Supplemental Methods
Measurement of ROS production by DCFDA oxidation. Effect of reduced mtDNA
content on reactive free radicals (ROS, RNS) production was measured by DCFDA
oxidation in a Spectrofluorimeter. Parental, mtDNA-reduced and reverted cells (5x104)
were incubated with 1µM DCFDA in PBS for 20min and fluorescence was measured at
Ex 488nm, Em 525nm. To verify the mitochondrial origin of the free radicals produced,
in parallel experiments cells were treated with either MitoQ (1µM) or MitoCP (1µM) 3h
before DCFDA addition.
Galactose Sensitivity Assay. Cells (1x 104) were seeded in 12-well plates in glucose-free
medium supplemented with 0.9 mg/ml galactose, 0.5mg/ml pyruvate and 10% dialyzed
FBS. Fresh galactose containing (glucose-free) medium was replaced every alternate day.
Cells grown in regular medium with glucose was used for comparison for each cell type.
Viable cells were counted using Guava Viacount at indicated time points (Suppl figure
S9).
Culturing and Immunostaining of three dimensional MCF10A mammospheres.
Three dimensional cultures of MCF10A cells were grown as described previously (1). In
brief, MCF-10A cells were suspended in DMEM/F-12 (1:1) medium supplemented with
2% FBS, 10 μg/ml insulin, 1 ng/ml cholera toxin, 100 μg/ml hydrocortisone, 50 U/ml
penicillin, 50 μg/ml streptomycin, and 5 ng/ml EGF. Individual wells of eight-well
chamber slides (Falcon; BD Biosciences Discovery Labware, Bedford, MA) were coated
with 50 μl of Matrigel, and 5000 cells were seeded per well in assay medium
supplemented with 2% Matrigel. Fresh medium containing 2% Matrigel was added every
3 days. For mtDNA reduced-MCF10A cells, the medium was replaced with assay
medium supplemented with 2% Matrigel and 50 ng/ml ethidium bromide in the presence
of 1 mM sodium pyruvate and 50 μg/ml uridine. Structures were stained and subject to
confocal analysis as described below.
The mammosperes were fixed with 4% Formalin for 20 min at room temperature. Fixed
structures were washed with phosphate-buffered saline (PBS)-glycine (130 mM NaCl, 7
1
mM Na2HPO4, and 100 mM glycine) three times for 10 min each. The structures were
blocked using IF buffer (130 mM NaCl, 7 mM Na2PO4, 7.7 mM NaN3, 0.1% bovine
serum, 0.2% Triton X-100, and 0.05% Tween 20) plus 2% goat serum for 1.5 h.
Structures were then incubated with a secondary blocking buffer (IF buffer supplemented
with 10% goat serum and 20 μg of goat anti-mouse F(ab′)/ml) for 1 h. Structures were
incubated with Texas-Red conjugated Phalloidin (Invitrogen) diluted in secondary
blocking buffer overnight at 4°C. After three 20-min washes with IF buffer, structures
were incubated with Alexa Fluor conjugated anti-rabbit secondary antibody (Invitrogen)
diluted in IF buffer and supplemented with 10% goat serum for 1 h at room temperature.
After incubation with secondary antibody, structures were washed three times with IF
buffer then incubated with 0.5 ng/ml 4′,6′-diamidino-2-phenylindole (DAPI) for 10 min
at room temperature. Structures were washed again for 5 min with IF buffer before
mounting with the antifade agent Prolong (Invitrogen). Z-stack images of the 3D
spheroids were captured using an upright Leica 2-photon confocal microscope using 40x
magnification (Leica Microsystems, Deerfield, IL). Images analysis was done using the
imaging software Velocity (Leica Microsystems). Bright field images of the spheroids
were captured using an inverted NIKON Eclipse TE300 microscope using 20x
magnification and processed using Metamorph analysis software. Images were converted
as Tiff and size adjustments were done using Adobe Photoshop.
Gene manipulation studies. For silencing Calcineurin Aα, predesigned siRNAs for
human CnAα (ID:5530) and negative control (scrambled siRNA, ID:SR30004) were
purchased from Origene Technologies. Three different Calcineurin Aα siRNAs were
screened in initial experiments. Cells (104cells/well) were transfected using SiTRAN
(Origene Technologies) with pre-annealed double-stranded siRNAs at a final
concentration of 10 nm. Total RNA was isolated 48 h after transfections using TRIzol
reagent (Invitrogen), and the level of silencing of CnAα was determined by real-time
PCR using human CnAα specific primers.
