Figure 7. Cer activates the UPR by... | Rockefeller University Press

Figure 7.

Cer activates the UPR by disrupting membrane fluidity and Ca2+homeostasis. (A and B) Western blot of p-IRE1α, XBP1s, p-PERK, and BiP in AML12 (upper panel) and agarose gel of Xbp1 cDNA amplicons (lower panel) from AML12 treated with Ctrl/CM (A) or Ctrl/CM-extracted lipids (B) with or without 5 mM MβCD. The diamond indicates the nonspecific band. (C and D) Laurdan staining (C) and histogram of the GP values of the plasma membrane and inner membrane (D) in AML12 treated with Ctrl/CM ± MβCD (5 mM) for 2 h. Scale bar, 20 μm. (E–H) (E and G) Representative confocal microscopic images of AML12 cells stained with anti-Cer (green), anti-Golgin-97 (red), and anti-calnexin (red) antibodies and Hoechst (blue). (E) Cells were treated with Ctrl/CM for the indicated time. (G) Cells were treated with Ctrl/CM lipids for 30 min. Quantifications of Cer fluorescence intensities in the left panel of (E) and in (G) measured by ImageJ are shown in (F) (n ≥ 5 fields) and (H) (n = 10 fields), respectively. The statistical analyses were performed using an unpaired t test, two-tailed. Data were shown as the mean ± SEM. ***P < 0.001, ****P < 0.0001. Scale bar, 20 μm. (I) Quantification of Cer content in microsome (μmol/g protein) by mass spectrometry. Microsome was isolated from AML12 treated with Ctrl/CM for 1 h. The statistical analyses were performed using an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 4. *P < 0.05. (J) Western blot of p-IRE1α, XBP1s, p-PERK, and BiP in AML12 treated with Ctrl/CM ± inhibitors. The diamond indicates the nonspecific band. Inhibitors are FB1 (10 μM), myriocin (10 μM), GW4869 (20 μM), and DPA (50 μM). (K) Western blot of p-IRE1α, XBP1s, p-PERK, and BiP in AML12 treated with Ctrl/CM ± 5 μM of CRT0066101 (CRT) for 6 h. The diamond indicates the nonspecific band. (L) SERCA activity determined in AML12 cell lysates. Cells were treated with Ctrl/CM for 2 h. The statistical analyses were performed using an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 3. *P < 0.05. (M) Measurement of Ca2+ signals in the cytosol of AML12 by 5 μM Fura-2 staining. Cells were treated with Ctrl/CM for 2 h. nCtrl = 74 cells; nCM = 120 cells. (N) SERCA activity determined in AML12 cell lysates. Cells were treated with Ctrl/CM-extracted lipid for 2 h. The statistical analyses were performed using an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 3. *P < 0.05. (O) SERCA activity determined in AML12 cell lysates. Cells were treated with 20 μM of C16:0 or C18:0 for 2 h. The statistical analyses were performed using an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 3. ***P < 0.001, ****P < 0.0001. (P) Measurement of Ca2+ signals in the cytosol of 293T by 4 μM Fluo-4 staining. Cells were treated with 20 μM of C16:0 or C18:0 for 2 h. nsolvent = 139 cells; nc16:0 = 144 cells; nc18:0 = 97 cells. Source data are available for this figure: SourceData F7. Refer to the image caption for details.

Cer activates the UPR by disrupting membrane fluidity and Ca ²⁺ homeostasis. (A and B) Western blot of p-IRE1α, XBP1s, p-PERK, and BiP in AML12 (upper panel) and agarose gel of Xbp1 cDNA amplicons (lower panel) from AML12 treated with Ctrl/CM (A) or Ctrl/CM-extracted lipids (B) with or without 5 mM MβCD. The diamond indicates the nonspecific band. (C and D) Laurdan staining (C) and histogram of the GP values of the plasma membrane and inner membrane (D) in AML12 treated with Ctrl/CM ± MβCD (5 mM) for 2 h. Scale bar, 20 μm. (E–H) (E and G) Representative confocal microscopic images of AML12 cells stained with anti-Cer (green), anti-Golgin-97 (red), and anti-calnexin (red) antibodies and Hoechst (blue). (E) Cells were treated with Ctrl/CM for the indicated time. (G) Cells were treated with Ctrl/CM lipids for 30 min. Quantifications of Cer fluorescence intensities in the left panel of (E) and in (G) measured by ImageJ are shown in (F) (n ≥ 5 fields) and (H) (n = 10 fields), respectively. The statistical analyses were performed using an unpaired t test, two-tailed. Data were shown as the mean ± SEM. ***P < 0.001, ****P < 0.0001. Scale bar, 20 μm. (I) Quantification of Cer content in microsome (μmol/g protein) by mass spectrometry. Microsome was isolated from AML12 treated with Ctrl/CM for 1 h. The statistical analyses were performed using an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 4. *P < 0.05. (J) Western blot of p-IRE1α, XBP1s, p-PERK, and BiP in AML12 treated with Ctrl/CM ± inhibitors. The diamond indicates the nonspecific band. Inhibitors are FB1 (10 μM), myriocin (10 μM), GW4869 (20 μM), and DPA (50 μM). (K) Western blot of p-IRE1α, XBP1s, p-PERK, and BiP in AML12 treated with Ctrl/CM ± 5 μM of CRT0066101 (CRT) for 6 h. The diamond indicates the nonspecific band. (L) SERCA activity determined in AML12 cell lysates. Cells were treated with Ctrl/CM for 2 h. The statistical analyses were performed using an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 3. *P < 0.05. (M) Measurement of Ca²⁺ signals in the cytosol of AML12 by 5 μM Fura-2 staining. Cells were treated with Ctrl/CM for 2 h. n_Ctrl = 74 cells; n_CM = 120 cells. (N) SERCA activity determined in AML12 cell lysates. Cells were treated with Ctrl/CM-extracted lipid for 2 h. The statistical analyses were performed using an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 3. *P < 0.05. (O) SERCA activity determined in AML12 cell lysates. Cells were treated with 20 μM of C16:0 or C18:0 for 2 h. The statistical analyses were performed using an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 3. ***P < 0.001, ****P < 0.0001. (P) Measurement of Ca²⁺ signals in the cytosol of 293T by 4 μM Fluo-4 staining. Cells were treated with 20 μM of C16:0 or C18:0 for 2 h. n_solvent = 139 cells; n_c16:0 = 144 cells; n_c18:0 = 97 cells. Source data are available for this figure: SourceData F7.