Supplementary figure legends
Supplementary Figure 1. Starvation–induced autophagy is involved in
apoptosis in 7702 cells but not in HCC cell lines. (a) The 7702, HepG2, Hep3B
and Huh7 cell lines were serum-deprived for 48 hours. Flow cytometry was
used to detect the number of cells with sub-G1 DNA content. (b and c) An
immunoblot assay was used to detect the effect of NH4Cl on starvation
(sta)-induced autophagy and apoptosis in cells. (d) Flow cytometry was used
to detect the effect of Bafilomycin A1 (BafA 1) treatment on starvation
(sta)-induced changes in the number of cells with sub-G1 DNA content. The
data are presented as the mean ± SD of three independent experiments in (a)
and (b).
Supplementary Figure 2. Detection of cells with sub-G1 DNA content using
flow cytometry. (a and b) Hep3B (a) and Huh7 (b) cells were starved (sta) for
48 hours with or without pre-treatment by infection with rAd-p53. (c and d)
Hep3B (c) and Huh7 (d) cells were infected with rAd-p53 with or without
pre-transfection with DRAM siRNA (DRAM si) and were then starved for 48
hours. The data are presented as the mean ± SD of three independent
experiments in (a), (b), (c) and (d).
Supplementary Figure 3. The effect of inhibiting the PI3K/AKT pathway via
LY294002 (LY) treatment on the expression of starvation (sta)-induced p-PI3K,
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p-AKT, LC3 I/II, PARP, DRAM and p53 (a) or p73 (b and c) was determined via
immunoblot assay using the indicated antibodies.
Supplementary Figure 4. Co-localization (yellow) of DRAM (green) and the
mitochondrial protein HSP60 (red) was determined by confocal microscopy in
7702, HepG2, Hep3B and Huh7 cells with or without starvation (sta) treatment.
Control: no starvation. Magnification, ×1000.
Supplementary Figure 5. 7702, HepG2, Hep3B and Huh7 Cells were
transfected with GFP-LC3 plasmids and subsequently starved for 48 hours.
Co-localization (yellow) of GFP-LC3 puncta (green) and the mitochondrial
protein HSP60 (red) was determined by confocal microscopy. Magnification,
×1000.
Supplementary Figure 6. Inhibition of PI3K via LY294002 (LY) induces the
translocation of DRAM to mitochondria and mitophagy in HepG2, Hep3B and
Huh7 cells. (a) HepG2, Hep3B and Huh7 cells were starved (sta) for 48 hours
with or without pre-treatment with LY294002 (LY). The cytoplasm (C) and
mitochondria (M) were extracted using Percoll gradient centrifugation. The
levels of DRAM, LC3 I/II and p-AKT were evaluated by immunoblot assay.
Anti-VDAC and anti-β-actin antibodies were used as controls for the extracted
mitochondria and cytoplasm. (b) Co-localization (yellow) of DRAM (green) and
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the mitochondrial protein HSP60 (red) was assayed by confocal microscopy in
cells starved for 48 hours with LY pre-treatment. Magnification, ×1000. (c)
Cells were transfected with GFP-LC3 plasmids and subsequently treated by LY;
these cells were then starvation for 48 hours. Co-localization (yellow) of
GFP-LC3 puncta (green) and the mitochondrial protein HSP60 (red) was
assayed by confocal microscopy. Magnification, ×1000.
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