Journal: PLoS Genetics

Article Title: TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage

doi: 10.1371/journal.pgen.1009260

Figure Lengend Snippet: A) Screnshoots of genome windows showing co-occurrence between TDP43 (red), DRIPc-seq (green), untreated (dark blue) and RNH-treated (light blue) DRIP-seq and RNA-seq (yellow) data. Genome location is indicated in the top left corner and scale in the top right. Coverage scale indicated in the top left of each track. Biological replicates are also indicated (R1, R2). B) Venn diagram showing correlation between genes bind by TDP-43 (TDP43; red circle), genes forming R loops (DRIPc-seq; green) and expressed genes (RNA-seq; yellow). Numbers refer to genes co-occurring between conditions. C) TDP-43 ChIP-seq average coverage (log10) across silent (-) and expressed (+) genes. ****, P < 0,0001 (Mann-Whitney U test, two-tailed). D) Metagene analysis showing TDP-43 ChIP-seq coverage (red line) along gene body (+/- 2kb) of silent (-) and expressed (+) genes. Mean coverage is plotted in the upper panel and heatmap intensities for the entire gene population is shown below. Scale is also indicated.

Article Snippet: SH-SY5Y cells were stably transfected using Fugene HD Transfection Reagent (Promega) with C-terminally GFP-tagged TDP-43 WT vector (PS100010 pCMV6-AC-GFP), C-terminally GFP-tagged TDP-43 A382T mutation vector (CW303334 mutated ORF of RC210639 at nt position 1144, changed from G to A, to obtain A382T, inserted in PS100001) and a C-terminally GFP-tagged TDP-43 G294V mutation vector (CW303287 mutated ORF of RG210639 at nt position 881, changed from G to T, to obtain G294V, inserted in PS100010), purchased from OriGene.

Techniques: RNA Sequencing Assay, ChIP-sequencing, MANN-WHITNEY, Two Tailed Test

Journal: PLoS Genetics

Article Title: TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage

doi: 10.1371/journal.pgen.1009260

Figure Lengend Snippet: A) IF of LCL-CTL, LCL-TDP382, LCL-SALS using an anti-TDP-43 antibody and an anti-S9.6 antibody after methanol fixation. The scatter plots show the increase of S9.6 signal intensity and the decrease in TDP-43 nuclear content in SH-TDP382. Median values are indicated. Scale bar: 25μm. *, P< 0,05 (Mann-Whitney U test, two-tailed). When no asterisk is shown indicates that is not significant. B) Flow cytometry plot reports the amount of R-loops in LCLs (blue) in comparison to LCL-TDP382 (orange) and LCL-SALS (blue). The RNaseH action on LCL-TDP382 decreases the presence of R-loops signal (orange peak vs green peak). Histogram shows S9.6 mean fluorescence of LCL-CTL, LCL-TDP382, LCL-SALS in presence (+) and in absence (-) of RNaseH. The value represented is the mean ± SEM of three biological experiments. ANOVA, Newman-Keuls Multiple Comparison Test, *P <0,05. When no asterisk is shown indicates that is not significant. C ) Model showing the link between TDP-43 and R loop metabolism. Functional TDP43 activity in the nucleus protects genome integrity by preventing R-loop accumulation. However, TDP-43 nuclear dysfunctions results in R loop-dependent DNA damage that eventually might lead to genome instability that could aggravate ALS phenotype.

Article Snippet: SH-SY5Y cells were stably transfected using Fugene HD Transfection Reagent (Promega) with C-terminally GFP-tagged TDP-43 WT vector (PS100010 pCMV6-AC-GFP), C-terminally GFP-tagged TDP-43 A382T mutation vector (CW303334 mutated ORF of RC210639 at nt position 1144, changed from G to A, to obtain A382T, inserted in PS100001) and a C-terminally GFP-tagged TDP-43 G294V mutation vector (CW303287 mutated ORF of RG210639 at nt position 881, changed from G to T, to obtain G294V, inserted in PS100010), purchased from OriGene.

