human crispr activation library sam-3 system  (Addgene inc)

Journal: Molecular Cancer

Article Title: Double vulnerability of active-NRF2 lung squamous cell carcinoma to NRF2 and TRIM24

doi: 10.1186/s12943-025-02401-y

Figure Lengend Snippet: sgRNA-library infection rescues cell survival in NRF2-depleted and OS-treated LUSC cells expressing active NRF2. (A) Representative images of HCC15 cells (library-infected or not) and NRF2-depleted or not (scale 50 μm) (arrows indicate viable cells). The flow cytometry plots (bottom, left) show the higher mortality of NRF2-depleted cells without library infection, compared to library-infected cells. The bar graph (bottom, right) shows the percentage of PI-negative viable cells in HCC15 cells infected or not with the library (and NRF2-depleted or not). Upon doxy-induced NRF2 depletion there is a higher percentage of viable cells in the library-infected cells versus control infection. (B) NRF2 and KEAP1 protein levels in HCC15 cells treated or not with doxycycline. The graph shows protein signal corrected for β-Actin loading and normalized to the HCC15 control (100%). (C) mRNA expression levels of indicated genes from cell extracts, as in (B) , represented as RQ values referred to GAPDH mRNA levels (in CTR group). (D) The samples from three independent experiments were collected to compare CTR groups (sgRNA library infected and RSL3-treated HCC15 cells) and TESTs (the same but NRF2-depleted). Genomic DNA extracted and the sgRNA sequences amplified by PCR. These sequences were examined by NGS. VOLCANO plot showing the Log2 fold change of sgRNAs expression changes resulting from the NGS library analysis (x-axis) versus -Log2 (FDR) (y-axis). Some of the genes are indicated. (E) The round graphic shows the first functional annotation groups from David analysis ( https://david.ncifcrf.gov ) of the 180 genes showing an FDR < 0.05 and an increase in sgRNA levels upon NRF2 depletion. The area of each term is proportional to the number of genes (grouped by decreasing P values). (F) GO term clusters organized according to enrichment scores (x-axis) and the number of genes in the term (y-axis). (G) List of top genes resulting from the CRISPRa analysis. Additional criteria for gene selection was CRISPhieRmix and MaGECK analysis of gene lists, an FDR < 0.05, a change in gene levels > 1.66-fold. (H) mRNA levels of the selected genes in the CTR and TEST samples, represented as RQ values normalized to those of GAPDH . Values were referred to those in CTR group (considered 1). Mean ± SD. Unpaired t-test (one or two-tailed) (*) p < 0.05, (**) p < 0.01, (***) p < 0.001

Article Snippet: The human CRISPR activation library (SAM-3 system, Addgene) including 70,290 sgRNAs covering 23,430 target human genes was amplified as previously described [ ].

Techniques: Infection, Expressing, Flow Cytometry, Control, Amplification, Functional Assay, Selection, Two Tailed Test