Journal: Cell Death & Disease

Article Title: Human frataxin, the Friedreich ataxia deficient protein, interacts with mitochondrial respiratory chain

doi: 10.1038/s41419-023-06320-y

Figure Lengend Snippet: List of antibodies used in this work.

Article Snippet: , Anti – FXN , Proteintech; 14147-1-AP , 1:500 in TBST 1 × 5% milk – O/N, 4 °C , .

Techniques: Milk, FLAG-tag

Journal: bioRxiv

Article Title: mtDNA breaks compromise mitochondrial membrane ultrastructure and trigger an integrated stress response

doi: 10.1101/2022.10.01.510431

Figure Lengend Snippet: a , Western blot analysis of TFAM in MTA2 GFP11 cells expressing ApaLI, ApaLI*, and no ApaLI, and treated with Dox and/or ddC. b , Percentage of GFP+ cells determined by flow cytometry in cells as per Extended Data . Data are means ± s.d. of n = 2 biological replicates; two-way ANOVA. c , Subcellular fractionation followed by Western blot analysis of indicated proteins on MTA2 GFP11 cells with indicated treatments, including cell sorting based on GFP. p, precursor isoform; m, mature isoform; Long exp., long exposure of Western blots. d , Western blot analysis of three isoforms of frataxin (FXN) transfected to GFP- and GFP+ populations of MTA2 GFP11 cells expressing ApaLI and treated with Dox for 2 days. Mitochondrial uncoupler CCCP (Carbonyl cyanide m -chlorophenyl hydrazone) was used at 20 μM for 6 hours. i, intermediate isoform. e , Western blot quantification of frataxin isoforms in Extended Data , normalized to the total level of all isoforms. f , Representative image of TMRM (Tetramethylrhodamine, methyl ester) staining in RRBP1 GFP11 cells expressing mito-GFP1-10 and ApaLI and treated with Dox for 2 days. Yellow and white boxes depict examples of GFP+ and GFP-cells, respectively. g-h , Flow cytometry analysis of TMRM staining in RRBP1 GFP11 cells expressing mito-GFP1-10 and ApaLI (or ApaLI*). Analysis was carried out 2 days post-Dox treatment. g , Representative histograms generated from FlowJo software showing TMRM intensity on the X-axis, unit area on the Y-axis; the frequencies of GFP+ cells are labeled. h , Percentage of cells with low TMRM signal (TMRM low cells) gated based on untreated ApaLI* cells (left). Data are means ± s.d. of n = 4 biological replicates; two-way ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. i , Western blot analysis of nucleus-encoded OXPHOS proteins in the mitochondrial fraction of GFP- and GFP+ populations from MTA2 GFP11 cells expressing ApaLI and treated with Dox for 2 days. j , Western blot analysis of LC3B-I/II in sorted GFP+ population of Dox-treated MTA2 GFP11 cells expressing ApaLI.

Article Snippet: Frataxin plasmid (pCMV6-FXN-Myc-FLAG) was purchased from Origene (RC204880).

Techniques: Western Blot, Expressing, Flow Cytometry, Fractionation, FACS, Transfection, Staining, Generated, Software, Labeling

Journal: Disease Models & Mechanisms

Article Title: A novel GAA-repeat-expansion-based mouse model of Friedreich’s ataxia

doi: 10.1242/dmm.018952

Figure Lengend Snippet: Transgene copy number. (A) Two TaqMan copy-number assays were applied: Hs05092416-cn assay (ATX 416; represented in black) was designed to amplify a 106-bp fragment of FXN within intron 3, and Hs02407730-cn assay (ATX 730; represented in grey) was designed to amplify an 80-bp fragment of FXN within intron 1 and exon 2. Human mammary epithelial cell (HMEC) with copy numbers of two served as a calibrator. Error bars=s.d. n =2. (B) Determination of the integration site of the transgenic FXN gene by FISH. Biotin-labelled RP11-265B8 and digoxigenin-labelled RP11-876N18 probes were hybridised onto interphase and metaphase chromosomes (DAPI stained) of YG8sR cells. YG8sR showed one hybridisation signal, indicating a single integration site of the FXN transgene containing one copy of the FXN gene. Scale bars: 10 μm.

