

Article Snippet: Colella S, Shen L, Baggerly KA, Issa JP, Krahe R. Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites.

Techniques: Methylation, Gene Expression, Control, Quantitation Assay, Expressing