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. Author manuscript; available in PMC: 2014 Mar 13.
Published in final edited form as: Oncogene. 2012 Oct 22;32(37):4397–4405. doi: 10.1038/onc.2012.461

Figure 2. NANOGP8 is Prevalent Form of NANOG Expressed in CRC Lines.

Figure 2

(a–b) Total RNA was extracted and qRT-PCR was performed for embryonic stem cell TFs. The results were normalized to GAPDH and HCC 2998 monolayers. Mean % ± SD.

(c–d) Restriction endonuclease digestion of 260 nt length of NANOG by RT-PCR amplifies a region where there is a SNP that identifies NANOGP8. Digestion with AlwNI reveals that even in monolayer culture NANOGP8 is frequently expressed in CRC lines (c) as well as in spheroids (d). The relative NANOGP8 expression is calculated as the ratio between densitometry reading of NANOGP8 and total NANOG by using Image J software. Direct sequencing confirmed that NANOGP8 is consistently upregulated in Clone A and CX-1 spheroids (e). Numbers in (e) stand for the numbers of NANOG or NANOGP8 in sequenced clones.

(f) MS/MS spectrum of a NANOGP8 tryptic peptide GKQPTSAENSVAK. This peptide is unique to NANOGP8, which includes the shift from Lys (K) in NANOG to Asn (N) in NANOGP8 at codon 82. Abbreviations: M, 100bp DNA ladder marker; bp, base pair; UD, undigested; D, digested with AlwNI.