SUPPLEMENTARY METHODS
Electron microscopy. Neo and BI-1 cells were collected and prepared for transmission
electron microscopy (TEM) by fixation in a phosphate buffered solution (Sorensen's
phosphate, pH 5.8) of 2.5% glutaraldehyde with 0.15% sucrose and 2% mannitol to
maintain proper osmosis for 12-24 hours at 4 °C. Cells were post-fixed in 2% osmium
tetroxide in the same buffer for 1 hour. After rinsing, the cells were exposed to 2% uranyl
acetate (aqueous) and subsequently embedded in 2% agar. Samples cut from the agar
blocks were dehydrated in a graded ethanol series, infiltrated with Spurr's resin,
embedded, and cured at 70 °C for 24 hours. Ultra-thin sections were cut and stained with
uranyl acetate and Reynold's lead citrate prior to observation on a Zeiss EM10
transmission electron microscope.
Supplementary Figure 1 N-terminal tagged BI-1 cells show metastasis. pEF vector, BI1 and CΔBI-HA-tagged pEF vectors were transfected into B16F10 melanoma cells and
expression was confirmed (a; right). SCI mice received a single tail vein inoculation with
2.5 x 104 melanoma transfectants. Four weeks later, the lungs from the mice were
isolated and photographed (a; left). The number of lung nodules was quantified (b). The
data shown represents the mean ± S.E. (n = 15). *p < 0.05; significantly different from
pEF vector alone. pEF: polypeptide chain Elongation Factor, BI-1: BI-1-N-terminal HAtagged pEF vector-expressing cells, CΔBI-1: CΔBI-1-N-terminal HA-tagged pEF vector-
expressing cells.
Supplementary Figure 2 The expression of BI-1 does not increase cell proliferation.
Neo, BI-1 and CΔBI-1 cells were cultured (1 × 104 cells/well) and cell number
determined at the indicated times. *p < 0.05; significantly different from Neo cells
Supplementary Figure 3 BI-1 is localized into mitochondria, leading to the decrease of
mitochondria size. (a) Electron microscopy was performed in Neo and BI-1 cells. (b)
Mitochondria size in Neo and BI-1 cells was quantified. *p < 0.05; significantly different
from Neo cells
Supplementary Figure 4 BI-1 induces acidic extracellular pH. Neo, BI-1 and CΔBI-1
cells were incubated with high glucose medium (20 mM). Thirty six hours later, the
medium was photographed.
Supplementary Figure 5 Acidic pH increases MMP2/9 expression and activity.
HT1080 cells were incubated with high glucose medium for the indicated periods. Neo,
BI-1, and CΔBI-1 cells were incubated with high glucose medium (20 mM) for 12 h.
Infiltration (a) and wound healing assays (b) were performed. (c) With the cell lysates,
immunoblotting was performed with anti-MMP-2/9 antibodies (right). With the cell
medium, gelatin zymography was performed (left). *p < 0.05; significantly different
from pH 7.4.
Supplementary Figure 6 EIPA significantly regulates intra (a) and extracellular pH (b)
in BI-1 cells. *p < 0.05; significantly different from Neo cells