Temporally Monitoring Autophagy

Transcription

1 Supporting Information An In Situ Intracellular Self-Assembly Strategy for Quantitatively and Temporally Monitoring Autophagy Yao-Xin Lin,, Sheng-Lin Qiao,, Yi Wang,, Ruo-Xin Zhang, Hong-Wei An,, Yang Ma,,, R P. Yeshan J. Rajapaksha, Zeng-Ying Qiao, Lei Wang *, and Hao Wang CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST). Beijing, , P. R. China University of Chinese Academy of Sciences, Beijing, , P. R. China *,,

2 Supporting Figures. Figure S1. The schematic illustration of 4 th PAMAM-OH.

3 Figure S2. 1 H NMR spectrum of BP in d 6 -DMSO. Concentration: 10 mg/ml.

4 Figure S3. FL spectrum of DPBP (The ratio of BP to PAMAM in feed is 1:1). FL spectrum was measured in the range from 375 to 650 nm and showed very weak fluorescence (525 nm) in the presence of ATG4B.

5 Figure S4. FL spectrum of DPBP (The ratio of BP to PAMAM in feed is 10:1). FL spectrum were measured in the range from 375 to 650 nm and showed very strong fluorescence (525 nm) in the absence of ATG4B.

6 Figure S5. HPLC characterized the responsive peptide incubation with ATG4B for 5 min a) and 120 min b). Acetonitrile (0.1% TFA) /water (0.1% TFA) gradient from 5/95% to 70/30% in 30 min flux 1 ml/min, 220 nm.

7 Figure S6. 1 H NMR spectrum of BP-Peptide in d 6 -DMSO. Concentration, 10 mg/ml.

8 Figure S7. 1 H NMR spectrum of BPDP in d 6 -DMSO. Concentration, 10 mg/ml.

9 Figure S8. 1 H NMR spectrum of C-DPBP in d 6 -DMSO, dotted boxes represent the BP-peptide characteristic peaks. Concentration, 10 mg/ml.

10 Figure S9. FL spectra of BP (5 µm) in DMSO/H 2 O mixture. Excitation wavelength, 350 nm.

11 Figure S10. Time-dependent FL spectra of DPBP (100 µg/ml) in the presence of ATG4B. The fluorescence reached a plateau in 140 min.

12 Figure S11. Cell viabilities of DPBP and C-DPBP were measured in MCF-7 Cells by CCK-8 assay. MCF-7 cells were treated with DPBP/C-DPBP (concentration ranges from 0 to 200 µg/ml) for 24 h, respectively.

13 Figure S12. Time-course cell uptake of DPBP-Cy5. CLSM images of MCF-7 cells incubated with DPBP-Cy5 (100 µg/ml) at different time (0 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h and 6.0 h). 633: λ ex = 633 nm, λ em = 670 ± 35 nm.

14 Figure S13. CLSM images of intracellular trafficking of internalized DPBP-Cy5 monitored by co-localization with caveolin-1. MCF-7 cells were incubated with DPBP-Cy5 (100 µg/ml) for 0.5 or 2.0 h and then co-cultured with caceolin-1 antibody overnight. Green signal was from anti-caceolin-1 green-labeled secondary antibody overnight. Red fluorescence showed the location of DPBP-Cy5. 488: λ ex = 488 nm, λ em = 520 ± 25 nm; 633: λ ex = 633 nm, λ em = 670 ± 35 nm; BF: Bright field.

15 Figure S14. CLSM images of intracellular trafficking of internalized DPBP-Cy5 monitored by co-localization with clathrin. MCF-7 cells were incubated with DPBP-Cy5 (100 µg/ml) for 0.5 or 2.0 h and then co-cultured with clathrin antibody overnight. Green signal was from anti- clathrin green-labeled secondary antibody overnight. Red fluorescence showed the location of DPBP-Cy5. 488: λ ex = 488 nm, λ em = 520 ± 25 nm; 633: λ ex = 633 nm, λ em = 670 ± 35 nm; BF: Bright field.

16 Figure S15. CLSM images of intracellular trafficking of internalized DPBP-Cy5 monitored by co-localization with early endosome marker. MCF-7 cells were incubated with DPBP-Cy5 (100 µg/ml) for 2.0 h and then co-cultured with early endosome marker. Green signal was from early endosome marker. Red fluorescence showed the location of DPBP-Cy5. 488: λ ex = 488 nm, λ em = 520 ± 25 nm; 633: λ ex = 633 nm, λ em = 670 ± 35 nm; BF: Bright field.

17 Figure S16. Colocalization of lysosome with DPBP-Cy5. MCF-7 cells were incubated with DPBP-Cy5 (100 μg/ml) for 2 h and then co-cultured with Lyso-Tracker Green DND-26. Green fluorescence showed lysosomes staining with Lyso-Tracker Green DND-26. Red fluorescence showed the location of DPBP. 488: λ ex = 488 nm, λ em = 520 ± 25 nm; 633: λ ex = 633 nm, λ em = 670 ± 35 nm; BF: Bright field.

18 Figure S17. Colocalization of endoplasmic reticulum (ER) with DPBP-Cy5. MCF-7 cells were incubated with DPBP-Cy5 (100 µg/ml) for 2 h and then co-cultured with ER-Tracker Green. Green fluorescence showed ER. Red fluorescence showed the location of DPBP-Cy5. 488: λ ex = 488 nm, λ em = 520 ± 25 nm; 633: λ ex = 633 nm, λ em = 670 ± 35 nm; BF: Bright field.

