Journal: Molecular Pharmaceutics

Article Title: High-Content Surface and Total Expression siRNA Kinase Library Screen with VX-809 Treatment Reveals Kinase Targets that Enhance F508del-CFTR Rescue

doi: 10.1021/acs.molpharmaceut.7b00928

Figure Lengend Snippet: RAF1 and CAMKK1 single siRNA validation in flow cytometry. RAF1 and CAMKK1 single Dharmacon ON- TARGETplus siRNAs with or without VX-809 treatment. Measured surface CFTR fluorescence with a BD Accuri flow cytometer with Intellicyt HTFC. The grayed out region represents VX-809s average surface fluorescence. VX-809 + siRNA significance is shown from VX-809 negative control. Data are shown as mean ± SEM (3–4 replicates). One-way ANOVA with multiple comparison for significant changes from VX-809 neg. ctrl (0 nM) (red P -values). *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05.

Article Snippet: Cell stripper was gently removed and 150 μL of HBSS with 500 nM MG-B-Tau dye was added to suspend cells and transfer them to a U-bottom 96-well plate, where they were allowed to incubate for 15 min in dye before being read on an Intellicyt HTFC BD Accuri flow cytometer.

Techniques: Flow Cytometry, Fluorescence, Negative Control

Journal: Molecular Pharmaceutics

Article Title: High-Content Surface and Total Expression siRNA Kinase Library Screen with VX-809 Treatment Reveals Kinase Targets that Enhance F508del-CFTR Rescue

doi: 10.1021/acs.molpharmaceut.7b00928

Figure Lengend Snippet: Measuring surface fluorescence changes using select kinase drug inhibitors with a plate reader and flow cytometry method. Kinase inhibitors, GW 5074 and STO-609, with VX-809 treatment showed an increase in surface fluorescence using a BD Accuri flow cytometer with Intellicyt HTFC. VX-809 + kinase inhibitor is shown normalized to DMSO-treated control cells. The grayed out region represents VX-809s average surface fluorescence. Each drug showed significant interaction effects. Data are shown as mean ± SEM (3 or more replicates). One-way ANOVA with multiple comparisons for significant changes from VX-809 control (0 nM) (red P -values). Two-way ANOVA was used for measuring interaction. **** P ≤ 0.0001; ** P ≤ 0.01.

Article Snippet: Cell stripper was gently removed and 150 μL of HBSS with 500 nM MG-B-Tau dye was added to suspend cells and transfer them to a U-bottom 96-well plate, where they were allowed to incubate for 15 min in dye before being read on an Intellicyt HTFC BD Accuri flow cytometer.

Techniques: Fluorescence, Flow Cytometry