grna cas9 expression vector made in  (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

Journal: bioRxiv

Article Title: DROSHA, DICER and Damage-Induced long ncRNA control BMI1-dependent transcriptional repression at DNA double-strand break

doi: 10.1101/2024.08.07.606960

Figure Lengend Snippet: A. RT-qPCR analysis of GFP mRNA levels in HeLa cells bearing an integrated GFP gene (HeLa-GFP) treated with control siRNAs (siCTRL) or siRNAs against DROSHA and DICER transcripts (siDROSHA/siDICER). Data are relative to uncut cells for each knockdown condition. Error bars represent SEM from three independent experiments. B. RT-qPCR analysis of GFP mRNA levels in cut and uncut HeLa-GFP cells treated with siCTRL or siRNAs against BMI1 transcript (siBMI1). Data are relative to uncut cells for each knockdown condition. Error bars represent SEM from three independent experiments. C. RT-qPCR analysis of GFP mRNA levels in cut and uncut cells treated with control ASOs or ASOs targeting dilncRNAs generated at the GFP locus. Error bars represent SEM from three independent experiments. Data are relative to uncut cells for each condition. Statistical test used was paired Student t-test. D. RT-qPCR analysis of GFP mRNA levels in cut and uncut HeLa-GFP stably expressing an inducible Cas13d. Cells were treated with a control Cas13 RNA guide (CTRL Cas13g) or dilncRNAs-targeting guides (anti-dilncRNAs Cas13g). Error bars represent SEM from four independent experiments. E. ChIP-qPCR analysis for BMI1 at the GFP locus and an unrelated region, performed in uncut (scramble-guide-Cas9) and cut (GFP-guide-Cas9) HeLa-GFP cells treated with control ASOs or dilncRNAs-targeting ASOs. Data are relative to uncut cells after the subtraction of mock values for each treated condition. Error bars represent SEM from three independent experiments. F. RT-qPCR analysis of LIN54 (DSB at Chr4) mRNA levels in cut and uncut DIvA cells stably expressing an inducible Cas13d. Cells were treated with a control Cas13 RNA guide (CTRL Cas13g) or guides targeting dilncRNAs generated downstream the AsI cut site on Chr4 (Chr4 RNA Cas13g). Error bars represent SEM from three independent experiments. Statistical analyses in panels B, D, E and F were performed by One-Way ANOVA.

Article Snippet: For experiments in HeLa-GFP cells, Cas9 expressing vectors (Addgene) bearing either a non-targeting scrambled gRNA or a cut-inducing GFP-targeting guide gRNA (see for sequences of scramble gRNA and GFP-targeting gRNA) were transfected using Lipofectamine 2000 (Invitrogen; #11668-027) for 24 hours before cell fixation or RNA extraction.

Techniques: Quantitative RT-PCR, Control, Knockdown, Generated, Stable Transfection, Expressing, ChIP-qPCR