Figure 8.
SM restored membrane fluidity and inhibited Cer-induced UPR. (A) Western blot of UPR markers and agarose gel of Xbp1 cDNA amplicons (left panel) and quantification of protein amount (right panel) in AML12 treated with Ctrl/CM ± SM (100 μg/ml). The diamond indicates the nonspecific band. For western blot, lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. The statistical analyses were calculated by one-way ANOVA. Data were shown as the mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (B and C) Laurdan staining (B) and histogram of the GP values of the plasma membrane and inner membrane (C) in AML12 treated with Ctrl/CM ± SM (100 μg/ml) for 2 h. Scale bar, 20 μm. (D) SERCA activity determined in AML12 cell lysates. Cells were treated with Ctrl/CM ± SM (100 μg/ml) for 2 h. The statistical analyses were calculated by one-way ANOVA. Data were shown as the mean ± SEM, n = 4. *P < 0.05, ***P < 0.001. (E) Measurement of Ca2+ signals in the cytosol of 293T by Fluo-4 (4 μM) staining. Cells were treated with Ctrl/CM ± SM for 2 h. nCtrl = 61 cells; nCM = 54 cells; nCtrl+SM = 64; nCM+SM = 50 cells. (F) Representative confocal microscopic images of AML12 cells stained with anti-Cer antibody and Hoechst (blue). Cells were treated with Ctrl/CM ± SM (100 μg/ml) for 2 h. Scale bar, 20 μm. (G) Quantification of Cer fluorescence intensities in G measured by ImageJ, n ≥ 8 fields. The statistical analyses were calculated by one-way ANOVA. Data were shown as the mean ± SEM. **P < 0.01, ***P < 0.001. (H) RT-qPCR of Cer synthesis–related genes in AML12 treated with Ctrl/CM ± SM for 30 min. The statistical analyses were calculated by an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available for this figure: SourceData F8. Refer to the image caption for details.

SM restored membrane fluidity and inhibited Cer-induced UPR. (A) Western blot of UPR markers and agarose gel of Xbp1 cDNA amplicons (left panel) and quantification of protein amount (right panel) in AML12 treated with Ctrl/CM ± SM (100 μg/ml). The diamond indicates the nonspecific band. For western blot, lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. The statistical analyses were calculated by one-way ANOVA. Data were shown as the mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (B and C) Laurdan staining (B) and histogram of the GP values of the plasma membrane and inner membrane (C) in AML12 treated with Ctrl/CM ± SM (100 μg/ml) for 2 h. Scale bar, 20 μm. (D) SERCA activity determined in AML12 cell lysates. Cells were treated with Ctrl/CM ± SM (100 μg/ml) for 2 h. The statistical analyses were calculated by one-way ANOVA. Data were shown as the mean ± SEM, n = 4. *P < 0.05, ***P < 0.001. (E) Measurement of Ca2+ signals in the cytosol of 293T by Fluo-4 (4 μM) staining. Cells were treated with Ctrl/CM ± SM for 2 h. nCtrl = 61 cells; nCM = 54 cells; nCtrl+SM = 64; nCM+SM = 50 cells. (F) Representative confocal microscopic images of AML12 cells stained with anti-Cer antibody and Hoechst (blue). Cells were treated with Ctrl/CM ± SM (100 μg/ml) for 2 h. Scale bar, 20 μm. (G) Quantification of Cer fluorescence intensities in G measured by ImageJ, n ≥ 8 fields. The statistical analyses were calculated by one-way ANOVA. Data were shown as the mean ± SEM. **P < 0.01, ***P < 0.001. (H) RT-qPCR of Cer synthesis–related genes in AML12 treated with Ctrl/CM ± SM for 30 min. The statistical analyses were calculated by an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available for this figure: SourceData F8.

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