Supporting Information

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1 Copyright WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 212. Supporting Information for Adv. Funct. Mater., DOI:.2/adfm MnO Nanocrystals: A Platform for Integration of MRI and Genuine Autophagy Induction for Chemotherapy Yang Lu, Li Zhang, Jing Li, Yu-De Su, Yun Liu, Yun-Jun Xu, Liang Dong, Huai-Ling Gao, Jun Lin, Na Man, Peng-Fei Wei, Wei-Ping Xu, Shu-Hong Yu,* and Long-Ping Wen*

2 Supporting Information for DOI:.2/adfm Figure S1. MnO NCs specifically induced autophagy in comparison with magnetite and dextran and elicited a time and dose-dependent autophagy in the presence of CQ. a, Equal dose ( µg/ml) of MnO nanocrystals induced autophagy displayed specificity in comparison with magnetite and dextran (which are major components of Feridex). b-c, HeLa cells were treated with MnO NCs for indicated hours (b) and doses (c) in the presence of µm Chloroquine. GAPDH served as loading control.(*) indicates non-specific bands. Figure S2. MnO NCs induced autophagy through the lysosomal stage in various cell lines. a, HepG2 and HaCat cells were either treated with PBS (Cont) or Trehalose( mm) and MnO NCs ( µg/ml) for 18 hours in the presence or absence of µm Chloroquine (CQ). b, HaCat cells were either treated with PBS (Cont), MnO NCs ( µg/ml) or PVP (1 µg/ml) for 18 hours and then subject to Western blotting with anti-lc3 antibody. c, COS-7 cells were either treated with PBS, Trehalose or MnO NCs in the presence or absence of CQ. (*) indicates non-specific bands. S1

3 Figure S3. MnO NCs induced p3-independent autophagy in p3 -/- HCT116 cell line. p3 knockout (p3 -/- ) HCT116 cells were either treated with PBS or MnO NCs for 12 hours. a 2 1 ** Control 3-MA MnO NCs MnO NCs +3-MA c Control Wortmannin MnO NCs * MnO NCs +Wortmannin d Control 3-MA ** MnO NCs MnO NCs+3-MA Figure S4. MnO NCs induced cell viability reduction was rescued by autophagy inhibitors in HeLa and HepG2 cells. a, MTT assay of HeLa cells after 24 h treatment with PBS, MnO NCs ( µg/ml), MnO NCs plus 3-MA (2. mm) and 3-MA only. b, HeLa cells were either treated with PBS (Cont) or Wortmannin (. µm), MnO NCs and MnO NCs plus Wortmannin. c, MTT assay of HeLa cells after treatment with PBS, MnO NCs, MnO NCs plus Wortmannin or Wortmannin alone. d, MTT assay of HepG2 cells after treatment with PBS, MnO NCs, MnO NCs plus 3-MA or 3-MA only. (In all the experiments, mean ± s.e.m., n=, *p <., **p <.1, compared to control of each group, data are representative of three independent experiments with similar results). S2

4 Figure S. Dox at a relatively high dose caused apoptosis in a caspase-mediated manner. HeLa cells were treated with Dox (2 µg/ml) for 12 h in the presence or absence of 2 µm z-vad-fmk and then subject to immunoblotting with anti-cleaved caspase 3 antibody. Figure S6. Combined treatment of MnO NCs and nonocytotoxic dose of Dox resulted in significant cell death, whereas 3-MA or z-vad-fmk attenuated it. HeLa cells were treated with Dox (.7µg/mL), MnO NCs (µg/ml) alone, MnO NCs plus Dox or MnO NCs-Dox co-treated with 3-MA (2.mM) or z-vad-fmk (2 µm) for 24 h. Cell death was assessed by Hoechst 33342/PI staining and indicated as the PI-positive staining cells, representative fluorescence images were shown. Extra Experimental Methods Associated with the Supporting Data: Cell viability assay. Cell viability assays were performed by using MTT. Briefly, cells were collected by trypsinization, counted, and plated at a density of approximately cells per well in 96-well plates. After 24 h incubation, different materials with various concentrations in complete DMEM ( µl) with serum were applied to replace the culture medium and the cells were further cultured for another 24 h. Then µl MTT stock solution ( mg/ml in PBS, ph 7.4) was added to each well to achieve a final concentration at. mg/ml. Cells were then incubated at S3

5 37 C for additional 4 h. After removing the growth medium, the resultant formazan was dissolved in 1 µl DMSO, and the optical density of the solution was measured at 7 nm using ELx8 Absorbance Microplate Reader (BioTek Instruments, USA). The cell viability was normalized to that of cells cultured in DMEM treated with PBS. S4