Figure 3.
ERS transmission is independent of Cer biosynthesis or UPR activation in donor cells. (A) Ctrl/CM were derived from IRE1α pathway–deficient 3T3-L1 cells. To block IRE1α pathway, IRE1α KO (upper panel), 0.5 μM KIRA8 treatment (middle panel), and XBP1 knockdown by shRNA (lower panel) were used. The blockage efficiency was examined by western blot and PCR of Xbp1 cDNA amplicons shown on the left. The xbp1 splicing–inducing effect of CM in AML12 cells was shown on the right. U, unspliced Xbp1; S, spliced Xbp1. (B) Ctrl/CM were derived from PERK pathway–deficient 3T3-L1 cells. To block the PERK pathway, PERK KO (upper panel) and 1 μM GSK2606414 (GSK) treatment (lower panel) were used. The blockage efficiency was examined by western blot shown on the left. The xbp1 splicing–inducing effect of CM in AML12 cells was shown on the right. U, unspliced Xbp1; S, spliced Xbp1. (C) Ctrl/CM were derived from ATF6 pathway–deficient 3T3-L1 cells. Ceapin-A7 (10 μM) was used to block the activation and nuclear translocation of ATF6. The blockage efficiency was examined by western blot of nuclear and whole cell proteins shown on the left. The xbp1 splicing–inducing effect of CM in AML12 cells was shown on the right. U, unspliced Xbp1; S, spliced Xbp1. (D) Ctrl/CM were derived from 3T3-L1 cells that have all the three UPR pathways blocked with KIRA8, GSK2606414 (GSK), and ceapin-A7 in combination. The blockage efficiency was examined by western blot of p-IRE1α, XBP1s, p-PERK, BiP, ATF6, and nuclear ATF6 in 3T3-L1 shown on the left. The UPR induction effect of CM in AML12 cells was detected by western blot of p-IRE1α, XBP1s, p-PERK, and BiP and agarose gel of Xbp1 cDNA amplicons shown on the right. U, unspliced Xbp1; S, spliced Xbp1. The diamond indicates the nonspecific band. (E) Heatmap (shown as z-score) for proteostasis-related genes in 3T3-L1 with indicated treatment. For UPR inhibitors (IN), KIRA8, GSK2606414, and ceapin-A7 were used in combination. (F) Agarose gel of Xbp1 cDNA amplicons (upper panel) and ratio of the band intensities for S/(S+U) quantified using ImageJ (lower panel). cDNA was from AML12 treated with Ctrl/CM generated as in E. (G) Heatmap (shown as z-score) for Cer synthesis–related genes in 3T3-L1 treated as in E. (H) Ctrl/CM were derived from FB1 (10 μM)-, myriocin (10 μM)-, or GW4869 (20 μM)-pretreated 3T3-L1. Agarose gel of Xbp1 cDNA amplicons from AML12 treated with regular Ctrl/CM or Ctrl/CM as specified. (I) Western blot of XBP1s (upper panel) and agarose gel of Xbp1 cDNA amplicons (lower panel) from AML12 treated with regular or Cer inhibitor–pretreated Ctrl/CM. Cer inhibitors indicate a combination of FB1, myriocin, and GW4869. Source data are available for this figure: SourceData F3. Refer to the image caption for details.

ERS transmission is independent of Cer biosynthesis or UPR activation in donor cells. (A) Ctrl/CM were derived from IRE1α pathway–deficient 3T3-L1 cells. To block IRE1α pathway, IRE1α KO (upper panel), 0.5 μM KIRA8 treatment (middle panel), and XBP1 knockdown by shRNA (lower panel) were used. The blockage efficiency was examined by western blot and PCR of Xbp1 cDNA amplicons shown on the left. The xbp1 splicing–inducing effect of CM in AML12 cells was shown on the right. U, unspliced Xbp1; S, spliced Xbp1. (B) Ctrl/CM were derived from PERK pathway–deficient 3T3-L1 cells. To block the PERK pathway, PERK KO (upper panel) and 1 μM GSK2606414 (GSK) treatment (lower panel) were used. The blockage efficiency was examined by western blot shown on the left. The xbp1 splicing–inducing effect of CM in AML12 cells was shown on the right. U, unspliced Xbp1; S, spliced Xbp1. (C) Ctrl/CM were derived from ATF6 pathway–deficient 3T3-L1 cells. Ceapin-A7 (10 μM) was used to block the activation and nuclear translocation of ATF6. The blockage efficiency was examined by western blot of nuclear and whole cell proteins shown on the left. The xbp1 splicing–inducing effect of CM in AML12 cells was shown on the right. U, unspliced Xbp1; S, spliced Xbp1. (D) Ctrl/CM were derived from 3T3-L1 cells that have all the three UPR pathways blocked with KIRA8, GSK2606414 (GSK), and ceapin-A7 in combination. The blockage efficiency was examined by western blot of p-IRE1α, XBP1s, p-PERK, BiP, ATF6, and nuclear ATF6 in 3T3-L1 shown on the left. The UPR induction effect of CM in AML12 cells was detected by western blot of p-IRE1α, XBP1s, p-PERK, and BiP and agarose gel of Xbp1 cDNA amplicons shown on the right. U, unspliced Xbp1; S, spliced Xbp1. The diamond indicates the nonspecific band. (E) Heatmap (shown as z-score) for proteostasis-related genes in 3T3-L1 with indicated treatment. For UPR inhibitors (IN), KIRA8, GSK2606414, and ceapin-A7 were used in combination. (F) Agarose gel of Xbp1 cDNA amplicons (upper panel) and ratio of the band intensities for S/(S+U) quantified using ImageJ (lower panel). cDNA was from AML12 treated with Ctrl/CM generated as in E. (G) Heatmap (shown as z-score) for Cer synthesis–related genes in 3T3-L1 treated as in E. (H) Ctrl/CM were derived from FB1 (10 μM)-, myriocin (10 μM)-, or GW4869 (20 μM)-pretreated 3T3-L1. Agarose gel of Xbp1 cDNA amplicons from AML12 treated with regular Ctrl/CM or Ctrl/CM as specified. (I) Western blot of XBP1s (upper panel) and agarose gel of Xbp1 cDNA amplicons (lower panel) from AML12 treated with regular or Cer inhibitor–pretreated Ctrl/CM. Cer inhibitors indicate a combination of FB1, myriocin, and GW4869. Source data are available for this figure: SourceData F3.

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