A Near Infrared Fluorescent Probe Based on ICT for Monitoring Mitophagy in Living Cells

Electronic Supplementary Material (ESI) for Analyst. This journal is © The Royal Society of Chemistry 2019 A near infrared fluorescent probe based on ICT for monitoring mitophagy in living cells WenqingTanga,c, Youzhi Daib,d*, Biao Guc, Mengqin Liuc, ZhengjiYic, Zhongliang Lia, Zhimin Zhangc, Huiyan Hec, Rongying Zengc* a School of Chemical Engineering, Xiangtan University, Xiangtan, Hunan 411105, People’s Republic of China b College of Environmental and Resources, Xiangtan University, Xiangtan, Hunan 411105, People’s Republic of China c Key Laboratory of Functional Organometallic Materials of College of Hunan Province, College of Chemistry and Material Science, Hengyang Normal University, Hengyang, 421008, People’s Republic of China d Hunan 2011 Collaborative Innovation Center of Chemical Engineering & Technology with Environmental Benignity and Effective Resource Utilization *Corresponding author. Tel.: +86 0734 8484932;Fax:+86 0734 8484932 E-mail: [email protected] Tel.: +86 18975283398; E-mail: [email protected] Br OH O O CHO O O O O NH HN 2 N N N I O2N O2N N I O O O O O2N O N I HO O NH2 I N NH2 Scheme S1. Synthetic routes for Cy-NH2. Figure S1. 1H-NMR spectrum of 2-bromocyclohex-1-ene-1-carbaldehyde Figure S2. 13C-NMR spectrum of 2-bromocyclohex-1-ene-1-carbaldehyde Figure S3. 1H-NMR spectrum of 6-methoxy-2,3-dihydro-1H-xanthene-4- carbaldehyde Figure S4. 13C-NMR spectrum of 6-methoxy-2,3-dihydro-1H-xanthene-4- carbaldehyde Figure S5. 1H-NMR spectrum of 2,3,3-trimethyl-3H-indole Figure S6. 13C-NMR spectrum of 2,3,3-trimethyl-3H-indole Figure S7. 1H-NMR spectrum of 2,3,3-trimethyl-5-nitro-3H-indole Figure S8. 13C-NMR spectrum of 2,3,3-trimethyl-5-nitro-3H-indole 1 Figure S9. H-NMR spectrum of Cy-NH2 13 Figure S10. C-NMR spectrum of Cy-NH2 384 #19 RT: 0.29 AV: 1 NL: 5.62E7 T: + c ESI Full ms [ 170.00-1000.00] 399.15 100 95 90 85 80 75 70 65 e 60 c n a d 55 n u b 50 A e v i t 45 a l e R 40 35 30 25 20 15 10 242.20 413.16 5 384.20 457.19 758.01 258.25 511.24 568.27 637.29 675.37 848.22 881.31 948.24 0 200 300 400 500 600 700 800 900 1000 m/z Figure S11. Mass spectrum of Cy-NH2 Figure S12. The absorption spectra of 10 μM probe in different pH contains 1.0% DMSO. Figure S13. The fluorescence spectra of 10 μM probe in different pH contains 1.0% DMSO. Figure S14. Sigmoidal fitting the pH-dependent fluorescence intensity at 670 nm. Figure S15. The absorption spectra of 10 μM probe in different solvents Figure S16. The fluorescence spectra of 10 μM probe in different solvents. (Em: 670 nm) Figure S17. The fluorescence spectra of 10 μM probe in different compounds.1. pH=4.0; 2. Cys (200 μM); 3. Hcy (200 μM); 4. GSH (5.0 mM); 5. Leu (100 μM);6. - + 2+ + H2O2 (100 μM); 7. ClO (100 μM); 8. Na (100 mM); 9. Fe (100 μM) ); 10. K (5.0 mM) and 11. Cl- (100 mM). Figure S18. Cell viability estimated by MTT assay. MCF-7 cells were incubated with different concentrations of Cy-NH2 (0-50 μM) for 24 h. Figure S19. Confocal fluorescence images of cells before (a-c) and after starvation- induced autophagy at 2 h (d-e). (λex= 488 nm, λem= 530-580 nm; λex= 639 nm, λem= 650-750 nm; scale bar=20 μm.) Figure S20. Confocal fluorescence images of cells incubated with NH2-Cy in normal medium (a-c) and the autophagy caused by rapamycin(d-f) ((a and d, λex= 488 nm, λem= 530-580 nm), (b and e, λex= 639 nm, λem= 650-750 nm), (c and f are merging of the green channel, red channel and bright field) scale bar=20 μm.) Figure S21. Flow Cytometric Assay. The green line represents normal culture of cells after incubating the probe; the purple line represents starvation-induced autophagy for 1 h; the red line represents starvation-induced autophagy for 2 h. Figure S22. The fluorescent spectra of 10 μM probe in different pH contains 1.0% DMSO.

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