Techniques: MANN-WHITNEY, Two Tailed Test, Flow Cytometry, Fluorescence, Functional Assay, Activity Assay

Journal: BMC Bioinformatics

Article Title: SimplySmart_v1, a new tool for the analysis of DNA damage optimized in primary neuronal cultures

doi: 10.1186/s12859-024-05947-8

Figure Lengend Snippet: Compared with WT TDP-43, TDP-43 mutant A315T induced more DNA damage, as calculated with a one-sided t test. A Representative images of mouse cortical neurons stained with TDP-43 and DAPI. The selection of cells treated with puromycin resulted in many apoptotic nuclei; however, staining for TDP-43 revealed surviving transduced neurons. B The upper panel shows DNA damage areas identified using SimplySmart_v1 in neuronal nuclei expressing A315T (color-mapped regions). The graphs show that the average number of DNA damage foci in the WT TDP-43 group was significantly lower than that in the A315T group (* p < 0.05, one-sided t test), whereas the foci area was unchanged. The images were analysed with SimplySmart_v1 with a threshold value = 140, a marker area range of 1000–5000 pixels, and a minimal size > 2 pixels. Forty-nine nuclei from the WT group and 38 from the A315T group were analysed from 3 technical replicates

Article Snippet: Primary mouse cortical neurons were transduced with lentiviruses carrying wild-type TDP-43 or TDP-43 mutant A315T. pLVX-Puro-TDP-43-A315T was a gift from Shawn Ferguson (Addgene plasmid # 133755; https://www.addgene.org/133755/ ; RRID: Addgene_133755).

Techniques: Mutagenesis, Staining, Selection, Expressing, Marker

Journal: Bio-protocol

Article Title: High-throughput Flow Cytometry Assay to Investigate TDP43 Splicing Function

doi: 10.21769/BioProtoc.3594

Figure Lengend Snippet: A. The fluorescent protein (FP)-based splicing module and BFP-tagged TDP43 variants are expressed from the same plasmid via a bi-directional promoter (pBI). B. Production of a dually-fluorescent GFP-mCherry fusion protein depends on the presence of functional TDP43, which is required to mediate the skipping of CFTR gene-derived exon (labeled E2).

Article Snippet: Cell lines HEK-293T wild-type cells (ATCC, catalog number: CRL-3216) HEK-293T TARDBP knock-out cells (available upon request) Cell culture 1.5 ml microtubes (Eppendorf, catalog number: 022363204) 60 μm scepter tips for cell counting (Milipore-Sigma, catalog number: PHCC60050) 10 cm tissue culture plates for propagating cell lines (Thermo Scientific, catalog number: 12-556-002) 6-well tissue culture plates for splicing assays (Thermo Scientific, catalog number: 14-832-11) 70 μm cell strainer (Corning, Falcon, catalog number: 08-771-2) 5 ml round-bottom polystyrene tubes (Corning, Falcon, catalog number: 14-959-1A) SH800S flow cytometer setup beads (Sony Biotechnology, catalog number: LE-B3001) HyClone DMEM high glucose (GE Healthcare, catalog number: SH30081FS) supplemented with: 10% FBS (Atlanta Biologicals, catalog number: S11150H) 1 mM sodium pyruvate (Gibco, catalog number: 11-360-070) 2 mM L-glutamine (Gemini Biosciences, catalog number: 400106) 1x MEM non-essential amino acids (Gibco, catalog number: 11-140-076) 40 U/ml penicillin and 40 μg/ml streptomycin (Gemini Biosciences, catalog number: 400109) Sterile 1x PBS at pH 7.4 (Sigma-Aldrich, catalog number: P3813-5x10PAK) Trypsin-EDTA (Gemini Biosciences, catalog number: 400150) X-tremeGENE9 transfection reagent (Roche, catalog number: 06365787001) OptiMEM (Gibco, catalog number: 31985-070) Plasmids (available at https://www.addgene.org/Rajat_Rohatgi/ , unless otherwise noted) Splicing reporter only: pHBS1389 IBB-GFP-mCherry3E (Addgene, catalog number: 118803) Positive control: pHBS1503 [IBB-GFP-mCherry3E]-[BFP-TDP43 WT] (Addgene, catalog number: 133327) Negative control: pHBS1501 [IBB-GFP-mCherry3E]-[BFP-TDP43 RRM] (Addgene, catalog number: 133325) Extensive collection of TDP43 CTD mutants in the splicing reporter ( e.g. , Addgene, catalog numbers: 133328-133333) Backbone for subcloning of CTD mutants into splicing reporter: pHBS1224 [IBB-GFP-mCherry3E]-[BFP-TDP43 CTD EcoRV] (available upon request) Compensation probes: mammalian expression vectors with BFP, GFP and mCherry inserts ( e.g. , Addgene, catalog numbers: 54665, 54759 and 54563)

