lenticrisprv2 vector expressing streptococcus pyogenes cas9 spcas9  (Addgene inc)

Journal: bioRxiv

Article Title: SMARCA2 is an essential and potent cofactor for a specific subset of the glucocorticoid response in A549 cells

doi: 10.1101/2025.11.13.688306

Figure Lengend Snippet: A. CRISPR screening timeline. Cells were transduced with the Brunello genome-wide CRISPR screening library at low MOI, selected with puromycin, and passaged for eight days. They were then treated with 100 nM dexamethasone or 0.1% ethanol as a vehicle control for four hours, stained with an anti-GILZ antibody, and sorted into high and low expression bins containing the top and bottom 10% GILZ-expressing cells.. gDNA was collected from cells, gRNA sequences were amplified, and enrichment in high and low bins vs. the bulk population was determined (n = 5 replicates per drug condition). B,C. Differential gRNA enrichment in the high and low GILZ expression bins in B. the vehicle control condition and C. the 100 nM dexamethasone treatment condition. Log 2 fold changes are an average of high bin vs. bulk unsorted and low bin vs. bulk unsorted. Negative sign indicates a gRNA was depleted from the high bin and enriched in the low bin. (n = 5 replicates per drug condition) D,E. Validation of top gRNAs from the screen using D. qPCR or E. flow cytometry to determine the impact of knocking out the target gene on glucocorticoid-induced GILZ expression (n = 3 independent transductions). * p < 0.05, ** p < 0.01, *** p <0.001, one-tailed student’s t-test. Samples transduced with non-targeting control gRNAs are shown in gray, positive control gRNAs targeting NR3C1 and TSC22D3 are in blue, and gRNAs targeting SMARCA2 and BPTF in red. F,G. Validation of top hits from the screen using siRNAs to knock down the genes of interest and assess their impact on glucocorticoid-induced GILZ expression using F. qPCR (n = 2 to n = 3 independent transfections) or G. flow cytometry (n = 4 independent transfections) * p < 0.05, ** p < 0.01, *** p <0.001, one-tailed student’s t-test

Article Snippet: The protospacers from the top enriched gRNAs found in the screen were ordered as oligonucleotides from IDT and cloned into a lentiviral gRNA expression vector (lentiCRISPR v2, Addgene Plasmid #52961).

Techniques: CRISPR, Transduction, Genome Wide, Control, Staining, Expressing, Amplification, Biomarker Discovery, Flow Cytometry, One-tailed Test, Positive Control, Knockdown, Transfection

Journal: Cell Death & Disease

Article Title: MEK5/ERK5 inhibition sensitizes NRAS -mutant melanoma to MAPK-targeted therapy by preventing Cyclin D/CDK4-mediated G1/S progression

doi: 10.1038/s41419-025-08036-7

Figure Lengend Snippet: A : Representative immunoblots of n = 3 experiments done using total lysates from BLM wild-type (Wt), BLM empty vector (EV)-infected, or two different CRISPR/ Cas9 -mediated MEK5 k.o. single cell clones (SCC) of BLM treated with diluent (ctrl) or 25 nM Tram for 14 days, showing expression of selected cell cycle proteins. ERK5 phosphorylation and DUSP4 suppression confirm Tram functionality. Successful MEK5 gene disruption was analysed by MEK5 immunoblot and functionally evaluated by immunoblotting for ERK5, which confirmed absent ERK5 autophosphorylation. Tubulin served as loading control. B : Representative cell cycle profiles of n = 2 experiments, as determined by flow cytometric analysis of the indicated PI-stained conditions with percentages of S-phase cells indicated. C : Cell doubling time analysis representative of n = 2 experiments with running times of 2–5 weeks performed with Wt BLM or the indicated MEK5 k.o. BLM SCCs cultured in absence or presence of Tram. The shown experiment run over a period of five weeks.

Article Snippet: BLM, lentiCRISPR_zeo, a derivative of the lentiviral dual gRNA/ Cas9 expression vector lentiCRISPRv2-Blast (Addgene_83480) replacing the blasticidin R gene by a zeocin R gene was used.

Techniques: Western Blot, Plasmid Preparation, Infection, CRISPR, Clone Assay, Expressing, Phospho-proteomics, Disruption, Control, Staining, Cell Culture