Fig. 2
SNG photosensitization mechanism and phototoxicity in living cells a Phototoxicity of SNG (black box, black solid line) compared to SNR (black box, black dashed line) and miniSOG (grey box, grey dashed line) targeted to matrix mitochondria in HeLa cells. HeLa cell death was counted for 7 h every 1 h post-irradiation with 2 W/cm2 excitation light for 2 min. EGFP- (grey diamond, grey solid line) and mCherry- (light grey diamond, light grey dashed line) expressing cells are treated with the same power and time of irradiation as negative control for excitation light. Time for significant cell death to occur was analysed between each hour post-irradiation to t0. miniSOG, SNG and SNR showed significant cell death after 3 h, 5 h and 6 h respectively (p < 0.05, one-way analysis of variance (ANOVA), Tukey, n = 144 cells for SNR, 128 cells for SNG, 73 cells for miniSOG. Cells were calculated from 8 images per construct). No significant cell death occurred for EGFP and mCherry (p > 0.05, one-way ANOVA, Tukey, n = 117 cells for EGFP, 149 cells for mCherry. Cells were calculated from 8 images per construct). b Time course of SNG and miniSOG 1O2 measurement. miniSOG irradiation with 440 nm light caused significant ADPA bleaching compared to SNG (p < 0.001, t test, n = 4 replicates for miniSOG, 6 for SNG; each replicate came from independently purified protein samples). 1O2 (c) and O2•- measurement (d) in HeLa cells expressing SNG or miniSOG in mitochondrial matrix using Si-DMA and MitoSOX respectively. After 10 s irradiation with 4 W/cm2 excitation light, significant Si-DMA fluorescence intensity increase was observed for HeLa cells expressing miniSOG (compared to control, independent t test, p < 0.001, n = 100 cells), but no significant difference observed between SNG and control (p = 0.185). MitoSOX fluorescence intensity increased significantly in cells expressing miniSOG and SNG (compared to control, independent t test, p < 0.001, n = 100 cells), but there was no significant difference between SNG and miniSOG (p = 0.659). e, f and g show O2•-, H2O2 and •OH quenching experiment on HeLa cells expressing miniSOG and SNG in mitochondrial matrix by 200 U/mL SOD, 1000 U/mL catalase and 60 mM mannitol respectively. HeLa cells expressing SNG treated (+) with SOD (e) and catalase (f) showed significant reduction in cell death after 2 W/cm2 excitation light irradiation for 2 min compared to non-treated (−) cells (p < 0.05, t test, n = 45 cells for SNG (−) SOD, 38 cells for SNG (+) SOD; 40 cells for SNG (−) catalase, 94 cells for SNG (+) catalase; cells were calculated from 4 images for each condition), while miniSOG showed no reduction in cell death (p > 0.05, t test, n = 59 cells for miniSOG (−) SOD, 62 cells for miniSOG (+) SOD; 67 cells for miniSOG (−) catalase, 93 for miniSOG (+) catalase; cells were calculated from 4 images for each condition). Same result was observed for mannitol experiment (g) (p < 0.05 for SNG, t test, n = 220 cells for SNG (−) mannitol, 185 cells for SNG (+) mannitol, 155 cells for miniSOG (−) mannitol, 180 cells for miniSOG (+) mannitol; cells were calculated from 4 images for each condition). Error bar represents ± standard error of the mean (SEM). Supporting numeric data are provided in Additional file 2