Supplemental figure 1 ASCL2 and MT3 are highly expressed in the
sphere-forming subpopulation in synovial sarcoma cell lines.
Quantitative real-time PCR analysis of upregulated genes in microarray results shows
that the higher expression of ASCL2 and MT3 is observed the floating sphere (Sphere)
than spindle shape cell (Normal) in all three synovial sarcoma cell lines. Data represent
the relative mRNA expression and are means ± s.d. of values from three independent
experiments. *P<0.05 and ***P<0.001 (Student's t-test) versus the samples under the
normal culture condition.
Supplemental figure 2 Analysis of CXCR4 transcriptional variant 1 and variant 2.
(A) Semi quantitative RT-PCR analysis shows that the higher expression of CXCR4
transcription variant 1 (CXCR4V1) is observed in LA and SP condition than N and NS
condition, respectively.
(B) Growth curves of CXCR4 variant 1 (black square), CXCR4 variant 2 (black circle),
or empty vector stable infectant of SYO-1 (black triangle), respectively are shown. Data
represent the number of viable cells and are means ± s.d. of values from three
independent experiments. *P<0.05 and ***P<0.001 (Student's t-test) versus the empty
vector stable infectant.
†††
P<0.001 (Student's t-test) versus the CXCR4 variant 1
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stable infectant.
(C) Statistical analysis of mouse xenografts is shown no significant difference of tumor
weights and volumes in three stable infectants. Data represent the weight and volume of
xenografts and are means ± s.d. of values from three independent experiments.
(D) Microscopic analysis by H&E stain shows no significant difference of tumor
morphology in three stable infectants.
Supplemental figure 3 CXCR4-positive cells are induced under the sphere culture
condition.
(A) Quantitative real-time PCR analysis of CXCR4 expression of SYO-1 shows that the
highest expression is observed under the sphere culture condition (Sphere) compared
with the normal and the non-coat 10% FBS culture conditon.
(B) Growth curves of SYO-1 under the normal (black square), the non-coat 10% FBS
(black circle), or sphere culture condition (black triangle), respectively are shown. Data
represent the number of viable cells and are means ± s.d. of values from three
independent experiments. ***P<0.001 (Student's t-test) versus the normal culture
condition.
†
P<0.05 and††P<0.01 (Student's t-test) versus the non-coat 10% FBS
culture condition.
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(C) Flow cytometry analysis of CXCR4 in SYO-1. Black bars and percentage indicate
the ratio of CXCR4-positive subpopulation. From the left side, it is shown the results of
isotype matched control stain, normal culture condition, non-coat 10% FBS condition,
and sphere culture condition of 2 days (upper panels) and 4 days (lower panels), after
seeding, respectively.
(D) Representative micrographs of phase contrast and immunocytofluorescence
microscopy. From the left side, it is shown phase contrast, ACTIN stain (red), SOX2
stain (green), and high magnification of SOX2 stain respectively (scale bars 100 m).
Sox2 expression was only detected in the sphere-forming subpopulation of FUJI and
SYO-1.
Supplemental figure 4 In silico pathway analysis of CXCR4 positive cells.
Representative scheme of JAK-STAT signaling pathway by KEGG_PATHWAY Chart
shows upregulated genes associated with MAPK signaling pathway, apoptosis, and cell
cycle (red star) inCXCR4 positive cells.
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