Journal: bioRxiv

Article Title: YTHDC1 m6A-dependent and m6A-independent functions converge to preserve DNA damage response

doi: 10.1101/2024.03.25.586632

Figure Lengend Snippet: A) Schematic representation of p21 endogenously tagged reporter system in A549 cell line. B) Overview of the CRISPR-screening design. A library containing 1,828 guide RNAs was cloned into a lentiviral system and used to infect A549 reporter cells. After 4 days of infection, cells were exposed to Nutlin-3a to allow for p53 protein stabilization and subsequent reporter gene activation. Cells were sorted by FACS based on the level of reporter gene activation. C) Scatter plot of Log2 Fold Change for sgRNA enrichment in p53-enhanced-response (Y-axis) and p53-attenuated-response (X-axis) populations. The three top candidates for each population are labelled and highlighted in the plot. D) Functional annotation of the top 50 candidates for p53-enhanced and p53-attenuated populations based on their molecular activity. E) In vitro validation of the top candidate for p53-enhanced and p53-attenuated populations. Each gene was independently silenced by siRNA. After silencing, mVenus signal was taken as readout of p21-reporter gene activation. Statistical analysis was performed by paired two-tailed Student’s t-test KD versus scramble (SCR)control. F) Pearson correlation score between p53-downstream-effector misregulation (p53 pathway) compared to the expressionof the 407 CRISPR-Screening candidates in Lung carcinoma patients from TCGA (LUAD + LUSC patients) with p53 wild-type genotype versus healthy patients. Data information: All data are shown are representative of at least three independent experiments. Data are presented as mean ± s.d. ns, not significant (P ≥ 0.05), *(P ≤ 0.05), ** (P ≤ 0.01), paired two-tailed student’st-test was performed in E.

Article Snippet: We further modified p21-reporter cells to stably express SpCas9 endonuclease , and transduced these cells with human lentiviral-based CRISPR pooled library in CRISPseq-BFP-backbone vector (Addgene).

Techniques: CRISPR, Clone Assay, Infection, Activation Assay, Functional Assay, Activity Assay, In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: YTHDC1 m6A-dependent and m6A-independent functions converge to preserve DNA damage response

doi: 10.1101/2024.03.25.586632

Figure Lengend Snippet: A) Representative Immunoblot of p53, with GAPDH as loading control. Cells were treated with Nutlin-3a or untreated as negative control. B) Nutlin-3a dose response treatment. P21-Reporter cells were treated with increasing doses of Nutlin-3a or untreated as negative control. After treatment, reporter gene activation was measured by flow cytometry. The percentage of cells showing reporter gene activation was calculated and presented in the plot of the signal distribution. C) Representative immunoblot of spCas9, with a-tubulin as loading control. Cells were transduced with a lentivirus carrying spCas9 or an empty vector as negative control. D) Flow cytometry quantification of P21-Reporter gene activation and CRISPR library infection for the two replicates used in the screening. E) Scatter plot of log2 fold change for sgRNA enrichment in p53-enhanced and p53-attenuated populations. Each individual sgRNA for the three top candidates for each population is labelled and highlighted in the plot. Non-targeting sgRNAs used as negative controls in black. F) Scatter plot of log2 fold change for gene enrichment in p53-enhanced and p53-attenuated populations. Previously reported negative or positive regulators of p53 activity are labelled in the plot in red and green, respectively. Functional annotation of the top 50 candidates for p53-enhanced and p53-attenuated populations based on theirmolecular substrate. H) RNA level quantification of mature mRNA by RT-qPCR for YTHDC1 and ASH2L . Cells were transfectedwith two independent siRNA against YTHDC1 (DC1-1 and -2), two independent siRNA against ASH2L (ASH2L-1 and -2) or Scramble (SCR) as negative control. Data information: All data are shown are representative of at least three independent experiments, except for C. Data are presented as mean ± s.d. *** (P ≤ 0.001), **** (P ≤ 0.0001), paired two-tailed student’s t-testwas performed in H.

Article Snippet: We further modified p21-reporter cells to stably express SpCas9 endonuclease , and transduced these cells with human lentiviral-based CRISPR pooled library in CRISPseq-BFP-backbone vector (Addgene).

Techniques: Western Blot, Negative Control, Activation Assay, Flow Cytometry, Transduction, Plasmid Preparation, CRISPR, Infection, Activity Assay, Functional Assay, Quantitative RT-PCR, Two Tailed Test