For Tfam silencing experiments, human Tfam and Gfp shRNAs cloned in pLKO.1
lentiviral vectors were used. Five independent Tfam shRNA constructs were used for the
initial screening experiments. Cells were transduced using 48h and 72h viral titre and
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cells expressing shRNA constructs were selected by screening for puromycin (2.0μg/ml)
resistance. Cells expressing Tfam shRNA were maintained in medium with added uridine
(50 μg/ml) and sodium pyruvate (1mM).
For ESRP1 overexpression (rescue) experiments, MCF10A and MCF7 cells with reduced
mtDNA were transduced with either pMXS-IRES- Puro- EGFP empty vector or pMXSIRES-Puro-Emerald-2xFLAG-ESRP1. ESRP1 overexpression was confirmed by nuclear
localization of GFP.
Ex Vivo Pulmonary Metastasis Assay (PuMA). Ex-Vivo PuMA was done as described
before (2). Healthy parental, mtDNA-reduced and reverted MCF10A or MCF7 cells (2 ×
105) were delivered by tail-vein injection to SCID/beige mice (Charles Rivers). Fifteen
minutes after the injection, the mice were euthanized by CO2 inhalation. The sternum was
exposed and the lung was infused with 1.2 ml of well-mixed culture medium 1 % agarose
solution (40°C). The lungs were then carefully removed and immediately placed in a cold
solution of PBS containing 100 U/ml penicillin and 100 μg/ml streptomycin at 4°C for 20
minutes to solidify the agarose/medium solution. Sections (1–2 mm in thickness) were
made from each lobe yielding ~15 lung sections. Five lung sections were placed on a
single 1.5 × 0.7-cm sterile Gelfoam (Pfizer-Pharmacia & Upjohn Co.) section that had
been pre-incubated for 2 hours with culture medium. Lung sections were incubated at
37°C in humidified conditions of 5% CO2. Fresh culture medium was replaced and lung
tissue sections were turned over with a sterile iris thumb forceps on alternate days.
Formalin-fixed, paraffin-embedded mouse lung tissue sections were prepared at 5 μm on
a transverse plain. Sections were stained with H&E and mounted on slides for imaging.
Immunofluorescence and Imaging. Mitochondrial morphology in all cell types was
analyzed by staining cells with antibody against mitochondrial protein Cytochrome
Oxidase (CcOIVi1). Immunostaining for CD24, CD44 was done using respective primary
antibodies (BD biosciences) and Alexa Fluor conjugated secondary antibodies. Cells
were imaged using a Leica SP5 confocal microscope under 40x magnification and images
were processed using Volocity software. Bright field images for cellular morphology
were captured using Nikon E600 upright fluorescence microscope.
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Real-Time PCR. Real Time PCR was carried out using 100ng cDNA, reverse
transcribed from 1 µg RNA using the High Capacity cDNA Reverse Transcription kit
(Applied Biosystems) as described before (3). Gapdh was used as an internal control for
normalization. Target gene expression levels are represented as relative quantification
compared to their parental levels.
Legend for the Movies
Time lapse recordings for “scratch wound healing” migration assay: Parental (Video
1), mtDNA-reduced (Video 2) and reverted (Video 3) MCF10A cells were grown to
confluence and wounded with a pipette tip and wound healing as a measure of cell
motility was followed over 20 hours.
Supplemental References
(1) Haenssen KK, Caldwell SA, Shahriari KS, Jackson SR, Whelan KA, Klein-Szanto
AJ, et al. ErbB2 requires integrin alpha5 for anoikis resistance via Src regulation of
receptor activity in human mammary epithelial cells. J Cell Sci 2010;123:1373-82.
(2) Mendoza A, Hong SH, Osborne T, Khan MA, Campbell K, Briggs J, et al.
Modeling metastasis biology and therapy in real time in the mouse lung. J Clin
Invest 2010;120:2979-88.
(3) Guha M, Pan H, Fang JK, Avadhani NG. Heterogeneous nuclear ribonucleoprotein
A2 is a common transcriptional coactivator in the nuclear transcription response to
mitochondrial respiratory stress. Mol Biol Cell 2009;20:4107-19.
