Journal: Molecular and Cellular Biology

Article Title: Loss of Mitochondrial Localization of Human FANCG Causes Defective FANCJ Helicase

doi: 10.1128/MCB.00306-20

Figure Lengend Snippet: Development of the FANCGR22P stable cell line. The R22P construct was stably integrated into the genome of the FANCG parental cell by Lentivector pLJM1-EGFP (Addgene). (A) GFP expression confirms the stable expression of R22P in FANCG parental cells. (B) Cells were treated with MMC (lanes 1, 2, and 3) or not treated (lanes 4, 5, and 6), and cell lysates were used for Western blotting with FANCG antibody. The expression of FANCGR22P was confirmed in the stable cell (lanes 2 and 5). (C) FANCD2 monoubiquitination studies of the FANCG corrected, FGR22P, and FG parental cells. Cells were treated with MMC (lanes 1, 2, and 3) or not treated (lanes 4, 5, and 6) and blotted with FAND2 antibody. FANCD2-L represents the monoubiquitinated proteins, and FANCD2-S represents the normal FANCD2 proteins.

Article Snippet: The viral particle protein-containing vectors pCMV-VSV-G, psPAX2, and FGR22P-pLJM1-EGFP constructs were transfected (1:1:3) into HEK293 cells by Turbofect (Fermentas).

Techniques: Stable Transfection, Construct, Expressing, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Loss of Mitochondrial Localization of Human FANCG Causes Defective FANCJ Helicase

doi: 10.1128/MCB.00306-20

Figure Lengend Snippet: Quantification of the amount of iron present in FANCJ protein of FG corrected, FG parental, and FGR22P cells. (A) The amount of 55Fe at 0 h was considered 100, and the relative amount of 55Fe was calculated at each time point. Each result is the mean from the minimum of three experiments. (ns, nonsignificant; *, 0.01 < P < 0.05; **, 0.0001 < P < 0.0009; and ***, P ≤ 0.0001, for α = 0.05). (B) Western blot of the cell lysates with FANCJ and GAPDH antibody. (C) mRNA expression of FXN and NFU1 at different time points. (D) Diagram of Fe-S cluster biogenesis and transfer to recipient proteins (53). (E) Colocalization of TOM70-EGFP and the MitoTracker in FANCGR22P fibroblast cells. (F) The graph represents the Pearson’s correlation coefficient for colocalization of TOM70-EGFP and the MitoTracker in FANCGR22P fibroblast cells under treated and untreated conditions.

Article Snippet: The viral particle protein-containing vectors pCMV-VSV-G, psPAX2, and FGR22P-pLJM1-EGFP constructs were transfected (1:1:3) into HEK293 cells by Turbofect (Fermentas).

Techniques: Western Blot, Expressing

Journal: Molecular and Cellular Biology

Article Title: Loss of Mitochondrial Localization of Human FANCG Causes Defective FANCJ Helicase

doi: 10.1128/MCB.00306-20

Figure Lengend Snippet: Model to explain the mitochondrial instability leads to genomic instability. (A) In a normal cell, FANCG prevents PRDX3 from calpain cleavage and maintains mitochondrial stability by reducing oxidative stress. Stable mitochondria maintain the helicase activity of FANCJ by providing the ISC domain. (B) In the FGR22P cell, FANCG fails to migrate to mitochondria, and PRDX3 is cleaved by calpain. Mitochondrial membrane potential (ΔΨ) is lost due to elevated oxidative stress, and ISC biosynthesis is reduced. FANCJ lost its helicase activity due to insufficient iron in its Fe-S domain.

Article Snippet: The viral particle protein-containing vectors pCMV-VSV-G, psPAX2, and FGR22P-pLJM1-EGFP constructs were transfected (1:1:3) into HEK293 cells by Turbofect (Fermentas).

Techniques: Activity Assay