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Legend for supplementary figure
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Figure S1. Pyrvinium reduces the total LC3 protein levels. HeLa cells were
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pre-treated with sicontrol or siAtg7 for 24 hr and then administrated with pyrvinium
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(200 nM) and vehicle control (1‰ DMSO) for another 24 hr. Cell lysates were
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analyzed by western blotting using the indicated antibodies. Actin served as a loading
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control.
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Figure S2. Pyrvinium decreases autophagic flux. (a) GFP-LC3-expressing HeLa and
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HEK293 cells were treated with pyrvinium (200 nM) and vehicle control (1‰ DMSO)
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in the presence or absence of E64 (10 µg/ml) or pepstatin A (PEPS A, 10 µg/ml) for
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24 hr and the cell lysates were analyzed by western blotting. (b) GFP-LC3-expressing
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HeLa cells in response to etoposide alone, or together with pyrvinium (200 nM) in the
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presence or absence of NH4Cl (20 mM) as indicated for 24 hr and the cell lysates
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were analyzed by western blotting. Actin served as a loading control throughout. Band
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intensity was calculated using ImageJ software and the ratio of LC3-II/Actin
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expression was normalized and values were indicated.
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Figure S3. Pyrvinium is identified as a lipophilic cation and pyrvinium-regulated
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autophagy is AMPK/mTOR independent. (a) Fluorescence microscopy of HEK293
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cells incubated with 200 nM pyrvinium for 4 hr. (b) Confocal microscopy of HEK293
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cells incubated with 200 nM pyrvinium (red) for 4 hr, then fixed and immunostained
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for Lamp-1, LC3, Cytochrome c (cyt-c) or V-DAC or treated with MitoTracker to
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stain for mitochondria (green). (c) Fluorescence microscopy of HEK293 cells
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incubated with 200nM pyrvinium in the presence of FCCP or incubated in high K+
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buffer for 4 hr. Scale bars, 50 µm. (d) HeLa and HEK293 cells were treated with
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vehicle control (1‰ DMSO) and indicated concentration of pyrvinium for 24 hr and
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then cellular ATP levels were detected using ATP Assay Kit. The ATP level was
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expressed as a percentage of that of control cells. Values are the mean ± SD (n=3). (e,
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f) HeLa and HEK293 cells were treated with pyrvinium (200 nM) and vehicle control
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(1‰ DMSO) for 24 hr (e) or added with ATP to the medium at the last 4 hr (f) and the
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cell lysates were analyzed by western blotting using the indicated antibody. (g) HeLa
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cells were treated with pyrvinium (200 nM) and Compound C (10 mM) as indicated
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and the cell lysates were analyzed by western blotting. Actin was used as a loading
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control. Scale bars were presented as indicated. Error bars indicate STD.
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Figure S4. Pyrvinium inhibits the transcription of some autophagy genes. (a) HeLa
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cells were treated with pyrvinium (200 nM) and vehicle control (1‰ DMSO) for the
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indicated periods of time and the cell lysates were analyzed by western blotting. (b)
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Hela cells were treated with indicated concentration of pyrvinium for 24 hr and the
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mRNA expression of the LC3B, Atg5 or Atg7 was analyzed by Real-time PCR. All
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values were normalized to the mRNA level of Actin. (c) HeLa cells were treated with
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actinomycin (10 µM) and pyrvinium (200 nM) as indicated and the mRNA expression
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of the Beclin1 and LC3B were analyzed by Real-time PCR. All error bars indicated
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STD. (d) HeLa cells were transiently transfected with a pEGFP-N1 plasmid. 24 hr
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after transfection, pyrvinium (200 nM) was added and the Hela cells were incubated
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for another 24 hr. GFP expression was visualized by fluorescent microscopy or
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analyzed by immunoblot. (e) Hela cells were treated with indicated concentration of
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pyrvinium for 24 hr and the cell lysates were analyzed by western blotting using the
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indicated antibodies. Actin was used as a loading control.
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Figure S5. Pyrvinium can sensitize tumor cells to apoptosis under starvation
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condition. (a) HeLa and HCT116 cells were treated with vehicle control (1‰ DMSO)
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and indicated concentration of pyrvinium for 2 days, and representative morphology
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was imaged using a phase contrast microscope. (b) TUNEL assay was performed to
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measure the ratio of apoptotic cells. (C) HeLa and HCT116 cells were treated as in (a)
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under starvation condition for 2 days, and TUNEL assay was performed to measure
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the ratio of apoptotic cells. Percentage of apoptotic cells was shown. Results were
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means ± SD of 3 independent experiments. (d) Expression of Beclin1 in HeLa cells
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transfected with control siRNA or siRNA targeting Beclin1. Actin served as a loading
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control. Band intensity was calculated using ImageJ software and the ratio of
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Beclin1/Actin expression was normalized and values are indicated. (e) HeLa cells
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were treated with vehicle control, CQ, 3-MA or siBeclin1 as indicated under normal
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growth media or glucose-free media, and the cell viability was determined by MTT
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assay. All error bars indicated STD.
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Figure S6. Effect of pyrvinium and 2-DG, alone or in combination, on 4T1 cancer
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cells in vitro. (a-c) 4T1 cells were treated with vehicle control (1‰ DMSO) or
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pyrvinium (200 nM) for 24 hr in the presence or absence of starvation or 2-DG (10
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mM). (a) Cell viability was determined by MTT assay, (b) representative morphology
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imaged using a phase contrast microscope, (c) cell lysates were analyzed by western
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blotting. Actin served as a loading control. Error bars indicated STD.
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Figure S7. Toxicity assessment following combination chemotherapy in normal mice.
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(a) Immunohistochemical detection of cleaved caspase-3 in tumor specimens.
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Representative images were provided as indicated. Scale bars, 20 µm. (b) Total body
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weight was measured every three days during the study. (c-f) Weight of heart (c), lung
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(d), kidney (e) and liver (f) was measured in normal mice after treatment with
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pyrvinium and 2-DG, alone or in combination. Data were presented as mean ± s.e.m.
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(n= 6 mice in each group). (g) H&E-stained sections of liver, heart, lung and kidney
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samples. Scale bars, 50 µm.
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