Figure 5. Ceapins are selective inhibitors of the ATF6α branch of the UPR.

(A) U2-OS cells were treated without (DMSO) or with ER stressor (100 nM Tg or 2.5 μg/mL Tm) in the absence or presence of Ceapin analogs (6 μM each) for two hours. One hour prior to lysis, proteasomal inhibitor (10 μM MG132) was added to prevent the degradation of the cleaved ATF6α-N fragment. Cells were harvested and analyzed by Western Blot for ATF6α, PERK, XBP1 and GAPDH (loading control). Arrowheads denote the positions of full-length (ATF6α), unglycosylated full-length (ATF6α*) and cleaved (ATF6α-N) variants of ATF6α and also PERK and phospho-PERK. Data shown is representative of three independent experiments. (B) U2-OS cells were treated without (DMSO) or with ER stressor (100 nM Tg or 2.5 μg/mL Tm) in the absence or presence of Ceapin analogs (6 μM each). After eight hours, cells were harvested and analyzed by Western Blot for BiP and GAPDH (loading control). (C) U2-OS cells were treated without (DMSO) or with ER stressor (100 nM Tg, black) in the absence or presence of ten-fold the IC₅₀ of either S1P inhibitor (Pf-429242, 3.2 μM, red) or Ceapin-A1 (35.7 μM, green). Four hours later cells were harvested and mRNA extracted. mRNA levels for XBP1s or DDIT3 were normalized to GAPDH for each well and then compared to unstressed controls. Data plotted are from duplicate qPCR reactions from duplicate wells, error bars are standard deviation. (D) U2-OS cells were treated without (DMSO) or with ER stressor (100 nM Tg or 2.5 μg/mL Tm) in the absence or presence of increasing concentration of Ceapin-A7 (0.6, 1.89, 6, 18.9 μM) or S1P inhibitor (5 μM Pf-429242) for four and a half hours. One hour prior to lysis, proteasomal inhibitor (10 μM MG132) was added to prevent the degradation of the cleaved ATF6α-N and ATF6β-N fragments. Cells were harvested and analyzed by Western Blot for ATF6α, ATF6β and GAPDH (loading control). Arrowheads denote the positions of full-length (ATF6α, ATF6β), unglycosylated full-length (ATF6α*, ATF6β*), cleaved membrane bound (ATF6α-M) and cleaved (ATF6α-N, ATF6β-N) variants of ATF6α and ATF6β. Data shown is representative of three independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.11878.016

Figure 5—figure supplement 1. Induction of XBP1s and DDIT3 mRNA is only partially inhibited by either the S1P inhibitor or Ceapin-A1 U2-OS cells were treated without (DMSO) or with ER stressor (2.0 μg/mL Tm, black) in the absence or presence of ten-fold the IC₅₀ of either S1P inhibitor (Pf-429242, 3.2 μM, red) or Ceapin-A1 (35.7 μM, green).

Four hours later cells were harvested and mRNA extracted. mRNA levels for XBP1s or DDIT3 were normalized to GAPDH for each well and then compared to unstressed controls. Data plotted are from duplicate qPCR reactions from duplicate wells, error bars are standard deviation.

DOI: http://dx.doi.org/10.7554/eLife.11878.017

Figure 5—figure supplement 2. ATF6β-N is generated in ER stressed cells treated with Ceapin-A7 but not ER stressed cells treated with the S1P inhibitor.

U2-OS cells were treated with ER stressor (100 nM Tg) in the absence (lanes 1, 2, 7 and 8) or presence of S1P inhibitor (5 μM Pf-429242, lanes 3, 4, 9 and 10) or Ceapin-A7 (6 μM, lanes 5, 6, 11 and 12) for four hours. One hour prior to lysis, proteasomal inhibitor (10 μM MG132) was added to prevent the degradation of the cleaved ATF6α-N and ATF6β-N fragments. Cells were harvested and separated by centrifugation into total (lanes 1, 3, 5, 7, 9 and 11) and nuclear fractions (lanes 2, 4, 6, 8, 10 and 12) and analyzed by Western blot for ATF6α, ATF6β, PERK and ATF4. Arrowheads denote the positions of full-length (ATF6α, ATF6β), cleaved membrane bound (ATF6α-M) and cleaved (ATF6α-N, ATF6β-N) variants of ATF6α and ATF6β. The transcription factor ATF4 is present in both total and nuclear extracts while membrane proteins PERK, ATF6α and ATF6β are present only in total extracts. In ER stressed cells faster migrating bands corresponding to ATF6α-N (lanes 1 and 2) and ATF6β-N (lanes 7 and 8) are found in both total and nuclear fractions. In ER stressed cells treated with the S1P inhibitor the faster migrating ATF6α and ATF6β bands found in the total extract are not present in the nuclear extract (compare lanes 3 and 4 for ATF6α and lanes 9 and 10 for ATF6β) indicating that neither ATF6α-N nor ATF6β-N were produced and these bands are likely ATF6α-M and ATF6β-M. In ER stressed cells treated with Ceapin-A7 ATF6α is not cleaved and no ATF6α-N is found in the nuclear extract (lanes 5 and 6). In contrast, ATF6β is cleaved and this faster migrating band is found in the nuclear extract indicating it is ATF6β-N (lanes 11 and 12). Data shown is representative of three independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.11878.018