Article Snippet: In brief, 20 ng of genomic DNA was combined with 2× TaqMan universal master mix, TaqMan copy number assay for human FXN (Hs05092416_cn or Hs02407730_cn), and TaqMan copy number reference assay for mouse Tert in a 20 μl reaction volume.

Techniques: Transgenic Assay, Staining, Hybridization

Journal: Disease Models & Mechanisms

Article Title: A novel GAA-repeat-expansion-based mouse model of Friedreich’s ataxia

doi: 10.1242/dmm.018952

Figure Lengend Snippet: Frataxin expression levels. (A–C) qRT-PCR analysis of transgenic FXN mRNA using mouse-human specific primers. (A) Analysis of males and females together. Analysis of (B) males and (C) females separately. Data were normalised to the mean FXN mRNA level of Y47R brain samples taken as 100%. Statistical differences between the YG8sR mutant and B6 controls are indicated by the top bar, whereas the bottom bar indicates the differences between the YG8sR mutant and Y47R controls. (D–F) Dipstick immunoassay of human frataxin protein. (D) Analysis of males and females together, or (E) males and (F) females separately. Data were normalised to the mean frataxin level of Y47R samples taken as 100%. Values represent mean±s.e.m. * P <0.05, ** P <0.01 and *** P <0.001. n =4–8.

Article Snippet: In brief, 20 ng of genomic DNA was combined with 2× TaqMan universal master mix, TaqMan copy number assay for human FXN (Hs05092416_cn or Hs02407730_cn), and TaqMan copy number reference assay for mouse Tert in a 20 μl reaction volume.

Techniques: Expressing, Quantitative RT-PCR, Transgenic Assay, Mutagenesis

Journal: Disease Models & Mechanisms

Article Title: A novel GAA-repeat-expansion-based mouse model of Friedreich’s ataxia

doi: 10.1242/dmm.018952

Figure Lengend Snippet: Transgene copy number. (A) Two TaqMan copy-number assays were applied: Hs05092416-cn assay (ATX 416; represented in black) was designed to amplify a 106-bp fragment of FXN within intron 3, and Hs02407730-cn assay (ATX 730; represented in grey) was designed to amplify an 80-bp fragment of FXN within intron 1 and exon 2. Human mammary epithelial cell (HMEC) with copy numbers of two served as a calibrator. Error bars=s.d. n =2. (B) Determination of the integration site of the transgenic FXN gene by FISH. Biotin-labelled RP11-265B8 and digoxigenin-labelled RP11-876N18 probes were hybridised onto interphase and metaphase chromosomes (DAPI stained) of YG8sR cells. YG8sR showed one hybridisation signal, indicating a single integration site of the FXN transgene containing one copy of the FXN gene. Scale bars: 10 μm.

Article Snippet: In brief, 20 ng of genomic DNA was combined with 2× TaqMan universal master mix, TaqMan copy number assay for human FXN (Hs05092416_cn or Hs02407730_cn), and TaqMan copy number reference assay for mouse Tert in a 20 μl reaction volume.

Techniques: Transgenic Assay, Staining, Hybridization

Journal: iScience

Article Title: Butyrate prevents visceral adipose tissue inflammation and metabolic alterations in a Friedreich’s ataxia mouse model

doi: 10.1016/j.isci.2023.107713

Figure Lengend Snippet:

Article Snippet: FXN shRNA scramble shRNA , OriGene Technologies , Cat# TL514670V.

Techniques: Recombinant, Red Blood Cell Lysis, SYBR Green Assay, Reverse Transcription, Transfection, XF Assay, DNA Extraction, Western Blot, Knock-In, Knock-Out, shRNA, Software, Sequencing, Expressing, Microscopy, Mass Spectrometry