cas9 grna dual expression vector  (Addgene inc)

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