Techniques: Plasmid Preparation, Functional Assay, Derivative Assay, Labeling

Journal: Bio-protocol

Article Title: High-throughput Flow Cytometry Assay to Investigate TDP43 Splicing Function

doi: 10.21769/BioProtoc.3594

Figure Lengend Snippet: A. BSC-A vs. FSC-A density plot showing all detected events and Gate 1 that selects for cells. B. FSC-H vs. FSC-A density plot and Gate 2 to select for single cells. C. FL1-A (BFP) histogram and Gate 3 to select for transfected cells. D. FL3-A (mCherry) vs. FL2-A (GFP) density plots for full-length, wild-type (WT) TDP43 and TDP43 lacking its RNA-binding domains (RRM).

Article Snippet: Cell lines HEK-293T wild-type cells (ATCC, catalog number: CRL-3216) HEK-293T TARDBP knock-out cells (available upon request) Cell culture 1.5 ml microtubes (Eppendorf, catalog number: 022363204) 60 μm scepter tips for cell counting (Milipore-Sigma, catalog number: PHCC60050) 10 cm tissue culture plates for propagating cell lines (Thermo Scientific, catalog number: 12-556-002) 6-well tissue culture plates for splicing assays (Thermo Scientific, catalog number: 14-832-11) 70 μm cell strainer (Corning, Falcon, catalog number: 08-771-2) 5 ml round-bottom polystyrene tubes (Corning, Falcon, catalog number: 14-959-1A) SH800S flow cytometer setup beads (Sony Biotechnology, catalog number: LE-B3001) HyClone DMEM high glucose (GE Healthcare, catalog number: SH30081FS) supplemented with: 10% FBS (Atlanta Biologicals, catalog number: S11150H) 1 mM sodium pyruvate (Gibco, catalog number: 11-360-070) 2 mM L-glutamine (Gemini Biosciences, catalog number: 400106) 1x MEM non-essential amino acids (Gibco, catalog number: 11-140-076) 40 U/ml penicillin and 40 μg/ml streptomycin (Gemini Biosciences, catalog number: 400109) Sterile 1x PBS at pH 7.4 (Sigma-Aldrich, catalog number: P3813-5x10PAK) Trypsin-EDTA (Gemini Biosciences, catalog number: 400150) X-tremeGENE9 transfection reagent (Roche, catalog number: 06365787001) OptiMEM (Gibco, catalog number: 31985-070) Plasmids (available at https://www.addgene.org/Rajat_Rohatgi/ , unless otherwise noted) Splicing reporter only: pHBS1389 IBB-GFP-mCherry3E (Addgene, catalog number: 118803) Positive control: pHBS1503 [IBB-GFP-mCherry3E]-[BFP-TDP43 WT] (Addgene, catalog number: 133327) Negative control: pHBS1501 [IBB-GFP-mCherry3E]-[BFP-TDP43 RRM] (Addgene, catalog number: 133325) Extensive collection of TDP43 CTD mutants in the splicing reporter ( e.g. , Addgene, catalog numbers: 133328-133333) Backbone for subcloning of CTD mutants into splicing reporter: pHBS1224 [IBB-GFP-mCherry3E]-[BFP-TDP43 CTD EcoRV] (available upon request) Compensation probes: mammalian expression vectors with BFP, GFP and mCherry inserts ( e.g. , Addgene, catalog numbers: 54665, 54759 and 54563)

