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. 2016 Jul 20;5:e15550. doi: 10.7554/eLife.15550

Figure 2. Counterscreening and transcriptional profiling identifies preferential activators of the ATF6 transcriptional program.

(A) Schematic of the XBP1-RLuc splicing reporter used to monitor activation of the IRE1/XBP1s arm of the UPR in HEK293T-Rex cells treated with small molecule ER proteostasis regulators. (B) Plot of ERSE-FLuc activation and XBP1-RLuc activation for the top 281 small molecule ER proteostasis regulators (6.8 µM; 18 hr) in HEK293T-Rex cells stably expressing ERSE-FLuc or XBP1-RLuc. Activation for each axis is shown as the percent signal relative to Tg treatment (500 nM; 18 hr). Prioritized ER proteostasis regulators described in Table 1 are shown in red. Error bars show standard deviation for n = 3. (C) Heat map of multiplex gene expression (MGE) profiling for the top 281 ER proteostasis regulator compounds. HEK293T-Rex cells were incubated with 10 μM of each proteostasis regulator for 6 hr, then cells were lysed and MGE profiling was performed. Genes probed constitute transcriptional targets preferentially induced by the ATF6, XBP1s or PERK arms of the UPR or other stress-responsive signaling pathways, as indicated. Heat map is shown with compounds clustered according to their activity. The green section represents global ER stressors while the pink sections represent molecules that preferentially activate the ATF6 transcriptional program. Prioritized ER proteostasis regulators identified in Table 1 are highlighted.

DOI: http://dx.doi.org/10.7554/eLife.15550.005

Figure 2—source data 1. Excel spreadsheet showing the structure, % activation ERSE-FLuc, and % activation XBP1-RLuc for the top 281 compounds identified through primary HTS screening and confirmation screening.
These data were used to prepare Figure 2B.
DOI: http://dx.doi.org/10.7554/eLife.15550.006
elife-15550-fig2-data1.xlsx (871.6KB, xlsx)
DOI: 10.7554/eLife.15550.006
Figure 2—source data 2. Excel spreadsheet showing the Multiplex Gene Expression (MGE) expression data used to prepare Figure 2C and Figure 2—figure supplement 1C.
DOI: http://dx.doi.org/10.7554/eLife.15550.007
elife-15550-fig2-data2.xlsx (186.4KB, xlsx)
DOI: 10.7554/eLife.15550.007

Figure 2.

Figure 2—figure supplement 1. XBP1-RLuc reporter dose response and Tg-normalized MGE profiling for 281 top ER proteostasis regulators.

Figure 2—figure supplement 1.

(A) Activation of Renilla luciferase (RLuc) luminescence in HEK293T-Rex cells stably expressing XBP1-RLuc treated with the indicated concentration of thapsigargin (Tg) for 18 hr. Error bars represent standard deviation for n = 3 replicates. The Z’ score is for [Tg] = 500 nM. (B) Activation of RLuc luminescence in HEK293T-Rex cells stably expressing XBP1-RLuc treated with the indicated concentration of tunicamycin (Tm) for 18 hr. Error bars represent standard deviation for n = 3 replicates. (C) Heat map of normalized gene expression from MGE profiling experiment shown in Figure 2C. The expression of the ATF6 (blue), XBP1s (green) and PERK (orange) target genes induced by the addition of the small molecule ER proteostasis regulators were normalized to the induction observed upon addition of Tg prior to plotting. The resulting heat map is shown with compounds clustered according to their activity. Molecules highlighted with green on the right are global UPR activators as they robustly induce genes regulated by the three arms of the UPR. Alternatively, molecules highlighted in pink preferentially activate ATF6.
DOI: http://dx.doi.org/10.7554/eLife.15550.008