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Zhou et al. J Cancer Metastasis Treat 2018;4:41 I http://dx.doi.org/10.20517/2394-4722.2018.16 Page 9 of 15

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Figure 4. FISH analyses of in vitro cultures of U251 with transient and long-term expression of ectopic EFEMP1. (A) representative FISH
interphase and metaphase nucleus images of U251 transduced by lentivirus of empty vector or doxycycline (Dox)-induction of ectopic
EFEMP1. Normal Chr7 was shown by a white arrow, abnormal Chr7 (with amplification of p-arm and deletion of q-arm) by a white
arrowhead; (B) comparison of Chr7-subpopulations in various U251 cultures with or without EFEMP1 overexpression

after 2, 14, and over 60 days of Dox-treatment. After lengthy induction of ectopic EFEMP1 by Dox, even after
withdrawal of Dox for a week, nearly 80% of cells in U251-EFEMP1 (+Dox) and U251-EFEMP1 (withdrawal
of Dox) carried similarly high percentages of 2-Chr7 (2n) cells. This demonstrated that the new steady state
of tumor subpopulation induced by EFEMP1 persisted for some time, even after the extent of EFEMP1
overexpression was eliminated or minimized. Long-term expression of ectopic of EFEMP1 changed the
steady state of U251 subpopulations with key subpopulation of 2-Chr7 (2n) of low tumorigenicity.

We then studied MS rate of this low tumorigenic 2-Chr7 (2n) subpopulation of U251, by analyzing two
single-cell lines derived from soft-agar colonies of U251-EFEMP1 (withdrawal of Dox) formed and expanded
with or without Dox-treatment. FISH analysis showed similarly high percentages of 2-Chr7 (2n) cells in
U251-EFEMP1 (withdrawal of Dox) and its derived single-cell lines, regardless of Dox-treatment [Figure 4B].
The lack of increase in cell population diversity in single-cell lines of U251-EFEMP1 (withdrawal of Dox)
suggests a lower CIN rate of cells with 2-Chr7 (2n) in U251, which is in striking contrast to that of single-cell
lines of high tumorigenic glioma cell lines, as described above and shown in Figure 2A.