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. 2012 Jul 25;2:77. doi: 10.3389/fonc.2012.00077

Figure 1.

Figure 1

Repercussions of wtDFNA5 and mutDFNA5 expression in BY4741 wild-type cells. (A) Western blot analysis of protein extracts of the BY4741 wild-type strain transformed with an empty plasmid (contr.) or constructs allowing for the expression of C-terminally HA-tagged and EGFP-tagged wtDFNA5 or mutDFNA5 as indicated. Immunodetection was performed using primary antibodies directed against the HA-tag, EGFP, or Adh2, which was used as loading control. The black arrow indicates full-length wtDFNA5 and the gray arrow full-length mutDFNA5. The small open arrowheads indicate a-specific bands detected by anti-EGFP. For mutDFNA5, the lane marked with [oe] represents an overexposure of the Western blot. (B) Growth of the BY4741 wild-type strain transformed with an empty plasmid (light gray circles) or a construct allowing for expression of C-terminally HA-tagged wtDFNA5 (dark gray squares) or mutDFNA5 (black triangles). (C) Visualization of ROS producing cells in control cultures (contr.) or cultures of cells expressing C-terminally HA-tagged wtDFNA5 or mutDFNA5 in post-diauxic phase. (D) Quantification of ROS accumulation using DHE staining in control cultures (contr.) or cultures of cells expressing C-terminally HA-tagged wtDFNA5 or mutDFNA5 when sampled at the mid-exponential growth phase (ME), at post-diauxic shift (PD), and in stationary phase (ST). (E) Quantification of the number of AV/PI positive cells in ME, PD, and ST phase of control cells or cells expressing HA-tagged wtDFNA5 or mutDFNA5. Significances were assayed using unpaired t-tests. For the AV/PI co-staining the following significances were obtained when compared to the control: for wtDFNA5 in ME: AV−/PI+ *; for wtDFNA5 in PD: AV+/PI+ ***, AV−/PI+ *; for wtDFNA5 in ST: AV+/PI− *, AV+/PI+ *, AV−/PI+ **; for mutDFNA5 in ME: AV+/PI− *, AV+/PI+ **, AV−/PI+ **; for mutDFNA5 in PD: AV+/PI− **, AV+/PI+ *, AV−/PI+ ***; for mutDFNA5 in ST AV+/PI− **, AV+/PI+ **, AV−/PI+ *. (* = p < 0.05; ** = p < 0.01; *** = p < 0.001).