Journal: bioRxiv

Article Title: A Novel C. elegans Model for Tau Spreading Reveals Genes Critical for Endolysosomal Integrity and Seeded Tau Aggregation

doi: 10.1101/2024.11.13.619586

Figure Lengend Snippet: (A) Schematic illustration of endolysosomal damage detection using the galectin-3 reporter (sfGFP::LGALS3) in HEK293T cells. (B) Representative collapsed confocal z-stacks (488 nm channel) of HEK293T cells stably expressing the sfGFP-LGALS3 reporter and the inducible dCas9 system, transiently transfected with either the empty vector control (pLG1) or pLG1 expressing sgRNAs targeting the indicated genes. Scale bar = 20 μm. (C) Quantification of Gal3 foci in HEK293T cells stably expressing the sfGFP-LGALS3 reporter and the inducible dCas9 system, transiently transfected with either the empty pLG1 vector or pLG1 expressing sgRNAs targeting the indicated genes. Downregulation of SERINC4 expression did not increase Gal3 foci formation, while targeting the expression of all other genes significantly increased Gal3 foci formation in the Gal3 reporter cells. Data are presented as boxplot, showing the mean, upper and lower quartiles, and the minimum and maximum values. Points outside the min-max range represent outliers. N = 4 with 10-11 images per gene and replicate. Each point represents one image. Statistical analysis was conducted using Two-Way mixed-model ANOVA on rank-transformed data, with pairwise comparisons of estimated marginal means with Bonferroni correction for multiple comparisons. Color-coding corresponds to p-values as indicated in the figure legend.

Article Snippet: The pLG1-non-targeting sgRNA (Addgene plasmid # 109002) was digested by Blp1 and BstX1 restriction enzymes.

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Control, Transformation Assay

Journal: Nature Communications

Article Title: PTPN23-dependent ESCRT machinery functions as a cell death checkpoint

doi: 10.1038/s41467-024-54749-2

Figure Lengend Snippet: a Gene essentiality of mouse PTPs in RN2 cells and NIH 3T3 cells. sgRNA frequencies at Day 2 and Day 14 were determined by next-generation sequencing, with average logarithmic frequency changes calculated. Yellow dots represent pan-essential genes spiked into the screen library. b Histogram of PTPN23 gene knockout effect in 1078 cancer cell lines (DepMap 22Q2). The dashed line indicates the median score. c GFP competition growth assays performed in RN2 cells ( n = 3 independent experiments). d Left, Human PTPN23 and ALIX full-length constructs and PTPN23 truncations used in rescue experiments. Right, Heatmap visualizing the complementation effects aligned with the corresponding numbers on D14 in ( e ). His: His domain; PRR: Proline-rich region, located at the N-terminus of the His domain. e GFP competition growth assays performed in RN2 stable cell lines expressing indicated cDNAs. Top, control sgRNAs; bottom, PTPN23 sgRNAs ( n = 3 independent experiments). f Left, immunoblot of NOMO-1 cells overexpressing empty vector (EV), sgRNA-sensitive (PTPN23-WT) or sgRNA-resistant (PTPN23-r2) PTPN23 cDNA ( n = 3 independent experiments). Right, GFP competition growth assays performed using NOMO-1 stable cell lines expressing either PTPN23-WT ( n = 3 independent experiments) or PTPN23-r2 ( n = 4 independent experiments). g Top, acute depletion of PTPN23 with dTAG system. NOMO-1 cells stably expressing sgRNA-resistant PTPN23 fused with dTAG and endogenous PTPN23 were depleted with two sgRNAs. Bottom, immunoblotting analysis of PTPN23 examined after 4-day treatment of dTAG-13 (100 nM). h CellTiter-Glo (CTG) luminescence cell viability assay measured on cells treated with either DMSO or dTAG-13. DMSO or 500 nM dTAG-13 were added to three PTPN23-dTAG NOMO-1 cell lines established in ( g ) ( n = 3 independent experiments). i Cell death indicated by Sytox Green positivity. PTPN23-dTAG NOMO-1 cells were treated with 200 nM dTAG-13 for 4 days, and stained with Sytox Green ( n = 60 fields captured, 3 independent experiments). Scale bar: 30 μm. Data are presented as mean ± SEM, statistical analysis for ( h ) by Two-way ANOVA, Sidak’s multiple comparisons test; ( i ) by One-way ANOVA, Tukey’s multiple comparisons test.

