Supplementary Materials
Supplementary FigureS1 Propionate and butyrate are cytotoxic to colon cancer cells.
The cytotoxic effects of propionate and butyrate on HCT116 and SW480 cells were examined by an
MTS assay 48 hours after indicated treatment. Propionate (C3); Butyrate (C4). The bars represent the
mean ± SE (n=4). SFM, serum free medium.
Supplementary FigureS2 Induction of autophagy by propionate in HCT116 and SW480 cells.
HCT116 cells with GFP-LC3 expression were treated with propionate (3mM) in the presence or
absence of CQ (5uM) for 48 hours. The cells were stained with LAMP-2 antibody (a) or P62 antibody
(b). Representative images were obtained by fluorescence microscopy. Magnification, X600. The
LAMP-2 expression levels were also determined by western blot (c). (d) A bulk population of SW480
cells with stable GFP-LC3 expression was treated with propionate for 36 hours or was starved within
serum free medium (positive control) for 24 hours and then visualized under fluorescence microscopy.
Cell culture medium was changed daily. Formation of acidic vesicular organelles was monitored by
LysoTracker DND-99 and MDC staining. Pictures are all taken at 400X. (e) Quantification of the
percentage of cells with more than 8 GFP-LC3 puncta per cell is represented as “GFP-LC3vac cells
(%)”. GFP-LC3 expressing cells were treated with the indicated concentrations of propionate for 36
hours. A minimum of 100 GFP-LC3 expressing cells were counted ** P<0.001, compared with a PBS
control at the corresponsding time. (f) Increased acidic vesicular organelle formation was reflected by
elevated GFP fluorescence and LysoTracker staining. The cells were stained with the dye and
subjected to flow cytometry analysis as described in “Materials and Methods”. The bars represent the
mean ± SE (n=4). (g) SW480 cells with stable GFP-LC3 expression were subjected to propionate
(3mM) treatment in the presence or absence of CQ (5uM) for the indicated times. Cell lysates were
immunoblotted against several autophagy markers.
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Supplementary FigureS3 Propionate induced autophagy activation is associated with mTOR
pathway inhibition.
SW480 cells were treated with propionate (3mM) for the indicated times. The phosphorylation status
of mTOR, and its major downstream substrates, 4E-BP1 and p70S6 Kinase, was detected by Western
blot. As for 4E-BP1 phosphorylation,  stands for the hyper-phosphorylated form of 4E-BP1, whereas
 and  stand for medium- and low-phosphorylated forms of 4E-BP1. The data represent as least three
independent experiments.
Supplementary FigureS4 mTOR pathway inhibition-induced autophagy is associated with AMPK
pathway activation.
SW480 cells were treated with propionate (3mM) for the indicated times. Immunoblotting analysis
revealed that the AMPKa phosphorylation status at Thr172 of α subunit was significantly elevated
following propionate treatment.
Supplementary FigureS5 Reduction of cellular ATP level during propionate treatment.
(a) The chromatography of AMP, ADP and ATP from standard mixture and cell samples. One single
run took 55 min which was sufficient to elute all detectable compounds. Retention times were AMP
10.4 min, ADP 18.5 min, and ATP 28.9 min. Detection limits were: AMP: 0.63 ng; ADP:0.32ng;
ATP:2.54 ng. Detection of quantitation: AMP: 2.11 ng; ADP:1.08 ng; ATP: 8.45 ng. (b) SW480 cells
were seeded into 96 well plates and treated with 3mM propionate for the indicated times. The
intracellular ATP level was measured by a CellTiter-Glo® Luminescent Cell Viability Assay kit as
described in “Materials and Methods”. The bars represent the mean ± SE (n=4). (c) HCT116 cells with
GFP-LC3 expression were treated with propionate (3mM) in the presence or absence of N-
2
acetylcysteine (NAC, 1mM) or cyclosporine A (CsA, 2 M) for 36 hours. Several autophagy markers
were examined by Western blot.
Supplementary FigureS6 Inhibition of autophagy potentiates propionate-induced apoptotic cell
death.
(a) HCT116 cells were infected with ATG7 shRNAs or scramble shRNA and the ATG7 level was
examined by western blot. The cells were subjected to propionate (3mM) for 36 hours and several
autophagy markers were determined by western blot (b). The cell viability was measured by trypan
blue exclusion based cell staining (ci) and apoptosis related caspases activation was also studied (cii).
(d) Knockdown of AMPK in HCT116 cells was achieved by infecting the cells with AMPKshRNA
and followed by doxycycline (Dox, 500nM) treatment. The cells were treated with propionate (3mM)
for 48 hours and the cell viability was measured by trypan blue exclusion based cell staining. SW480
cells pretreated with 3-MA (2mM) or infected with ATG5 shRNA were subjected to propionate (3mM)
for the indicated times. The cell viability was measured by trypan blue exclusion based cell staining
(e), and the apoptotic cell death was evaluated by phosphatidylserine (PS) based annexin V staining
(f). (g) Western blot analyses of caspase-7 and caspase-3 cleavage in SW480 cells treated with
propionate (3mM) for the indicated times in the absence or presence of autophagy inhibitors (3-MA
and CQ) or AMPK inhibitor (Compound C).
Supplementary FigureS7 Increased depletion of cellular ATP in HCT116 cells infected with ATG5
shRNA during propionate treatment.
HCT116 cells infected with ATG5 shRNA were treated with propionate (3mM) for the indicated times.
The intracellular ATP level was measured by CellTiter-Glo® Luminescent Cell Viability Assay as
described in “Materials and Methods”. The bars represent the mean ± SE (n=4).
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Supplementary FigureS8 Autophagy abrogation trigged ROS accumulation.
HCT116 cells were treated with propionate (3mM) in the presence or absence of CQ (5M) or 3MA
(2mM) for 36 hours. The cells were stained with H2DCFDA for ROS detection by flow cytometry
analysis (i). The proportion of cells with increased H2DCFDA and reduced MitoTracker Deep Red
staining was presented as bar graph (ii). The bars represent the mean ± SE (n=3).
Supplementary FigureS9 Propionate induced autophagy is independent of its HDAC inhibitor
activity.
HCT116 cells with GFP-LC3 expression were treated with Trichostatin A (TSA, 100nM) for the
indicated times. Several autophagy markers were examined by western blot.
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