Journal: Science signaling

Article Title: ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies

doi: 10.1126/scisignal.aac4380

Figure Lengend Snippet: (A) Representative immunoblot analysis (from three experiments) of the abundance and phosphorylation (p) of eIF2α kinases, eIF2α, and ATF4 in OCI-AML3 and JeKo-1 cells from a time course of treatment with ONC201 (5 μM for 36 hours). (B) Representative immunoblot analysis (from three experiments) of GCN2, eIF2α, and ATF4 in HL-60, MOLM-13, JVM-2, and MINO cells treated with control or ONC201 (5 μM) for 24 hours. (C) Representative immunoblot analysis (from three experiments) of mTORC1 target proteins in JeKo-1 cells from a time course of treatment with ONC201 (5 μM for 36 hours).

Article Snippet: The “ATF4 5: 5′ATF4.GFP” plasmid (#21852) was purchased from Addgene ( 52 ).

Techniques: Western Blot

Journal: Science signaling

Article Title: ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies

doi: 10.1126/scisignal.aac4380

Figure Lengend Snippet: (A) Immunoblot analysis of total and phosphorylated eIF2α in OCI-AML3 cells transfected with control shRNA and shRNAs against eIF2α (eIF2α_1 and eIF2α_2) and effects of eIF2α knockdown on apoptosis of cells treated with ONC201 (5 μM) for 72 hours. (B) Representative immunoblots (from three experiments) of ATF4 and eIF2α in OCI-AML3 cells transfected with control or eIF2α-targeting shRNA and treated with ONC201 (5 μM for 24 hours) or tunicamycin (Tm; 1 μM for 6 hours). (C) Green fluorescent protein (GFP) reporter assay of ATF4 translation. 4′,6-Diamidino-2-phenylindole (DAPI)–negative and GFP-positive cells were counted by flow cytometry after treatment with dimethyl sulfoxide (ctrl), ONC201 (5 μM for 27 hours), or thapsigargin (Tg; 1 μM for 1 or 6 hours) (n = 3 experiments). (D) Immunoblot verification of ATF4 knockdown in JeKo-1 (shATF4_1) and OCI-AML3 (shATF4_2) cells. (E) Specific apoptosis induced by ONC201 in control and ATF4 knockdown JeKo-1 (shATF_1) and OCI-AML3 (shATF_2) cells (n = 3 experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: The “ATF4 5: 5′ATF4.GFP” plasmid (#21852) was purchased from Addgene ( 52 ).

Techniques: Western Blot, Transfection, shRNA, Reporter Assay, Flow Cytometry

Journal: Science signaling

Article Title: ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies

doi: 10.1126/scisignal.aac4380

Figure Lengend Snippet: ONC201 appears to up-regulate ATF4 translation through a mechanism involving μORFs of the ATF4 gene that partially resembles that used by the classical ISR and UPR. However, ONC201-induced ATF4 up-regulation does not require an increase in the phosphorylation of eIF2a, indicating a mechanism of action of ONC201 that indirectly activates this pathway. ONC201-induced ATF4 serves as an apoptosis-promoting factor that also inhibits mTORC1 activity. ONC201 also reduces MCL-1 protein abundance, causing synergistic effects with the BCL-2 inhibitor ABT-199. AA, amino acids; dsRNA, double-stranded RNA.

Article Snippet: The “ATF4 5: 5′ATF4.GFP” plasmid (#21852) was purchased from Addgene ( 52 ).

Techniques: Activity Assay