Article Snippet: The pooled library containing phosphatase domain targeting and control sgRNAs (Supplementary Data ) was synthesized in duplicates or triplicates on an array platform (Twist Bioscience) and then cloned into the LRG2.1 vector with Gibson Assembly kit (NEB).

Techniques: Next-Generation Sequencing, Gene Knockout, Construct, Stable Transfection, Expressing, Control, Western Blot, Plasmid Preparation, Viability Assay, Staining

Journal: The EMBO Journal

Article Title: FBXL4 ubiquitin ligase deficiency promotes mitophagy by elevating NIX levels

doi: 10.15252/embj.2022112799

Figure Lengend Snippet: A Schematic depicting the CRISPR screening strategy. RPE1‐Cas9i‐mt‐mKeima cells were transduced with a lentiviral CRISPR sgRNA library targeting 606 E3 ligases. sgRNA‐expressing cells were selected for 7 days with puromycin (Puro) and Cas9 expression induced with doxycycline (Dox) for 9 days. Fourteen days post‐transduction, half the cells were treated with antimycin and oligomycin (AO; 1 and 10 μM respectively) for 24 h, then sorted alongside untreated cells (basal mitophagy) by FACS into “high” and “low mitophagy” populations based on mt‐mKeima fluorescence. Genomic DNA was extracted from sorted and unsorted reference cells, sgRNAs amplified by barcoded PCRs and samples analysed by next‐generation sequencing (NGS). B, C Volcano plot showing the average log 2 fold change and −Log 10 P ‐value of genes in low mitophagy versus unsorted cells in the AO‐induced and basal mitophagy screen for two independent biological replicates. Statistical thresholds of 2 and 3 standard deviations from the mean are indicated by dashed lines and colour coding. Indicated are high‐confidence (unbroken line) and lower‐confidence (dashed line) candidates shown in (D–F). D, E High‐confidence candidate list of positive (decreased) and negative (enhanced) regulators of Parkin‐independent‐induced mitophagy. Heatmap showing the Log 2 fold change of genes in low/high mitophagy versus unsorted cells in the induced and basal mitophagy screen for each of two independent biological replicates. Genes with P ‐values < 0.05 are indicated by an asterisk. F Venn diagram showing the overlap of genes listed in (D) and (E) that were identified in the basal and AO‐induced mitophagy screens as positive (green) and negative (magenta) regulators. Bold type indicates high‐confidence hits and regular type indicates lower‐confidence hits. Source data are available online for this figure.

Article Snippet: sgRNAs targeting 606 Ubiquitin E3 ligases, as well as 243 non‐targeting control (NTC) sequences, were designed by Azimuth2 (Doench et al , ) and the top four picks (on‐target scores > 0.6) were chosen per gene, extended by 5 and 3 prime 3Cs homology and obtained as a pool from Twist Bioscience (Dataset ).

Techniques: CRISPR, Transduction, Expressing, Fluorescence, Amplification, Next-Generation Sequencing

Journal: Cell reports

Article Title: Proinflammatory cytokines promote TET2-mediated DNA demethylation during CD8 T cell effector differentiation

doi: 10.1016/j.celrep.2021.109796

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: sgRNA mCherry:5′-CAAGUAGUCGGGGAUGTCGG - 3′ , Synthego , NA.

Techniques: Virus, Recombinant, Purification, DNA Methylation Assay, Methylation, Plasmid Preparation, Cloning, Cell Isolation, Software, Methylation Sequencing, Flow Cytometry