Figure S2. Lipid-mediated... | Rockefeller University Press

Figure S2.

$Lipid-mediated cell-nonautonomous UPR activation in different types of donor-receiving cells. (A) Agarose gel of Xbp1 cDNA amplicons in Ctrl/CM-treated cells 293T → 293T, medium fractions with M.W. > 30 kDa and/or 100°C heating were used. U, unspliced Xbp1; S, spliced Xbp1. (B–D) Quantification of protein levels in Fig. 2 A (B), Fig. 2 B (C), and Fig. 2 C (D). The statistical analyses were calculated by one-way ANOVA. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. Data were shown as the mean ± SEM, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. (E–I) Agarose gel of Xbp1 cDNA amplicons in receiving cells with different donor → receiving cell systems. Cleanascite-treated medium (E) and lipid extracts from medium (F–I) were used. (J) Quantification of protein levels in Fig. 2 D. The statistical analyses were calculated by one-way ANOVA. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. Data were shown as the mean ± SEM, n = 3. *P < 0.05; **P < 0.01; ns, not significant. (K) Agarose gel of Xbp1 cDNA amplicons from AML12 treated by various species of CM-extracted lipids. CM was derived from AML12. Lipids were fractionated as in Fig. 2 E. Other classes was a combination of FA+PhL+Residue+a+b+c+d. (L) Quantification of protein levels in Fig. 2 F. The statistical analyses were calculated by one-way ANOVA. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. Data were shown as the mean ± SEM, n = 3. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant. (M) Quantification of protein levels in Fig. 2 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. Each lane (3–12) was compared with lane 1, and only significant upregulation was denoted. Data were shown as the mean ± SEM, n = 3. *P < 0.05. (N) Western blot of BiP, XBP1s, and ATF4 (upper panel) and agarose gel of Xbp1 cDNA amplicons (lower panel) in AML12 treated with Cer. C18:0, Cer d18:1/18:0; C16:0, Cer d18:1/16:0. The diamond indicates the nonspecific band. (O and P) Agarose gel of Xbp1 cDNA amplicons in B16.F10 (O) and J774A.1 (P) treated with Cer for 6 h. (Q) Quantification of protein levels in Fig. 2 L. Lanes were numbered from left to right. The statistical analyses were calculated using an unpaired t test, two-tailed. Only lanes 6 and 8 are compared. Data were shown as the mean ± SEM, n = 3. *P < 0.05; ns, not significant. (R) Quantification of protein levels in Fig. 2 M. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. The statistical analyses were calculated using one-way ANOVA. Data were shown as the mean ± SEM, n = 3. *P < 0.05; **P < 0.01; ns, not significant. Source data are available for this figure: SourceData FS2. Refer to the image caption for details.$

Lipid-mediated cell-nonautonomous UPR activation in different types of donor-receiving cells. (A) Agarose gel of Xbp1 cDNA amplicons in Ctrl/CM-treated cells 293T → 293T, medium fractions with M.W. > 30 kDa and/or 100°C heating were used. U, unspliced Xbp1; S, spliced Xbp1. (B–D) Quantification of protein levels in Fig. 2 A (B), Fig. 2 B (C), and Fig. 2 C (D). The statistical analyses were calculated by one-way ANOVA. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. Data were shown as the mean ± SEM, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. (E–I) Agarose gel of Xbp1 cDNA amplicons in receiving cells with different donor → receiving cell systems. Cleanascite-treated medium (E) and lipid extracts from medium (F–I) were used. (J) Quantification of protein levels in Fig. 2 D. The statistical analyses were calculated by one-way ANOVA. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. Data were shown as the mean ± SEM, n = 3. *P < 0.05; **P < 0.01; ns, not significant. (K) Agarose gel of Xbp1 cDNA amplicons from AML12 treated by various species of CM-extracted lipids. CM was derived from AML12. Lipids were fractionated as in Fig. 2 E. Other classes was a combination of FA+PhL+Residue+a+b+c+d. (L) Quantification of protein levels in Fig. 2 F. The statistical analyses were calculated by one-way ANOVA. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. Data were shown as the mean ± SEM, n = 3. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant. (M) Quantification of protein levels in Fig. 2 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. Each lane (3–12) was compared with lane 1, and only significant upregulation was denoted. Data were shown as the mean ± SEM, n = 3. *P < 0.05. (N) Western blot of BiP, XBP1s, and ATF4 (upper panel) and agarose gel of Xbp1 cDNA amplicons (lower panel) in AML12 treated with Cer. C18:0, Cer d18:1/18:0; C16:0, Cer d18:1/16:0. The diamond indicates the nonspecific band. (O and P) Agarose gel of Xbp1 cDNA amplicons in B16.F10 (O) and J774A.1 (P) treated with Cer for 6 h. (Q) Quantification of protein levels in Fig. 2 L. Lanes were numbered from left to right. The statistical analyses were calculated using an unpaired t test, two-tailed. Only lanes 6 and 8 are compared. Data were shown as the mean ± SEM, n = 3. *P < 0.05; ns, not significant. (R) Quantification of protein levels in Fig. 2 M. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. The statistical analyses were calculated using one-way ANOVA. Data were shown as the mean ± SEM, n = 3. *P < 0.05; **P < 0.01; ns, not significant. Source data are available for this figure: SourceData FS2.