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Figure 1
Generation of HEK-293 stable cell line expressing wildtype and mutant RDH12. (A) Western blot analysis of HEK-293 cells transfected with GFP-tagged RDH12. p.Y226C and p.S13* cell lines show no RDH12 protein expression, while p.A109P shows reduced expression. (B) Co-staining of calnexin (red) and GFP (green) confirmed localisation of RDH12 to the endoplasmic reticulum. Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (blue). Scale bar = 10 µM. (C) RDH12 activity assay using HPLC showed no atROL was detected in untransfected cells, but it was found in WT cells, confirming active functional RDH12. (D) Enzyme activity of all mutant proteins was significantly reduced compared with WT RDH12. (E) RDH12 protects against atRAL-induced toxicity. Cells were dosed with increasing concentrations of atRAL for 24 h and cell viability was assessed by MTT assay. atRAL is toxic to untransfected cells, whereas cells expressing WT RDH12 were protected from atRAL-induced cell death. p.Y226C and p.S13* mutant RDH12 did not protect cells from atRAL toxicity, whereas p.A109P mutant protein offered significantly higher protection than untransfected cells at 100 µM atRAL concentration. Three independent experiments were performed. Data are expressed as mean ± SEM, and analysed using two-way ANOVA, followed by Dunnetts multiple comparison test. * p ≤ 0.05, *** p ≤ 0.001.