Journal: Scientific Reports

Article Title: Avicin G is a potent sphingomyelinase inhibitor and blocks oncogenic K- and H-Ras signaling

doi: 10.1038/s41598-020-65882-5

Figure Lengend Snippet: Avicin G perturbs SMases expression and localization, and EGFR recycling. ( A ) MDCK cells stably expressing SMPD1-, SMPD2-, or SMPD3-GFP were treated with 500 nM avicin G for 48 h, and cells were fixed with 4% PFA and imaged in a confocal microscope. Arrowheads indicate vesicular localization of SMPD. Scale bar 10 μm. ( B ) MDCK cells stably expressing SMPD1-, SMPD2-, or SMPD3-GFP were treated with avicin G for 48 h, and cell lysates were immunoblotted with an anti-GFP antibody to measure the total SMPD-GFP expression levels. Cropped representative blots are shown with actin blots being used as loading controls from three independent experiments. Full-length blots are shown in Fig. . ( C ) The graph shows the mean ± S.E.M. of SMPD expression levels from three independent experiments. Significant difference between control (DMSO-treated) and avicin G-treated cells were assessed using a one-way ANOVA test ( N.S . – not significant). ( D ) WT MDCK cells were grown in the presence of Lysosensor Yellow/Blue dextran probe for 48 h, and equilibrated with MES calibration buffer (pH 2–6) for 15 min at room temperature. Emissions of the probe at 452 nm and 521 nm were measured by a fluorescent plate reader using a 335 nm and 381 nm excitation wavelength, respectively. The mean 452/521 nm fluorescence ratio ± S.E.M. was plotted against pH from three independent experiments, and a best fit line was plotted. ( E ) WT MDCK cells were treated with various concentrations of avicin G for 48 h in the presence of Lysosensor Yellow/Blue dextran probe. Emissions at 452 and 521 nm were measured by a fluorescent plate reader using a 335 and 381 nm excitation wavelengths, respectively. The 452/521 nm fluorescence ratio ± S.E.M. from three independent experiments were used to determine the lysosomal pH using the calibration graph from ( D ). Significant difference between control (DMSO-treated) and avicin G-treated cells were assessed using a one-way ANOVA test ( N.S . – not significant). ( F ) CHO cells stably expressing EGFR-mGFP were treated with DMSO (vehicle) or 100 nM avicin G for 48 h. Cells were serum-starved for 2 h in the continued presence of DMSO or 100 nM avicin G, and incubated with 50 ng/ml of EGF on ice for 15 min. Excess EGF was washed away with ice-cold PBS. Cells were then incubated with fresh warm complete growth medium with DMSO or 100 nM avicin G at 37 °C and fixed at the indicated time points, followed by confocal microscopy. Representative images of DMSO- and avicin G-treated cells were shown from three independent experiments. Closed and open arrowheads indicate EGFR localized at the PM and perinuclear region, respectively. Scale bar 10 μm.

Article Snippet: The following antibodies used to measure housekeeping genes were purchased from Proteintech Group (Rosemont, IL): β-actin (Cat#60008-1-1 g) and GFP-tag (Cat#66002-1-lg; clone #1E10H7). cDNA of human SMPD1 (GenScript, Cat# OHu18710D), SMPD2 (GenScript, Cat# OHu30447D) and SMPD3 (Sino Biological, Cat# HG15755-G) were cloned into GFP-N1 vector.

Techniques: Expressing, Stable Transfection, Microscopy, Fluorescence, Incubation, Confocal Microscopy