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. Author manuscript; available in PMC: 2024 Jun 15.
Published in final edited form as: Mol Cell. 2023 Jun 15;83(12):2059–2076.e6. doi: 10.1016/j.molcel.2023.05.031

Figure 4. DELE1 activates an HRI-mediated ISR following iron deficiency.

Figure 4

(A, B) HEK293T cells were treated with DFO, DFP or CCCP for the indicated time periods (A) or were treated with the indicated concentrations of DFO for 16 hours (B). Cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies.

(C) ATF4 reporter (ATF4 uORF mApple)-expressing HeLa cells were treated with the indicated concentrations of DFO for 16 hours and were subjected to flow cytometry analysis. Data are shown as mean ± S.D. (N=4). ***P = 0.0001 (One-way ANOVA followed by Dunnett’s multiple comparison’s test).

(D) IB for lysates of endogenous DELE1-HA HEK293T cells treated with DFO with or without ferric ammonium citrate (FAC) for 16 hours.

(E) IB for lysates of HeLa cells treated with the indicated compounds for 16 hours.

(F) IB for lysates of endogenous DELE1-HA HEK293T cells treated with DFO for 16 hours in the presence or absence of hemin.

(G) 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.

(H) IB for lysates of HEK293T WT or DELE1 KO cell lines (clone #24 and #51) treated with DFO, 1 mM DFP, or CCCP for 16 hours. The lysates were analyzed by IB.

(I) Quantitative PCR for ISR target gene expression in HEK293T WT or DELE1 KO (clone #24) cells treated with DFO for 16 hours. Shown are mean ± S.D. (N=3). **** P<0.0001, *** P<0.001, and ** P<0.01 (One-way ANOVA followed by Turkey’s multiple comparison).

(J) DELE1(WT)-HA or DELE1(d246–272)-HA was expressed in DELE1 KO HEK293T cells (clone #24) through lentivirus infection for 40 hours. Cells were treated with DFO for the last 16 hours before harvest. Cell lysates were analyzed by IB.

See also Figure S3.