Supplementary Material and Methods
RNA isolation, estimation and quality check
Before isolating RNA all plastic wares and reagents were made RNase free by treating them
with 0.1% DEPC for overnight at room temperature followed by removal of DEPC by
autoclaving. RNA was isolated from cells at appropriate time points by TriReagent reagent
(Sigma-Aldrich, Germany). The RNA was treated with DNAse (MBI Fermentas, USA) and
quantified by NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). After
calculating the amount of RNA its quality was also checked by gel electrophoresis and for
that 1 gm of agarose was melted completely in 72 ml DEPC treated MQ water and then
cooled to 600C. 10 ml of 10x MOPS Electrophoresis buffer and 18 ml of 37% formaldehyde
(12.3 M) were added and mixed properly. The gel was poured in a horizontal chamber with
the well forming comb preplaced and allowed to solidify for 30 minutes. RNA sample was
prepared with 10 µl of RNA Gel-loading Buffer was added to 1 µg of RNA solution.
Ethidium bromide was added at a final concentration of 10 µg/ml, volume was made to 20 µl
with DEPC treated MQ water and mixed properly. The RNA mix was then heat denatured at
700C for 10 minutes and quickly chilled on ice. The RNA samples were loaded to the gel
and electrophoresed in 1x MOPS Electrophoresis buffer at constant volt of 50-60V.
Flow cytometry
To analyse cell cycle profile of cells undergone transfection, flow cytometry has been done
using propidium iodide, which when incubated with cells, goes into the nucleus and
intercalate with the minor groove of double stranded DNA. Initially the flask containing
experimented cells incubated at 370C for desired time points. After the incubation period,
medium from each flask was collected into microfuge tubes and centrifuged at a high speed
of 1000xg for 10 minutes to pellet the apoptotic bodies. Adhered cells were washed with PBS
and trypsinized as described before. Cells were then detached from the flask’s surface with
the supernatant medium from the microfuge tubes by gentle tapping and resuspended
thoroughly in that medium. Cell suspension was centrifuged at 200xg for 10 minutes in the
same microfuge tubes with pelleted apoptotic bodies. The supernatant was then discarded.100
µl of PBS was added to each tube and the pellets were resuspended thoroughly to prevent cell
clumping. 1.5 ml of cold 70% Ethanol was added to the tubes drop by drop on a vortex mixer
and mixed immediately by inversion. Fixed cells were kept overnight at –200C. Fixed cells
were pelleted and washed with PBS the very next day. The cell pellet was resuspended by
tapping in residual solution and 100 µl of PBS was added to the tubes. 400 µl of 4 mM
Citrate buffer was added followed by 100 µl of PI solution per tube. RNAse A was added to
final concentration of 10 µg/ml. Tubes were incubated at 40C for 1 hour and samples were
acquired in Flow Cytometer. Fluorescence was acquired in a BD Flow Activated Cell Sorter
(FACS). Data was analyzed using WinMDI software. Each set of experiment was repeated
thrice and quality of machine has been checked before starting experiment.
DAPI (4′,6-Diamidino-2-phenylindole) Staining
On day 0, forty thousand cells were plated per well in 6 well plate. On day 1, transfection was
carried out with. DAPI staining was done after 72 hrs of transfection to assess DNA
fragmentation which is a marker for apoptotic induction in the cells.
Gelatin Zymography
U87MG cells (2.5X105cells/flask) were plated into 25cm2 flask. Transfection was performed
on day 1 and after 72hrs, the conditioned medium was collected, clarified by centrifugation,
concentrated by Centricon YM-30 (10:1 concentration; Millipore, Billerica, MA), and
separated in non-reducing polyacrylamide gels containing 0.1% (wt/vol) gelatin. The gel was
incubated with 1XZymogram renaturing buffer for 30min at room temperature, 1X
Zymogram developing buffer for 30min at room temperature, and 1X Zymogram developing
buffer at 37°C O/N. The gel was then stained for protein with 0.25%(w/v) Coomassie Blue
R-250 and then destained with Coomassie R-250 destaining solution (Metahnol:Aceticacid:Water,50:10:40). Proteolysis was detected as a white zone in a dark field.