Figure 3.
Figure 3. d-cer-C6–induced Golgi membrane organization defects are mediated by SMS1 and SMS2. (A) HeLa cells were transfected with control (CTRL) siRNA or with SMS1 and SMS2 siRNA for 96 h. Total RNA was extracted, and the knockdown efficiency was monitored by RT-PCR using primers for SMS1, SMS2, or GAPDH and loading the products on an agarose gel. (B) HeLa cells grown on coverslips were transfected with control or SMS1 and SMS2 siRNA for 92 h. Then, the cells were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The cells were then fixed, and the localization of the TGN markers p230 and TGN46 was monitored by immunofluorescence microscopy. Bar, 5 µm. (C) Quantitation of the relative colocalization of p230 and TGN46 in the experiments shown in B, as measured by the Pearson’s correlation coefficient between the green and red channels. Bars show the mean values ± SEM of ≥10 cells counted from three independent experiments. **, P < 0.01.

d-cer-C6–induced Golgi membrane organization defects are mediated by SMS1 and SMS2. (A) HeLa cells were transfected with control (CTRL) siRNA or with SMS1 and SMS2 siRNA for 96 h. Total RNA was extracted, and the knockdown efficiency was monitored by RT-PCR using primers for SMS1, SMS2, or GAPDH and loading the products on an agarose gel. (B) HeLa cells grown on coverslips were transfected with control or SMS1 and SMS2 siRNA for 92 h. Then, the cells were treated with ethanol, 20 µM l-cer-C6, or 20 µM d-cer-C6 for 4 h. The cells were then fixed, and the localization of the TGN markers p230 and TGN46 was monitored by immunofluorescence microscopy. Bar, 5 µm. (C) Quantitation of the relative colocalization of p230 and TGN46 in the experiments shown in B, as measured by the Pearson’s correlation coefficient between the green and red channels. Bars show the mean values ± SEM of ≥10 cells counted from three independent experiments. **, P < 0.01.

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