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Legends for supplementary figures and tables:
Supplementary Figure S1. The images show ‘minus primary antibody’ negative control for
each immunocytochemical and immunohistochemical experiment. Images in each row
correspond to the main figure depicted on the left-side.
Supplementary Figure S2. (a) Confirmation of over-expression of LT antigen by
immunoblotting (top panel) and hTERT (by RT-PCR) in primary mammospheres transduced
with SV40ER and hTERT. (b) Graph represents the percentage of β-galactosidase positive cells
in untransduced, pCSCG empty vector, hTERT alone, SV40ER alone and SV40ER+hTERT
transduced NB130 mammosphere-derived cells. (c) and (d) show the bar-graphs representing the
number of passages in suspension of two independent primary mammosphere-derived cells from
tissues (NB74 and NNB1) transduced with the indicated genetic elements. The immortalized
NB74 and NNB1 tissue derived cells have been termed as NB74LE and NNB1LE, respectively.
(e) Image shows a representative RT-PCR analysis of expression of stemness-related genes in
primary tissue-derived mammospheres and NBLE-derived mammospheres; (n=3).
Supplementary Figure S3. (a) Immunocytochemical analysis for CK14 expression in NBLE
cells cultured in differentiating conditions compared to normal media conditions. (b) The graph
shows the quantification of CK14 positive cells grown in normal media or differentiation media
as described above. Each bar represents the percentage of CK14 positive cells; (n=3), error bars
indicate standard deviation.
Supplementary Figure S4. (a) Graph shows fold change in gene expression of indicated genes
(normalized to HPRT) in NBLE clones. (b) Representative RT-PCR data on NBLE clones for
CD44 expression.
Supplementary Figure S5. Hematoxylin and Eosin stained picture of NBLE-derived tumor
shows formation of an occasional keratin pearl (KP), (scale bar-100µm).
Supplementary Figure S6. Quantification of immunostaining data represented in Figure 3i.
Fluorescence intensity was measured on 20 randomly chosen cells using ImageJ software. Bar
graphs represent mean fluorescence intensity; error bars represent standard deviation (AUArbitrary Units).
Supplementary Figure S7. Validation of microarray results represented in Figures 4 by RTPCR analysis. The bar-graphs represent the densitometric quantification of RT-PCR data using
Multi-Gauge software. Expression of each gene was normalized to HPRT. (a) Validation of
competitive microarray comparing CD44+/CD24- fraction versus ‘rest’ fraction of NBLE cells.
(b) Validation of competitive microarray comparing CD44+/CD24- fraction versus ‘rest’ fraction
of CB272 cells. (The inset tables show fold changes observed in the respective microarray data).
Supplementary Figure S8. Heat maps showing high similarity between biological repeats of
microarray experiments performed on CD44+/CD24- versus ‘rest’ fractions of NBLE cells
(NBLE_Exp1, Exp2 and Exp3).
Supplementary Figure S9. (a) Comparison of NBLE gene expression with published data set of
tumor initiating breast cancer cells (TICs) comprising of 13 primary breast cancer samples
(Creighton et al.,2009), (see gene list in Table S6). (b) Significantly altered genes related to selfrenewal pathways (Notch, Wnt, Hedgehog, and TGFβ) and genes involved with stemness in
embryonic stem cells (see gene list Table S7).
Supplementary Figure S10. (a) Bar-graph shows the RT-PCR analysis as shown in Figure 4d,
revealing elevated expression of stemness and EMT markers, and decreased expression of
epithelial markers in NBLE-LP cells compared to NBLE cells. Gene expression was normalized
to HPRT (n=5). Error bars depict the standard deviation. (b) Microarray data analysis showing
up-regulation of markers of self-renewal pathways, stemness-related genes, EMT and
proliferation-related genes (red bars), while down-regulation of differentiation-inducing genes
(green bars) in NBLE-LP cells compared to NBLE cells.
Supplementary Table S1. The table contains the oligonucleotide sequence of all the primers
used for RT-PCR analysis as described in the main text. The primers were designed using online
Primer3 software. (FW-Forward primer, RV- Reverse primer).
Supplementary Table S2. The table shows the status of various genes analyzed by RT-PCR
analysis on NB130, NBLE, NB74LE and HMLE cells. Band intensities were densitometrically
quantified and normalized to RPL/HPRT. Gene expression was represented as High (Red), Low
(Yellow) and Undetected (Green).
Supplementary Table S3. The table summarizes the results of experiments on NBLE single cell
clones.
Supplementary Table S4.
Table contains the list of significantly regulated genes in
CD44+/CD24- fraction compared to ‘rest’ in three biological repeats of NBLE cells.
Supplementary Table S5. Table contains the common list of significantly regulated genes in
CD44+/CD24- fraction versus 'rest' fraction of NBLE and three primary cancer samples (CB272,
CB386 and CB361).
Supplementary Table S6. The list of genes that were commonly regulated in CD44+/CD24fraction versus 'rest' of NBLE and CD44+/CD24- versus 'others' from primary breast cancer
samples published literature from Creighton et al., 2009 .
Supplementary Table S7. List of genes involved in self-renewal pathways and ESC as
described in the main text (Figure 4c).
Supplementary Table S8. Table shows karyotype analyses of metaphase spreads of NBLE and
its derivatives. ‘+’: gain of Chr, ‘-’: loss of Chr, ‘add’: addition, ‘t’ : translocation, ‘del’: deletion,
‘BRK’: break-point and ‘Mar’: marker chromosomes.
Supplementary Table S9. The list of genes related to EMT commonly regulated in the
CD44+/CD24- fraction versus the 'rest'; (n=2).