Journal: bioRxiv

Article Title: A consensus set of genetic vulnerabilities to ATR inhibition

doi: 10.1101/574533

Figure Lengend Snippet: a, Clonogenic survival of RPE1-hTERT Flag-Cas9 TP53 -/- (WT) and two RPE1-hTERT Flag-Cas9 TP53 -/- APEX2 -/- clones treated with indicated concentrations of ATR inhibitor AZD6738. b, as in a using two CIP2A -/- clones. c, as in a using a POLE3 -/- clone. d, as in a using two POLE4 -/- clones. e, as in a using two C16orf72 -/- clones. Data are from three biologically independent experiments.

Article Snippet: GFP-POLE3 full length and GFP-POLE3 with amino acid residues 113-140 deleted (GFP-POLE3ΔC) were cloned between NheI and AgeI restriction sites of pCW57.1 (Addgene: 41393),

Techniques: Clone Assay

Journal: bioRxiv

Article Title: A consensus set of genetic vulnerabilities to ATR inhibition

doi: 10.1101/574533

Figure Lengend Snippet: a-c, Immunoblotting to assess loss of protein expression in clonal knockout cell lines. siRNA-mediated knockdown was used to control for antibody specificity in the case of POLE3 and POLE4 antibodies. Antibodies targeting alpha-tubulin (Tubulin) or GAPDH were used as loading controls. Numbers indicate molecular mass in kDa. Asterisks indicate unspecific bands. d, mRNA level analysis of APEX2 after siRNA mediated APEX2 knockdown as assay control and in APEX2 -/- clones. Clone #2 showed mRNA levels similar to parental (WT) cells but has frameshifting mutations (see panel D) and was sensitive to ATR inhibitor treatment (see ).

Article Snippet: GFP-POLE3 full length and GFP-POLE3 with amino acid residues 113-140 deleted (GFP-POLE3ΔC) were cloned between NheI and AgeI restriction sites of pCW57.1 (Addgene: 41393),

Techniques: Western Blot, Expressing, Knock-Out, Clone Assay

Journal: bioRxiv

Article Title: A consensus set of genetic vulnerabilities to ATR inhibition

doi: 10.1101/574533

Figure Lengend Snippet: a, Whole cell extracts from wild type RPE1-hTERT Flag-Cas9 TP53 -/- (WT) or the indicated POLE4 -/- clones treated with 1 μM camptothecin (CPT) were used for immunoblotting with indicated antibodies. pCHK1 and pRPA refer to phosphorylated proteins; brackets indicate modified amino acid residues. KAP1 served as loading control. b, Whole cell extracts from WT or the indicated POLE3 -/- clone expressing GFP, GFP-POLE3 or GFP-POLE3ΔC were used for immunoblotting with indicated antibodies. GAPDH served as loading control. c, Clonogenic survival of WT or the indicated POLE3 -/- clone expressing GFP, GFP-POLE3 or GFP-POLE3ΔC treated with indicated concentrations of ATR inhibitor AZD6738. Data are from three biologically independent experiments.

Article Snippet: GFP-POLE3 full length and GFP-POLE3 with amino acid residues 113-140 deleted (GFP-POLE3ΔC) were cloned between NheI and AgeI restriction sites of pCW57.1 (Addgene: 41393),

Techniques: Clone Assay, Western Blot, Modification, Expressing

Journal: bioRxiv

Article Title: A consensus set of genetic vulnerabilities to ATR inhibition

doi: 10.1101/574533

Figure Lengend Snippet: Cell survival of RPE1-hTERT Flag-Cas9 TP53 -/- (WT) or the indicated POLE3 -/- clone expressing GFP, GFP-POLE3 or GFP-POLE3ΔC treated with indicated concentrations of ATR inhibitor (AZD6738) was determined by monitoring growth in an Incucyte instrument. Data are from three biologically independent experiments.

Article Snippet: GFP-POLE3 full length and GFP-POLE3 with amino acid residues 113-140 deleted (GFP-POLE3ΔC) were cloned between NheI and AgeI restriction sites of pCW57.1 (Addgene: 41393),

Techniques: Expressing

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