19 . Figure S18. Autophagy measurement by western blot on LC3. MCF-7 cells were treated with DPBP (25, 50, 100, 200 µg/ml) for 24 h or Rapa (1 µm) for 2 h, respectively. The corresponding results of semi-quantitative analysis are shown in the lower panel.

20 Figure S19. Western blot on BECN1, LC3 and P62 expression in MCF-7 cells. MCF-7 cells were treated with DPBP (100 µg/ml) for 24 h or Rapa (1 µm) for 2 h, respectively. The corresponding results of semi-quantitative analysis are shown in the lower panel.

21 Figure S20. Western blot on LC3 expression in MCF-7 cells. MCF-7 cells were treated with PBS,Rapa (1 µm) alone, Rapa (1 µm) and DPBP (100 µg/ml) for 2 h, respectively. The corresponding results of semi-quantitative analysis are shown in the lower panel. Statistical significance: n.s.: no significance, one-way ANOVA for indicated comparison.

22 Figure S21. Dose-dependent fluorescence of DPBP in Rapa-treated cells. MCF-7 cells were treated with DPBP (0, 50, 100, 200 µg/ml) for 1 h and then incubated with Rapa (1 µm) for 2 h.

23 Figure S22. Validation of DPBP performance in the absence of Rapa. MCF-7 cells were treated with DPBP (100 µg/ml) or DPBP-Cy5 (100 µg/ml) for 1 h and then incubated with PBS or Rapa (1 µm) for 2 h.

24 Figure S23. Specificity confirmation of DPBP to autophagy by immunofluorescence method in living cells. CLSM images of Rapa-induced autophagic cells with DPBP (100 µg/ml) and LC3 immunofluorescence. Green signal was from BP aggregates, red signal was from anti-lc3 red-labeled secondary antibody. Cells were cultured as follows: in regular culture medium for 1 h and followed by 10 µl of PBS (lane 1), Rapa (lane 2) in regular culture medium for 2 h; followed by 100 µg/ml C-DPBP (lane 3) or DPBP (lane 4) for 1 h and 10 µl of Rapa in regular culture medium for 2 h. 405: λ ex = 405 nm, λ em = 525 ± 25 nm; 548: λ ex = 548 nm, λ em = 565 ± 15 nm; BF: Bright field.

25 Figure S24. The CLSM images of colocalization of DPBP-Cy5 with ATG4B immunofluorescence. MCF-7 cells were incubated with DPBP-Cy5 (100 µg/ml) for 1 h and then co-cultured with Rapa for 2 h. Green fluorescence showed anti-atg4b FITC-labeled secondary antibody. Red fluorescence showed the location of DPBP-Cy5. 488: λ ex = 488 nm, λ em = 520 ± 25 nm; 633: λ ex = 633 nm, λ em = 670 ± 35 nm; BF: Bright field.

26 Figure S25. Correlation plot of BP aggregates and ATG4B. MCF-7 cells were first cultured with Rapa for 2 h, and then followed by DPBP (100 µg/ml) for 2 h. Green signal was from BP aggregates, red signal was from anti-atg4b red-labeled secondary antibody. 405: λ ex = 405 nm, λ em = 525 ± 25 nm; 548: λ ex = 548 nm, λ em = 565 ± 15 nm. An overlap coefficient of 0.41 (0: no co-localization, 1: all pixels co-localized) confirmed that the BP spots from DPBP did not co-localize with ATG4B.

27 Figure S26. Cellular real-time monitoring and quantitatively analyzing the activity of ATG4B by DPBP. CLSM images of Rapa-treated MCF-7 cells and the corresponding quantitative analysis of fluorescence intensities. The cells were treated with DPBP for 1 h, and then incubated with Rapa (1.0 µm). 0 min was regarded as 100 (A control ), the fluorescence intensity of sample (A sample ) and control (A control ) were measured, respectively. Fluorescence intensity (%) was equal to A sample /A control 100.

28 Figure S27. CLSM images of MCF-7 cells in the absence of Rapa. MCF-7 cells were treated with DPBP (100 µg/ml) for 1 h, and then detected by CLSM.

29 Figure S28. CLSM images of starvation-induced cells. a) MCF-7 cells were incubated with EBSS for 0, 2, 4, 6, 12, 24 h in the presence or absence of NAC, followed by an incubation with DPBP (100 µg/ml) for 2 h. b) Quantitative analysis of fluorescence intensity: PBS treated group was regarded as 100 (A control ).

30 Figure S29. ATG4 activity detection in BECN1 knocks down cells. a) Western blot on BECN1 expression in MCF-7 cells. The corresponding results of semi-quantitative analysis are shown b). MCF-7 cells were treated with 10 μl of PBS in regular culture medium (lane 1); 10 μl Rapa (1 μm) (lane 2); transfection of a sirna against BECN1 (lane 3); transfection of a control sirna (lane 4). c) CLSM images of DPBP in BECN1 knock down cells, and followed by DPBP (100 µg/ml) for 2 h.

31 Figure S30. The measurement autophagic flux by LC3 immunofluorescence and western blot method. a) CLSM images of LC3 immunofluorescence. Green signal was from anti-lc3 green-labeled secondary antibody. b) Western blot on LC3 and P62 expressions in MCF-7 cells that were treated with Baf (100 nm) or mix of Baf (100 nm) and Rapa (1 µm) for 1 h.