Figure 4. Stable overexpression of FLAG-tagged Mic60 in differentiated SH-SY5Y cells.

(A) Non-transfected passage-matched Control SH-SY5Y cells and stable-expressing Mic60-FLAG, pcDNA3 Empty Vector, and mitoYFP SH-SY5Y cells were differentiated for 5 days, collected, and whole-cell lysate examined by Western blot using T3867 rabbit anti-Mic60. A FLAG-immunoreactive band was detected corresponding to the same molecular weight as Mic60. (B) Mic60 levels were quantified from Western blots with respect to the actin loading control, and presented as % of non-transfected Control cells (n=4; mean ± SEM; * = significant from non-transfected Control, pcDNA3, and mitoYFP, p<0.05). (C–E) Differentiated cells underwent 24 hr treatment with 250 μM DA or non-treated control media (C,D), or 48 hr treatment with 0.5 μM rotenone or DMSO vehicle (E). Viability was assessed by trypan blue exclusion. (C) Basal viability did not differ between empty vector pcDNA3, mitoYFP, and Mic60-FLAG stable transfected cells or passage-matched non-transfected Control cells in non-treated media (n=6). (D) Viability is presented as % cell death as compared to respective non-treated cells following 24 hr treatment with 250 μM DA (n=6; mean ± SEM; * = Mic60-FLAG cell death significant from pcDNA3, mitoYFP, and non-transfected Control cells, p<0.05). (E) Viability is presented as % cell death as compared to respective DMSO-vehicle treated cells following 48 hr treatment with 0.5 μM rotenone (n=6; mean ± SEM; * = Mic60-FLAG cell death significant from pcDNA3, mitoYFP, and non-transfected Control cells, p<0.05).