Figure 6. Determination of critical factors that are required for the iron deficiency-induced ISR activation.
(A) A schematic representation of DELE1 domain structures. See text for details.
(B) DELE1 (WT) or DELE1 deletion mutants that lacked indicated regions were expressed in DELE1 KO HEK293T cells through lentivirus infection for 40 hours. Cells were treated with DFO for the last 16 hours before harvest. Cell lysates were analyzed by immunoblotting (IB).
(C) The indicated DELE1 deletion mutants were transiently transfected in DELE1 KO HEK293T cells. After 24 hours, cells were lysed and each DELE1 was immunoprecipitated with anti-HA antibody. The samples were subjected to IB. IP; immunoprecipitation.
(D) DELE1 WT or d102-200 were transiently infected in DELE1 KO HEK293T cells (clone #24). Cells were subsequently treated with DFO for the last 20 hours before harvest. Each DELE1 was immunoprecipitated with anti-HA antibody. Samples were subjected to IB.
(E, F) DELE1 WT or DELE1 deletion mutants that lacked indicated regions (E) or linker-inserted DELE1(d102-200) mutants (F) were expressed in DELE1 KO HEK293T cells through lentivirus infection for 40 hours. Cells were treated with DFO for the last 16 hours before harvest. Cell lysates were analyzed by IB.
(G) A model for the role of 102–200 a.a. region of DELE1 in HRI activation during iron deficiency. See text for details.
(H) IB for lysates of HeLa cells transfected with indicated siRNAs for 72 hours, and treated with DFO for the last 16 hours.
(I) IB for lysates of HeLa cells transfected with indicated siRNAs for 72 hours, and treated with DFO, DFP, or CCCP for the last 16 hours.
(J) Knockdown efficiency of ABCB7 in (I) determined by quantitative PCR (qPCR).
(K) Endogenous DELE1-HA HEK293T cells were transfected with indicated siRNAs for 72 hours. Cells were treated with DFO for 16 hours, followed by the CHX chase for the last 30 min before harvest. Cell lysates were analyzed by IB. *; non-specific bands.
(L) Knockdown efficiency of ABCB7 in (K) determined by qPCR.