Figure S3. Cer is delivered in the form... | Rockefeller University Press

Figure S3.

$Cer is delivered in the form of lipoprotein but not sEV. (A) Transmission electron microscope of sEV extracted from 3T3-L1–derived Ctrl or CM by ultracentrifugation. Scale bar, 200 nm. (B) Agarose gel of Xbp1 cDNA amplicons in AML12 treated with sEV extracted from 3T3-L1–derived Ctrl or CM by ultracentrifugation. (C) Coomassie blue staining of FPLC fractions of Ctrl/CM numbered as in Fig. 5 D. Bands corresponding to ApoB and ApoA1 were denoted. (D) Quantification of protein levels in Fig. 5 F. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. The statistical analyses were calculated using an unpaired t test, two-tailed. Each lane (3–14) was compared with lane 1, and only significant upregulation was denoted. Data were shown as the mean ± SEM, n = 3. *P < 0.05. (E) FPLC of the remaining eluents after sEV extraction from AML12-derived Ctrl/CM. A total of 40 fractions were collected, and the cholesterol concentrations in each fraction were determined. (F and G) Agarose gel of Xbp1 cDNA amplicons (F) and western blot of p-IRE1α, XBP1s, and BiP (G) in AML12 treated with each fraction from (E) or fraction combinations denoted as I-VI as shown in F. The diamond indicates the nonspecific band. (H) Cholesterol content in FBS, fractions of VLDL/LDL and HDL, DFBS, and undetermined fractions isolated by ultracentrifugation. (I) Quantification of protein levels in Fig. 5 G. Lanes were numbered from left to right, with the ratio in lane 1 set as 1. The statistical analyses were calculated using an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (J) Quantification of protein levels in Fig. 5 I. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. The statistical analyses were calculated using an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 3. *P <0.05, ***P < 0.001. FPLC, fast-protein liquid chromatography; ApoB, apolipoprotein B; ApoA1, apolipoprotein A1. Source data are available for this figure: SourceData FS3. Refer to the image caption for details.$

Cer is delivered in the form of lipoprotein but not sEV. (A) Transmission electron microscope of sEV extracted from 3T3-L1–derived Ctrl or CM by ultracentrifugation. Scale bar, 200 nm. (B) Agarose gel of Xbp1 cDNA amplicons in AML12 treated with sEV extracted from 3T3-L1–derived Ctrl or CM by ultracentrifugation. (C) Coomassie blue staining of FPLC fractions of Ctrl/CM numbered as in Fig. 5 D. Bands corresponding to ApoB and ApoA1 were denoted. (D) Quantification of protein levels in Fig. 5 F. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. The statistical analyses were calculated using an unpaired t test, two-tailed. Each lane (3–14) was compared with lane 1, and only significant upregulation was denoted. Data were shown as the mean ± SEM, n = 3. *P < 0.05. (E) FPLC of the remaining eluents after sEV extraction from AML12-derived Ctrl/CM. A total of 40 fractions were collected, and the cholesterol concentrations in each fraction were determined. (F and G) Agarose gel of Xbp1 cDNA amplicons (F) and western blot of p-IRE1α, XBP1s, and BiP (G) in AML12 treated with each fraction from (E) or fraction combinations denoted as I-VI as shown in F. The diamond indicates the nonspecific band. (H) Cholesterol content in FBS, fractions of VLDL/LDL and HDL, DFBS, and undetermined fractions isolated by ultracentrifugation. (I) Quantification of protein levels in Fig. 5 G. Lanes were numbered from left to right, with the ratio in lane 1 set as 1. The statistical analyses were calculated using an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (J) Quantification of protein levels in Fig. 5 I. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. The statistical analyses were calculated using an unpaired t test, two-tailed. Data were shown as the mean ± SEM, n = 3. *P <0.05, ***P < 0.001. FPLC, fast-protein liquid chromatography; ApoB, apolipoprotein B; ApoA1, apolipoprotein A1. Source data are available for this figure: SourceData FS3.