Figure 1.

Citrin Protein Expression Levels in Mammalian Cell Lines Transfected with hCitrin-mRNA

(A and B) Modified mRNAs consisting of the open reading frame of the wild-type hCitrin nucleotide sequence (hCitrin-mRNA_WT) or EGFP (1 μg) were transfected into (A) HeLa and HepG2 cells or (B) CTLN2 patient fibroblasts. Twenty-four hours post-transfection, cell lysates were processed by capillary electrophoresis (CE) to assess protein expression levels. (C) Immunocytochemistry of exogenously expressed hCitrin in Hep3B cells. Cells were transfected with hCitrin-mRNA_WT and analyzed with confocal microscopy for co-localization with the mitochondrial marker TOM20. Non-transfected cells (wild-type Hep3B) showed very low levels of endogenous Citrin, whereas cells transfected with the hCitrin-mRNA_WT showed significantly higher protein levels, which colocalized with TOM20. The cells were also counterstained with DAPI (blue) for nucleus visualization. (D) Image analysis showed similar co-localization with mitochondrial markers for untransfected and hCitrin-mRNA_WT-transfected cells (Pearson coefficient values of 0.79, and 0.8, respectively). (E) Malate/aspartate transporting activity in isolated mitochondria prepared from HepG2 cells transfected with EGFP or hCitrin-mRNA. Data are represented as mean ± SEM (n = 3, independent experiments). Student’s t test (*p < 0.05 versus EGFP) was performed to assess statistically relevant differences between groups.