Supplemental Figure Legends
Figure S1. Effects of reduced mtDNA copy number on mitochondrial respiration
and ROS production. (A) Real Time PCR showing the relative mtDNA content between
MCF10A and MCF7 cells. Gapdh gene was used as an internal control. (B) Real time
PCR amplification curve of nuclear gene GAPDH in parental and mtDNA-reduced
MCF10A (top) and MCF7(bottom) cells. (C) Maximum respiratory capacity of parental,
mtDNA reduced and reverted MCF10A cells. Cells (50,000) were seeded in XF24
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seahorse plate overnight. Oxygen consumption rates were measured under basal
condition and after addition of 2µg/ml oligomycin and 75µM DNP as described in Figure
1. (D) Extracellular acidification rate (ECAR), an indicator of glycolysis was measured
by the Seahorse XF24 analyzer in the presence of glucose (10mM), oligomycin (2µg/ml)
and 2- deoxyglucose (100mM) respectively.
Figure S2 (A) Transmembrane potential was measured in parental and mtDNA-reduced
(either by EtBr or Tfam-silenced) cells using TRME dye as described in Materials and
Methods. (B) ROS production in mtDNA reduced MCF7 and MCF10A cells: Relative
DCF fluorescence in control, mtDNA reduced and reverted cells incubated with 1µM
DCFDA. MitoQ (1µM) or MitoCP (1µM) was added 3h before DCFDA addition.
Figure S3. Altered cytoskeletal organization in mtDNA reduced- MCF10A cells.
Texas Red-Phalloidin and DAPI stained image of three dimensional spheroids grown in
matrigel formed by parental and mtDNA-reduced MCF10A cells imaged using 2 photon
confocal microscope.
Figure S4. Self Renewal capacity of cells in response to reduced mtDNA content. (A)
Cell Viability of floater cell populations in parental, mtDNA-reduced and reverted MCF7
cells assessed by annexin staining and quantitated using a Guava personal cytometer. (B)
Sphere-forming capacity of the MCF7 floater populations were analyzed by counting the
spheres formed by equal number of viable cells. (C) Representative immunoblot pattern
showing CD44 and CD24 levels in parental, mtDNA reduced and reverted MCF10A
cells. (Di, Ei) Flow cytometry plot showing the distribution of CD44+/CD24-,
CD44+/CD24+, CD44-/CD24+ and CD44-/CD24- expressing cells. Unstained cells were
used to set the gating. (Dii, Eii) Quantitative representation of the FACS analysis in Di
and Fi showing the percentage of cells expressing CD44 and CD24 cells.
Figure S5. (A) H&E staining of ex vivo lung sections showing invading parental,
mtDNA-reduced and reverted MCF10A epithelial cells (marked by arrows) after 7 days.
(B) H&E staining of lung sections (PuMA) at Day 0 showing intact lung architecture
5
mouse lung epithelial cells (indicated by arrows) lining the bronchioles. Images were
taken at 10x magnification.
Figure S6. (A) Western Immunoblot showing calcineurin Aα levels in total cell extracts
(50µg protein) from parental, mtDNA-reduced and reverted MCF7 cells. The immnublot
was reprobed with βactin to confirm equal protein loading. (B) Immunofluorescence
images (40x magnification) of parental, mtDNA-reduced and reverted MCF10A cells
stained with vimentin. (C) Bright field images showing fibroblast like morphological
alterations in mtDNA-depleted A549 cells. (D) Real time PCR analysis showing
increased expression of EMT marker genes in mtDNA-reduced A549 cells.
Figure S7. (A, B) Real time PCR analysis showing the relative mRNA levels of
epithelial and mesenchymal marker genes involved in EMT MCF10A and MCF 7 cells
with varying amounts of decrease in mtDNA content (as indicated) relative to parental
cells.
Figure S8. (A and B) Fluorescent images (40x magnification) showing GFP expression
in mtDNA-reduced MCF10A and MCF7 cells transduced with either GFP-empty vector
or GFP-ESRP1. (C) Reverse transcribed, PCR amplification products showing Alternated
Splicing patterns of ESRP target genes involved in tumor progression and EMT in
mtDNA reduced MCF10A cells compared to parental cells.
Figure S9. Galactose Sensitivity in Tfam silenced mtDNA-reduced cells. Viable cells
cultured in glucose-free, galactose containing medium indicating galactose sensitivity
(tolerance) in parental and TFAM-silenced MCF10A (A) and MCF7 (B) cells.
6