Techniques: Transfection, RNA Binding Assay

Journal: Bio-protocol

Article Title: High-throughput Flow Cytometry Assay to Investigate TDP43 Splicing Function

doi: 10.21769/BioProtoc.3594

Figure Lengend Snippet: A. Outline of the analysis pipeline. A flow cytometer is used to measure and deconvolute the mCherry, GFP and BFP fluorescence signals of individual cells in a population. Using custom scripts, the splicing efficiency is calculated by taking the ratio of the mCherry to GFP signal of a given cell (Figure 1) and compared to the TDP43 level of the same cell (BFP signal). B. Screenshot highlighting where to change the gate boundaries and cut-offs in the ‘CSA_Gating.nb’ script. C. Screenshot indicating where to provide the file path to the fcs raw data in the ‘CSA_Gating.nb’ script. D. Example density plots generated during evaluation of the ‘CSA_Gating.nb’ script to visualize the gates defined for analysis. E. Screenshot illustrating where in the ‘CSA_Plotting.nb’ script to provide the file path to the csv files generated during evaluation of the ‘CSA_Gating.nb’ script. F. Screenshot highlighting where to adjust the plot scales in the ‘CSA_Plotting.nb’ script. G. Example density plot generated by the ‘CSA_Plotting.nb’ script that depicts splicing efficiency vs. TDP43 levels for wild-type (WT) and RRM TDP43 variants (top) and example violin plot highlighting the splicing efficiencies in the defined BFP signal range (bottom). H. Screenshot of the ‘CSA_Plotting.nb’ script indicating where to define the BFP signal range for the violin plots comparing splicing efficiency.

Article Snippet: Cell lines HEK-293T wild-type cells (ATCC, catalog number: CRL-3216) HEK-293T TARDBP knock-out cells (available upon request) Cell culture 1.5 ml microtubes (Eppendorf, catalog number: 022363204) 60 μm scepter tips for cell counting (Milipore-Sigma, catalog number: PHCC60050) 10 cm tissue culture plates for propagating cell lines (Thermo Scientific, catalog number: 12-556-002) 6-well tissue culture plates for splicing assays (Thermo Scientific, catalog number: 14-832-11) 70 μm cell strainer (Corning, Falcon, catalog number: 08-771-2) 5 ml round-bottom polystyrene tubes (Corning, Falcon, catalog number: 14-959-1A) SH800S flow cytometer setup beads (Sony Biotechnology, catalog number: LE-B3001) HyClone DMEM high glucose (GE Healthcare, catalog number: SH30081FS) supplemented with: 10% FBS (Atlanta Biologicals, catalog number: S11150H) 1 mM sodium pyruvate (Gibco, catalog number: 11-360-070) 2 mM L-glutamine (Gemini Biosciences, catalog number: 400106) 1x MEM non-essential amino acids (Gibco, catalog number: 11-140-076) 40 U/ml penicillin and 40 μg/ml streptomycin (Gemini Biosciences, catalog number: 400109) Sterile 1x PBS at pH 7.4 (Sigma-Aldrich, catalog number: P3813-5x10PAK) Trypsin-EDTA (Gemini Biosciences, catalog number: 400150) X-tremeGENE9 transfection reagent (Roche, catalog number: 06365787001) OptiMEM (Gibco, catalog number: 31985-070) Plasmids (available at https://www.addgene.org/Rajat_Rohatgi/ , unless otherwise noted) Splicing reporter only: pHBS1389 IBB-GFP-mCherry3E (Addgene, catalog number: 118803) Positive control: pHBS1503 [IBB-GFP-mCherry3E]-[BFP-TDP43 WT] (Addgene, catalog number: 133327) Negative control: pHBS1501 [IBB-GFP-mCherry3E]-[BFP-TDP43 RRM] (Addgene, catalog number: 133325) Extensive collection of TDP43 CTD mutants in the splicing reporter ( e.g. , Addgene, catalog numbers: 133328-133333) Backbone for subcloning of CTD mutants into splicing reporter: pHBS1224 [IBB-GFP-mCherry3E]-[BFP-TDP43 CTD EcoRV] (available upon request) Compensation probes: mammalian expression vectors with BFP, GFP and mCherry inserts ( e.g. , Addgene, catalog numbers: 54665, 54759 and 54563)

Techniques: Flow Cytometry, Fluorescence, Generated