35 mm glass bottom dishes

Cells were seeded at a density of 1 × 10⁵ in 35-mm glass bottom dishes (IWAKI, Newport, UK) and cultured overnight in RPMI medium. The cells were then fixed in 3% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 4 min, and blocked with 1% BSA for 20 min. Cells were incubated with anti-paxillin antibody (Ab) (BD Transduction Laboratories) overnight at 4°C, and then with a secondary antibody conjugated with AlexaFluor488 (Invitrogen) for 1 h at room temperature. F-actin was visualized by phalloidin conjugated with AlexaFluor594. GST-PAK-RBD was used as a probe to detect the active form of Rac, followed by incubation with an anti-GST antibody. Images were acquired using a confocal laser-scanning microscope (FV-300; Olympus, Tokyo, Japan).

SK-N-AS neuroblastoma (cat.no. 94092302) and HeLa cervical carcinoma (Cat. No. 93021013) cell lines were obtained from European Collection of Authenticated Cell Cultures (ECACC). Cells were cultured and frozen down to form a low passage working stock. Subsequent working stocks were used for no more than 10 passages. Working stocks were screened to ensure the absence of mycoplasma every 3 months using LookOut Mycoplasma PCR Detection Kit (Cat. No. D9307 Sigma, UK). For confocal fluorescence microscopy and immuno-cytochemistry, SK-N-AS and HeLa cells were plated on 35 mm glass-bottom dishes (Iwaki, Japan and Greiner, Germany) at 1x10⁵ cells per dish in 3 ml medium. HeLa cells were plated at 5x10⁴ cells per dish in 3 ml medium. 24 hr post-plating, the cells were transfected with the appropriate plasmid(s) using Fugene 6 (Boehringer Mannheim/Roche, Germany). The optimized ratio of DNA:Fugene 6 used for transfection of HeLa or SK-N-AS cells was 2 µg DNA with 4 µl Fugene 6 and 0.8 µg DNA with 1.2 µl Fugene 6 respectively.
For Co-IP assays, SK-N-AS cells were plated on 100 mm tissue culture dishes (Corning, USA) at 4.5x10⁶ cells per dish in 10 ml medium. For western blotting, semi-quantitative and quantitative PCR, HeLa and SK-N-AS cells were plated on 60 mm tissue culture dishes (Corning, USA) at 5x10⁵ and 1x10⁶ cells respectively per dish in 5 ml medium.

A549 cells were obtained from RIKEN Cell Bank (Tsukuba, Japan). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (low glucose)(DMEM, SIGMA-ALDRICH, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (Moregate BioTech, Bulimba, Australia), 1% penicillin and streptomycin, and 2% GlutaMax™ (GIBCO, Dublin, Ireland), and incubated in a humidified atmosphere of 5% CO₂ at 37 °C. For caspase-3/7 activity assays, cells were seeded at a density of 5 × 10³ cells/cm² in 35-mm glass bottom dishes (IWAKI, Japan) and treated with various concentrations of apigenin. For extracting total RNA, cells were seeded at a density of 1 × 10⁵ cells/well in 6-well plates and were treated with 50 μM apigenin or dimethyl sulfoxide (DMSO, as control) for 48 h. We chose this concentration (50 μM) and exposure time (48 h) based on the 50% lethal dose.

Internalization and distribution of LM nanocapsules in cancer cells were analysed
using confocal laser microscopy (LSM 5 PASCAL; Carl Zeiss, Oberkochen, Germany).
HeLa cells were pre-seeded in 35 mm glass-bottom dishes (Iwaki,
Tokyo, Japan) and incubated for 24 h before the experiment, washed
with phosphate-buffered saline (PBS; Gibco), and incubated with fresh RPMI
containing 100 μg ml⁻¹ LM
nanocapsules for 24 h. Cells were then washed twice with PBS and
mounted in fresh PBS for observation.

Real-time monitoring of cancer cell elimination using an optical microscope
equipped with a laser irradiation set-up was performed as follows. HeLa cells
were pre-seeded in 35-mm glass-bottom dishes (Iwaki) for 24 h, then
washed with PBS, and incubated in fresh medium containing
200 μg ml⁻¹ LM
nanocapsules for 24 h. Subsequently, cells were washed twice with PBS
and mounted in fresh PBS for observation. The same laser irradiation set-up as
transformation of LM in microfluidic devices was used for this experiments.
To assess cell viability with the WST-1 assay, HeLa cells were pre-seeded in
96-well plates at 3 × 10⁴ cells
well⁻¹ for 24 h. Cells were treated with
samples (DSPE-PEG₂₀₀₀-Amine-DC(8,9)PC-LM encapsulating carmofur,
DSPE-PEG₂₀₀₀-Amine-DC(8,9)PC-LM without carmofur or
DSPE-PEG₂₀₀₀-Amine-DC(8,9)PC encapsulating carmofur), irradiated
with a fibre-coupled CW laser at 785 nm for 3 min at
maximum power (1 W,
∼80 mW mm⁻²), and
incubated for 24 h. The following day, cells were washed with fresh
RPMI, and viability was analysed using the WST-1 assay. Concentrations of LM and
carmofur were adjusted to 200 and
250 μg ml⁻¹,
respectively. Statistical analyses were performed using an analysis of variance
with Tukey's test (ANOVA), and a P value of less than 0.05 was
considered significant.