(a) Tumor growth curves of human SCCs infected with
lentivirus expressing short hairpin RNA (shRNA) against SOX2 along with nuclear
red fluorescent protein (H2B-RFP; shSOX2) or scrambled control shRNA along with
nuclear green fluorescent protein (H2B-GFP; shSCR) followed by transplantation
onto Nude recipient mice. Data are represented as mean with error bars
indicating ± s.e.m. (n=6, *P<0.05, Student’s t-test).
(b) Doxycycline-inducible knockdown of SOX2 in established
human SCC xenografts. Data are represented as mean with error bars indicating
± s.e.m. (n=10, *P<0.05, Student’s t-test).
(c–e) Quantitative analysis of proliferative Ki67
(c), mitotic phospho-Histone H3(Ser10) pH3 (d),
and apoptotic activated Caspase-3 (Casp3) (e) positive cells in
shSOX2;H2B-RFP or shSCR;H2B-GFP transduced A431 xenografts. Scatter plots
illustrate the percentage of infected parenchymal cells that are positive for
the respective marker in n>200 microscopic fields. Horizontal bars
indicate mean ± 95% CI. (*P<0.05 Mann-Whitney
non-parametric t-test). (f–g) Analysis of clonal growth
competition assay at 2 and 4 weeks after intradermal transplantation when
1–2% of A431 cells have been transduced with shSOX2;H2B-RFP and
shSCR;H2B-GFP (n=6). (f) Scatter plots illustrate clone size
distributions. Horizontal lines represent mean with error bars indicating
± 95% CI. (g) Line graphs showing average clone
size as a function of time (± s.e.m). (f–g) P
values were obtained by Mann-Whitney non-parametric t-tests.