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. 2012 Nov 23;31(24):4535–4546. doi: 10.1038/emboj.2012.317

Figure 4.

Figure 4

D-Ceramide-C6 inhibits membrane fission. (A) GF17 cells stably expressing PKD1-KD-GST-Flag were placed at 20°C to induce tubulation of the TGN. Cells were pretreated with ethanol (control) or D-ceramide-C6 for 30 min at 37°C, then shifted to 20°C and, at indicated time points, fixed and stained with anti-TGN46 and anti-GST antibodies. (B) NRK cells were pretreated for 30 min with 20 μM L- or D-ceramide-C6, then washed and incubated with 140 μM IQ or DMSO for 1 h at 37°C. The cells were fixed with PFA and visualized by fluorescence microscopy with anti-TGN38 antibody. (C) Quantitation of cells with a fragmented (vesiculated) Golgi apparatus in 100 randomly selected cells per experiment. Bars show the mean±s.e.m. of three independent experiments (N=3) P<0.01 (**). (D) Post IQ treatment, NRK cells were treated with 20 μM L- or D-ceramide-C6. After 30 min, IQ was removed and the cells incubated in medium containing L- or D-ceramide-C6, respectively, and observed by fluorescence microscopy. (E) NRK cells were treated as in (B), fixed, and thin sections visualized by electron microscopy. Size scale bars (A), (B), and (D)=10 μm and (E)=500 nm.