Journal: Molecular Therapy Oncolytics
Article Title: CRISPR-Cas knockout of miR21 reduces glioma growth
doi: 10.1016/j.omto.2022.04.001
Figure Lengend Snippet: Reduced proliferation, migration, and invasion in miR-21 KO cells (A) Schematic overview of the in vitro assays used to study proliferation and colony formation (A1, A2), migration (A3), and invasion (A4). (B) Proliferation rates of CT2A miR-21 WT and KO cells were measured using the WST reduction assay. Cell viability was measured every 24 h for 5 days. The results display the ratio of miR-21 KO compared with WT cells. miR-21 WT cells proliferated at a significantly higher rate compared with the miR-21 KO cells. Data represent triplicates and are presented as the mean with SEM (error bars). ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, multi-comparison two-way ANOVA. (C) Proliferation rates of U87 miR-21 WT and KO cells were measured using the WST reduction assay. Cell viability was measured every 24 h for 5 days. The results display the ratio of miR-21 KO compared with WT cells. miR-21 WT cells proliferated at a significantly higher rate compared with the miR-21 KO cells. Data represent triplicates and are presented as the mean with SEM (error bars). ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, multi-comparison two-way ANOVA. (D) Proliferation rates of GL261 miR-21 WT, KO-3, and the KO-3 restored cell were measured using the WST reduction assay. Cell viability was measured every 24 h for 5 days. The results display the ratio of GL261 miR-21 KO and the GL261 miR-21 restored cells compared with GL261 WT cells. WT and restored cells proliferated at a significantly higher rate compared with the miR-21 KO-3 clone. Data represent triplicates and are presented as the mean with SEM (error bars). ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, multi-comparison two-way ANOVA. (E) Representative images and quantification of colony-formation assay show a significantly lower number of colonies grown of the CT2A, U87, and GL261 miR-21 KO cells and larger colonies for miR-21 restored cells, as compared with the WT cells. miR-21 KO shows a significantly lower number of colonies compared with WT cells. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, one-way ANOVA. Data represent three independent experiments and are presented as the mean with SEM (error bars) (F and G) Representative images and quantification of cell migration and invasion measured over 24 h across a transwell membrane with 8 μm pores with (invasion) or without (migration) Matrigel. Both migration (F) and invasion (G) ability of CT2A, U87, and GL261 miR-21 KO cell lines were significantly reduced compared with the miR-21 WT cells. GL261 KO cells were significantly reduced compared with GL261 miR-21 restored cells (∗∗∗∗p < 0.0001, multi-comparison two-way ANOVA). Histograms show the number of cells that grew out as clones and number of cells that crossed the cell membrane. Experiment was repeated three times, and data are presented as the mean with SEM (error bars). ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, one-way ANOVA.
Article Snippet: Cell proliferation was assessed in vitro by the WST reduction assay to determine cell viability (cell counting kit-8 [CCK-8]; Dojindo, Rockville, MD).
Techniques: Migration, In Vitro, Comparison, Colony Assay, Membrane, Clone Assay
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