Supporting Information for Mechanical Stress-Dependent Autophagy Components Release via Extracellular Nanovesicles in Tumor Cells Kaizhe Wang,, Yuhui Wei,, Wenjing Liu,, Lin Liu,, Zhen Guo,, Chunhai Fan,, Lihua Wang,,,* Jun Hu,,,* and Bin Li,,* Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China University of Chinese Academy of Sciences, Beijing 100049, China Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China School of Chemistry and Chemical Engineering, and Institute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China *Corresponding authors. E-mail addresses: libin@sinap.ac.cn (Bin Li); hujun@sinap.ac.cn (Jun Hu); wanglihua@sinap.ac.cn (Lihua Wang). This file includes: Figure S1: ASS induced autophagy through an mtor independent pathway in tumor cells. Figure S2: Press stress induced autophagy and promoted extracellular vesicles release in tumor cells.
Figure S3: ASS promoted autophagy components release via extracellular nanovesicles under suspension condition in tumor cells. Figure S4: Blocking EVs release induced AP-MVB compounds accumulation and increased apoptosis via the caspase3/parp pathway under shear stress. Figure S5: Autophagy-associated proteins released via EVs in LC3 over-expressed HeLa was mechanical stress-dependent. Figure S6: Acute shear stress induced Ca 2+ influx in tumor cells. Figure S1. ASS induced autophagy through an mtor independent pathway in tumor cells. (A-D) Western blot analysis of mtor and P70S6K phosphorylation in HeLa and MDA-MB-231 cells after exposed to shear stress (10 dyn/cm 2 ) or treated with rapamycin (1 μm) for 30 min. Data were represented as the mean ± SD from three independent experiments. *, **, and *** denote p < 0.05 p < 0.01 and p < 0.001 in comparison to control, respectively. Figure S2. Press stress induced autophagy and promoted extracellular vesicles
release in tumor cells. A LC3 I/II levels of cells compressed under 0, 0.12, 0.23 and 0.33 kpa for 30 min. Cells were lysed and LC3 levels detected by western blot. B EVs were isolated from serum-free cultural supernatants after compressed and the concentration of EVs were obtained by NTA. Data were represented as the mean ± SD from four independent experiments. * and ** denote p < 0.05 and p < 0.01 in comparison to control, respectively. Figure S3. ASS promoted autophagy components release via extracellular nanovesicles under suspension condition in tumor cells. (A)HeLa and MDA-MB- 231 cells were culture in suspension condition and then exposed to shear stress for 60 min. Whole cell lysates were collected and protein levels of LC3 were analyzed by western blot. GAPDH was used as a loading control. (B) LC3 and LAMP1 in EVs were determined by western blot.
Figure S4. Blocking EVs release induced AP-MVB compounds accumulation and increased apoptosis via the caspase3/parp pathway under shear stress. (A) Blocking EVs release induced AP-MVB accumulation and then increased apoptosis via the caspase3/parp pathway. (B) EGFP-LC3 HeLa cells were pretreated with GW4869 (10 μm) (an inhibitor of neutral sphingomyelinase 2 which inhibit exosomes release) for 4 h and then exposed to ASS (10 dyn/cm 2 ) for 60 min. Immunofluorescence of MVB marker CD63 (red) was performed; scale bar: 20 µm. (C-D) The number of LC3II -positive puncta puncta and yellow puncta (Co-localization of LC3II and CD63) were quantified. Data were
represented as the mean ± SEM. (E) HeLa cells were pretreated with GW4869 (10 μm) or Bafilomycin A1 (100 nm) for 4 h and then exposed to ASS (10 dyn/cm2) for 60 min, the level of cleaved PARP and cleaved caspase3 were detected by western blot, GAPDH was used as a loading control. Figure S5. Autophagy-associated proteins released via EVs in LC3 over-expressed HeLa was mechanical stress-dependent. (B) Wild type HeLa cells were stained with anti-lc3 antibody. The number of LC3 puncta in wild type and LC3 overexpressed HeLa (EGFP-LC3 HeLa) cells was quantified. Scale bar 10 μm. Data were represented as the mean ± SEM. (C) LC3 over express HeLa cells (EGFP- LC3) were exposed to ASS (10 dyn/cm 2 ) for 60 min. EVs were isolated form serumfree culture. LC3 and LAMP1 in EVs were detected by western blot (D). Data were represented as mean ± SD. * and ** denote p < 0.05 and p < 0.01 in comparison to control, respectively.
Figure S6. Acute shear stress induced Ca 2+ influx in tumor cells. HeLa cells were exposed to acute shear stress (10 dyn/cm 2 ) for 0, 5, 15 min and loaded with 1 μm Fluo4-AM. Cells were immediately analyzed by fluorescence microscopy, scale bar 30 μm.