Journal: Nucleic Acids Research

Article Title: RNA/DNA-binding protein TDP43 regulates DNA mismatch repair genes with implications for genome stability

doi: 10.1093/nar/gkaf920

Figure Lengend Snippet: Differential expressions of MMR genes in iPSC-derived neuronal cells and their regulation by TDP43. ( A ) WB images and histogram illustrate a quantitative comparison of MMR expression in control and siTDP43-treated human iPSC-derived neural lineage progenitor stem cells (NPSCs). Quantitation of protein levels normalized to that of GAPDH. ( B ) Schematic of iPSC, NPSC, and terminally differentiated motor neurons (iMN) utilized in this study. ( C ) IF images revealing the expression of TDP43; and MLH1, MSH2, MSH3, MSH6, and PMS2, and nuclear DNA (DAPI) after siTDP43-mediated KD of TDP43 in iPSC-derived iMNs. ( D ) WB images show the expression of select MMR factors across the following states of cellular differentiation: iPSC, NPSC, and iMN. Error bars indicate mean ± SEM from three independent experiments. Significance values (P-values) are as follows P > 0.5 (ns), P <0.5 (*), P <0.001 (***) and P <0.0001 (****).

Article Snippet: For human-induced pluripotent stem cell (iPSC) to motor neuron differentiation, control iPSC clones KYOU-DXR0109B (201B7) (ATCC) were initially plated on a 60-cm dish, then passaged to a T-25 flask with neuronal basic medium (mixture of 50% Neurobasal medium and 50% DMEM/F12 medium, with N2 and B27 supplements without vitamin A), following collagenase type IV digestion.

Techniques: Derivative Assay, Comparison, Expressing, Control, Quantitation Assay, Cell Differentiation

Journal: Nucleic Acids Research

Article Title: RNA/DNA-binding protein TDP43 regulates DNA mismatch repair genes with implications for genome stability

doi: 10.1093/nar/gkaf920

Figure Lengend Snippet: MMR expression modulates TDP43 proteinopathy-induced DNA damage. ( A ) MTT assay showing cellular viability of HEK293 cell cultures treated with 10 mM (also change in figure) 6-TG relative to nontreated controls. Experimental groups included scrambled siRNA control (siControl), siRNA KD TDP43 (siTDP43), siRNA KD of MLH1 and MSH2 (siMMR), OE of WT TDP43 (OE-TDP43), and MMR factors MLH1 and MSH2 (OE–MMR), and siTDP43 with OE–MMR. ( B ) Alkaline single-cell gel electrophoresis (comet assay) in TDP43ΔNLS (NLS) showing increased levels of DNA damage relative to controls, which is partially rescued by siRNA KD of MSH2. The histogram shows quantification of the mean olive tail moment across each condition. ( C ) IB analysis with quantitation histograms of NLS expressing cells (Dox 2.5 mg/ml for 5 days) shows increased levels of DNA damage marker \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\gamma$\end{document} H2AX that decrease following siRNA KD of MSH2. ( D ) IB analysis of MMR and DNA damage markers with indicated antibodies in two distinct ALS patient-derived iPSC-derived NPSC lines carrying TDP43 mutations at G287S and G298S, and an isogenic mutation-corrected control line (S287G). GAPDH served as the loading control for total protein and H2AII as the control for γH2AX level normalization. Quantitation of γH2AX levels. Error bars indicate mean ± SD, derived from at least three biological and technical replicates. ( E ) IB analysis of control or TDP43 A382T+C9ORF72 patient cells with or without siMMR (MLH1+MSH2). GAPDH was the loading control. And quantitation of γH2AX levels. Significance values ( P -values) are as follows: P >0.5 (ns), P <0.5 (*), P <0.001(***), and P <0.0001(****). Error bars indicate standard error mean ± SD, derived from at least three biological and technical replicates.

Article Snippet: For human-induced pluripotent stem cell (iPSC) to motor neuron differentiation, control iPSC clones KYOU-DXR0109B (201B7) (ATCC) were initially plated on a 60-cm dish, then passaged to a T-25 flask with neuronal basic medium (mixture of 50% Neurobasal medium and 50% DMEM/F12 medium, with N2 and B27 supplements without vitamin A), following collagenase type IV digestion.

Techniques: Expressing, MTT Assay, Control, Alkaline Single Cell Gel Electrophoresis, Single Cell Gel Electrophoresis, Quantitation Assay, Marker, Derivative Assay, Mutagenesis