Iron loss triggers mitophagy through induction of mitochondrial ferritin (2020)
Figure 2

Figure 2. Induction of mitochondrial ferritin via hypoxia inducible factor 1α-specific protein 1 axis.

(A) Cytoplasmic and mitochondrial iron content was measured by a ferrozine-based assay for Huh7 cells with DFP, DFX or DFO treatment (0.01 mM) for 24 h (n=5, biological replicates). The cytoplasmic iron content was measured by subtracting the iron content of the mitochondrial fraction from the iron content of the whole lysate. The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.

(B) Cytoplasmic and mitochondrial iron content was measured for Huh7 cells with DFP treatment (0, 0.01, 0.1, 0.5 or 1.0 mM as indicated) for 24 h. (n=5, biological replicates) The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.

(C) Immunoblots (left panel) for mitochondrial ferritin (FTMT), frataxin, mitoferrin, heat shock protein 60 (HSP60), ferritin heavy chain (FTH1) and transferrin receptor (TfR) and β-actin using the mitochondrial fraction or whole cell lysate of Huh7 cells following treatment with DFP (0, 0.01, 0.1, 0.5 or 1.0 mM as indicated). HSP60 and β-actin were used as loading controls for mitochondrial and whole cell lysates, respectively. The expression level of FTMT was normalized to HSP60 (n=4, biological replicates, right panel). The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05.

(D) The mRNA expression of FTMT in Huh7 cells was quantified by real-time RT-PCR before and after DFP (1.0 mM) treatment (n=4, biological replicates). The expression of FTMT mRNA level was normalized to GAPDH mRNA. The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. **: P<0.01.

(E) The mRNA expression of FTMT in Huh7 cells was quantified by real-time RT-PCR before DFP (1.0 mM) treatment and 3, 6, 12, and 24 h after DFP treatment (n=5, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01, ***: P<0.001.

(F) DNA-binding activity of six transcription factors (specific protein 1 [SP1], cAMP response element binding protein [CREB], Ying yang 1 [YY1], GATA2, forkhead box protein A1 [FoxA1] and CCAAT-enhancer-binding protein β [ C/EBPβ]) to the FTMT promoter region was compared by chromatin immunoprecipitation before and after DFP (1.0 mM) treatment in Huh7 cells (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. *: P<0.05.

(G) The DNA-binding activity of SP1 to the FTMT promoter region was assayed by chromatin immunoprecipitation following treatment with DFP (0, 0.01, 0.1, 0.5 or 1.0 mM) as indicated in Huh7 cells (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01.

(H) FTMT mRNA expression was quantified for Huh7 cells by real-time RT-PCR before and after DFP (1.0 mM) treatment with or without SP1 knockdown by siRNA (n=5, biological replicates). mRNA expression of FTMT level was normalized to GAPDH. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. NC: negative control siRNA, **: P<0.01.

(I) Immunoblots for SP1 using the cytoplasmic fraction and nuclear fraction before and after DFP (1.0 mM) treatment. β-actin and Lam B were used as loading control for the cytoplasmic and nuclear fractions, respectively.

(J) Immunoblots for HIF1α using Huh7 cells and HepG2 cells before and after DFP (0.1 mM) treatment. β-actin was used as the loading control.

(K) Immunoblots for FTMT, HIF1α and SP1 using the mitochondrial fraction lysate and whole cell lysate of Huh7 cells before and after DFP (1.0 mM) treatment with or without HIF1α or SP1 knockdown by siRNA. HSP60 and β-actin were used as loading controls for the mitochondrial fraction and whole cell lysates, respectively.