Supplementary Material
1. Evaluation of organelles purity, recovery and integrity using western blot analysis
For identification of isolated fractions, verification of fraction purity,
quantification of organelle recovery and evaluation of organelle integrity, the resulting
samples were examined by western blot analysis. The organelle-specific markers
employed were as follows: LAMP-1 (acidic organelles) and VDAC1 (mitochondria).
As one of the most powerful tools for preparing membrane proteins [1]. Triton X-114
was applied to extract LAMP-1 and VDAC1 proteins.
The extraction of membrane proteins in Triton X-114 has been described
previously [1]. Briefly, the obtained mitochondria and acidic organelles after isolation
were resuspended in 500 µl 1% Triton X-114 extraction buffer (1 mM DTT, 2 mM
EDTA in 50 mM Tris/HCl, pH 7.4). After an incubation period of 30 min on ice and
10 min at 37°C the samples were spanned at 10000 × g for 3 min to separate the
detergent and aqueous phases. To achieve a complete extraction, 500 µl 1% Triton
X-114 extraction buffer and 500 µl 0.06% Triton X-114 wash buffer (1 mM DTT, 2
mM EDTA in 50 mM Tris/HCl, pH 7.4) were added to the aqueous phase and the
detergent phase, respectively. The incubation and centrifugation steps were repeated.
The detergent phases from the consecutive extractions were pooled and the proteins
were precipitated in cold acetone. Afterward, all protein pellets were dissolved in 1%
SDS solution (Generay Biotech Co., Ltd, Shanghai, China).
Equivalent amounts of samples were separated by 12% sodium dodecyl sulfate
polyacrylamide
gel
electrophoresis
(SDS-PAGE)
and
transferred
onto
a
polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). The
membranes were incubated with mouse anti-human VDAC1 antibody (1:2000;
Abcam, Cambridge, UK) or mouse anti-human LAMP-1 antibody (1:1000; Abcam,
Cambridge, UK) followed by HRP-conjugated goat anti-mouse IgG (1:5000;
MultiSciences Biotech. Co., Ltd., Hangzhou, China). The proteins were then detected
using enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA) according to
the manufacturer’s protocol.
The integrity of the purified acidic organelles and mitochondria was additionally
assessed by estimation of the amount of cathepsin D and cytochrome C translocated
to the cytoplasmic fraction. After the separation of cytoplasmic fraction and organelle
samples by SDS-PAGE, the membranes were exposed overnight at 4ºC to a
monoclonal mouse anti-human cathepsin D antibody (1:1000; Abcam, Cambridge,
UK) and a monoclonal mouse anti-human cytochrome C antibody (1:1000; Abcam,
Cambridge, UK), respectively. The visualization of proteins was performed as
described above.
2. Fluorescence Microscopy
MCF-7/ADR cells were incubated in 10 cm dish and exposed to 5 μM DOX or
TRF-DOX for 24 h at 37°C in cell culture media without fetal calf serum. Then the
cells were washed with PBS, prior to an addition of 4 ml of fresh PBS. Finally, the
cells were imaged using a Leica microscope fitted with a Halogen display lamp
(OSRAM, Munich, BRD). The combination of BP560/40 excitation filter, 595
dichroic mirror, and BP 645/75 suppression filter was used for imaging. The
fluorescence was detected by a DFC450C CCD digital camera (Leica Microsystems
Wetzlar GmbH, Germany) and images were acquired and analyzed using software
LAS 4.2 (Leica Microsystems Wetzlar GmbH, Germany).
References
[1] Pryde JG, Partitioning of Proteins in Triton X-114. New Jersey: Humana Press Inc., 1998.