Extended Data Fig. 6. The SIFI complex silences the cellular response to mitochondrial import stress.

a. The SIFI complex limits signal duration, not amplitude. Wildtype or ΔUBR4 cells were treated with 5 μM sodium arsenite and cell lysates were analyzed for ATF4 levels by Western blotting over time. Quantification of 4 independent experiments shown in Fig. 3e. b. The SIFI complex also limits signal duration after ISR activation with 5 μM antimycin A (AM). Cell lysates were analyzed as described above. Similar results in n = 3 independent experiments. c. The SIFI complex, not the GADD34 phosphatase, mediates stress response silencing in response to arsenite. WT or ΔUBR4 cells were depleted of GADD34, as indicated, and treated with 5 μM arsenite. At different times, samples were analyzed for ATF4 expression by Western blotting. Similar results in n = 2 independent experiments. d. The SIFI complex, not the CReP phosphatase, mediates stress response silencing after antimycin A treatment. WT or ΔUBR4 cells were depleted of CReP, as indicated, and treated with 0.6 μM antimycin A. At different times, samples were analyzed for ATF4 expression by Western blotting. Similar results in n = 2 independent experiments. e. The SIFI complex, not the GADD34 phosphatase, mediates stress response silencing in response to antimycin A. WT or ΔUBR4 cells were depleted of GADD34, as indicated, and treated with 0.6 μM antimycin A. At different times, samples were analyzed for ATF4 expression by Western blotting. Similar results in n = 2 independent experiments. f. The SIFI complex does not mediate degradation of GADD34, as shown by a GADD34 stability reporter in flow cytometry. Similar results in n = 2 independent experiments. g. The SIFI complex does not mediate degradation of CReP, as shown by a CReP stability reporter in flow cytometry. Similar results in n = 2 independent experiments. For gel source data, see Supplementary Fig. 1.