Journal: International Journal of Molecular Sciences

Article Title: Antisense Dipeptide Repeat Proteins Drive Widescale Purine Metabolism Aberration in C9orf72 Amyotrophic Lateral Sclerosis via ADA

doi: 10.3390/ijms27041953

Figure Lengend Snippet: Gain-of-function mechanisms are responsible for purine metabolism dysfunction in C9orf72 ALS. ( A ) Representative images and ( B ) densitometry analysis of C9orf72 KO HeLa adenosine deaminase (ADA) Western blot. ( C ) Representative images and ( D ) densitometry analysis of 38-repeat sense and 39-repeat antisense expressing Neuro2a (N2a) ADA Western blot. ( E ) Representative images and ( F ) densitometry analysis of 36-repeat glycine-alanine (poly-GA), glycine-arginine (poly-GR) and proline-arginine (poly-PR) expressing HeLa ADA Western blot as fold change to JetPRIME mock transfection. ( G ) ADA activity and ( H ) inosine output in 38-repeat sense and 39-repeat antisense expressing N2a. ADA relative mRNA expression in ( I ) untreated, empty vector (EV) or poly-PR expressing HEK293T and ( J ) 1000-repeat polyGR and poly-PR pan-neuronally expressing Drosophila . Data presented as mean and standard deviation of three to five biological replicates. Statistical analysis by unpaired t -test ( B ), Brown–Forsythe and Welch ANOVA test ( D , I , J ) RM one-way ANOVA ( G ) or ordinary one-way ANOVA ( H ). * p ≤ 0.05, ** p ≤ 0.01. Where p value is not indicated results were non-significant. Uncropped Western images can be found in .

Article Snippet: Transfections were performed using either jetPRIME transfection kit (Polyplus, Strasbourg, France) per the manufacturer’s instructions, Xfect transfection reagent (Takara Bio, Kusatsu, Japan) per the manufacturer’s instructions, polyethyleneimine (PEI) (2.5 μL/μg plasmid) with Opti-MEM (50 μL/0.7 μg plasmid) or 0.5 M calcium chloride with HBS buffer (280 mM NaCl, 100mM HEPES, 1.5mM Na 2 HPO 4 , pH 7.1).

Techniques: Western Blot, Expressing, Transfection, Activity Assay, Plasmid Preparation, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Antisense Dipeptide Repeat Proteins Drive Widescale Purine Metabolism Aberration in C9orf72 Amyotrophic Lateral Sclerosis via ADA

doi: 10.3390/ijms27041953

Figure Lengend Snippet: Inhibiting antisense DPR translation via SRSF1 knockdown and RuvBL2 overexpression restores ADA expression. ( A ) Adenosine deaminase (ADA) Western blot representative images and ( B ) densitometry analysis of HEK293T lines with a PEI mock transfection or co-expressing control shRNA and either sense or antisense HREs analysed as fold change to mock transfection. ( C ) V5 Western blot representative images and ( D ) densitometry analysis of HEK293T lines with a PEI mock transfection or co-expressing control or serine/arginine-rich splicing factor 1 (SRSF1) shRNA and either sense or antisense HREs compared as fold change to mock transfection. ( E ) ADA Western blot representative images and ( F ) densitometry analysis of HEK293T lines with a PEI mock transfection or co-expressing control or SRSF1 shRNA and either 45-repeat sense or 43-repeat antisense hexanucleotide repeat expansions (HREs) analysed as fold change to mock transfection. ( G ) ADA Western blot representative images and ( H ) densitometry analysis of HeLa expressing empty vector (EV), EV and 45-repeat sense HRE or 45-repeat sense HRE and RuvB-like 2 (RuvBL2) analysed as fold change to EV average. ( I ) Adenosine level, ( J ) deoxyadenosine level, ( K ) inosine level, ( L ) deoxyinosine level and ( M ) dATP level in HEK293T cells that had undergone a mock transfection or co-transfection with 45-repeat sense HRE and either control shRNA or SRSF1 shRNA. Data presented as mean and standard deviation of three biological replicates normalised to a PEI mock transfection. Analysis performed by a Friedman test ( B , D , F , I – L , M ) or ordinary one-way ANOVA ( H ). * p ≤ 0.05, ** p ≤ 0.01. Where p value is not indicated results were non-significant. Uncropped Western images can be found in .

Article Snippet: Transfections were performed using either jetPRIME transfection kit (Polyplus, Strasbourg, France) per the manufacturer’s instructions, Xfect transfection reagent (Takara Bio, Kusatsu, Japan) per the manufacturer’s instructions, polyethyleneimine (PEI) (2.5 μL/μg plasmid) with Opti-MEM (50 μL/0.7 μg plasmid) or 0.5 M calcium chloride with HBS buffer (280 mM NaCl, 100mM HEPES, 1.5mM Na 2 HPO 4 , pH 7.1).

Techniques: Knockdown, Over Expression, Expressing, Western Blot, Transfection, Control, shRNA, Plasmid Preparation, Cotransfection, Standard Deviation

Journal: bioRxiv

Article Title: S-SELeCT: A Human-Evolved Serine Integrase System for Efficient Large-Cargo Genome Integration

doi: 10.64898/2026.01.30.702954

Figure Lengend Snippet: Localization optimization ( A ) Split-GFP site A integration efficiency assay. Wildtype C31 attP was placed at both site A loci in HEK293 cells. Downstream of each attP, a splice acceptor, 3’ segment of GFP, and transcription-termination sequence were also introduced. To enable detection of site-specific integration, a donor plasmid was constructed that contains the elements needed to form a complete GFP-expression cassette: CMV promoter, 5’ GFP segment, splice donor, wildtype attB site. After co-transfection of the donor and integrase-expression plasmids, cells where site-specific integration has occurred can be identified by looking for green fluorescence. Integration can happen at one (panels i and ii) or both loci (panel iii). ( B ) WT C31-int dMad7 fusion protein expression cassette used to test impact of different gRNA on site A localization efficiency. A 5’ mCherry segment fused to a trans-splicing intein domain was co-expressed and separated from int-dMad7 via a 2A-skipping peptide. In the donor plasmid, the remaining 3’ mCherry segment fused to the complementary trans-splicing intein domain was co-expressed with the 5’ GFP segment mentioned in (A), and these two proteins were separated via 2A peptide ribosome skipping. ( C ) Results of site A localization experiments for the indicated guide RNAs and combinations, in transfected cells that express the fusion protein most strongly. Assay was performed for 72 hours in HEK293 cells that stably express the fusion protein from the H11 locus. ( D ) Expression (mCherry) and GFP gating. The rightmost mCherry gate was used to analyze the cells summarized in (C).

Article Snippet: The poly beta-amino ester transfection reagent Xfect (Takara 631318) was used for all plasmid delivery, at a 0.3 uL per 1 ug ratio.

Techniques: Sequencing, Plasmid Preparation, Construct, Expressing, Cotransfection, Fluorescence, Transfection, Stable Transfection