Figure 6
Characterization of PPO knock-in clones. R38/1, W227/1, U-2 OS, and N2 cell lines were transfected with the PPOX-GFP construct and corresponding knock-in cell lines denoted R38/1-KI, W227/1-KI, U-2 OS-KI and N2-KI, respectively. Control, PPO-KO and PPO-KI samples are highlighted by green, orange and blue background, respectively. (a) Expression levels of PPO-GFP and ALAS-1 were analyzed by Western blotting. Notice markedly higher PPO-GFP expression levels compared to endogenous PPO. Alpha-tubulin was used as a loading control. (b) PPO enzymatic activity in cell lysates was determined by following conversion of protoporphyrinogen IX to protoporphyrin IX. PPO oxidase activity is shown as ratio to the endogenous PPO activity in parent U-2 OS cells (100%). Data represent mean (± S.D.); n = 2. (c) Localization of PPO-GFP (green channel) was assessed by live-cell confocal microscopy. Mitochondria were visualized by vital staining using Mitotracker dye (red channel), cell nuclei were stained by Hoechst 33258 (blue channel). Scale bar 10 µm. (d) Intracellular concentrations of heme and protoporphyrinogen/protoporphyrin IX were determined by RP-HPLC. Data represent mean (± S.D.); n = 2. (e) The oxygen consumption rate was measured by Seahorse. ATP production, maximal respiration, and spare respiratory capacity were determined upon the addition of 1 µM oligomycin, 1 µM CCCP, and 0.5 µM rotenone + 0.5 µM antimycin A, respectively. Statistical significance was calculated by one-way ANOVA and assigned by ***(P < 0.001), **(P < 0.01), *(P < 0.05) and ns (non-significant; P > 0.05).
