All iPSC markers except NANOG were also expressed in the PTS. tumorspheres in vitro. We demonstrate the presence of two putative CSC subpopulations within HNmMM, which may be a novel restorative target in the treatment of this aggressive tumor. = 20. Expected staining patterns were shown in the human being positive control MTEP hydrochloride cells: seminoma for OCT4 (Number S2A) and NANOG (Number S2B), normal pores and skin for SOX2 (Number S2C), breast carcinoma for KLF4 (Number S2D), and normal colon for c-MYC (Number S2E). The isotype-matched antibodies offered appropriate negative settings (Number S2F). In order to compare the protein manifestation of different iPSC markers, we performed cell counting analysis of 19 IHC slides (one case was excluded due to dense melanin pigmentation, refer to Methods) of HNmMM, where all marker-positive cells in both the TNs and PTS were counted and recorded as a proportion of the total quantity of cells in the field of view. When comparing the total proportion of positively stained cells within the TNs and PTS for each marker, post-hoc statistical analysis shown a hierarchy of manifestation of these markers with increasing abundance as follows: NANOG < OCT4 < KLF4 < c-MYC < SOX2 (Number 2). All comparisons were highly statistically significant between markers (< 0.0005) except for the comparisons between NANOG and OCT4, which was significant (< 0.05), and between c-MYC and SOX2, which was not significant. Open in a separate window Number 2 Graph demonstrating mean percentage positive manifestation of induced pluripotent stem cell markers NANOG, OCT4, KLF4, c-MYC, MTEP hydrochloride and SOX2 by cells within the tumor nests and the peritumoral stroma on immunohistochemical sections of head and neck metastatic malignant melanoma. Error bars symbolize 95% confidence intervals of the mean. Three replicates from each of the 19 patient cells samples MTEP hydrochloride were utilized for an Analysis of Variance (ANOVA), thus giving a sample size of 57 for each of the following markers: OCTs, SOX2, KLF4, and c-MYC (= 57). Similarly, for NANOG, three replicates from each of the two patient cells samples were utilized for an ANOVA, thus giving a sample size of 6 (= 6). ***, < 0.0005; *, < 0.05. 3.2. Rabbit Polyclonal to NEIL3 Subpopulations of CSCs Expressing OCT4, NANOG, SOX2, KLF4, and c-MYC are Present in HNmMM Cells Samples To investigate localization of two iPSC markers simultaneously, IF staining was performed on two representative HNmMM cells samples. IF staining shown manifestation of OCT4 (Number 3ACC, green), SOX2 (Number 3B,E, reddish), KLF4 (Number 3C,F, reddish), and c-MYC (Number 3DCF, green) from the cells within the TNs (solid arrows) and the PTS (arrowheads). NANOG (Number 3A,D and inset, reddish) was present in one sample that showed NANOG manifestation on IHC staining and was absent in the additional sample that did not show NANOG manifestation on IHC staining. OCT4 was indicated within the NANOG+ (Number 3A and inset, reddish), the SOX2+ (Number 3B and inset, reddish), and the KLF4+ MTEP hydrochloride (Number 3C and inset, reddish) cells within the TNs and the PTS. c-MYC was also indicated from the NANOG+ (Number 3D and inset, reddish), the SOX2+ (Number 3E and inset, reddish), and the KLF4+ (Number 3F and inset, reddish) cells within the TNs and the PTS. Interestingly, some c-MYC+ (Number 3D and inset, green) cells within the TNs were NANOG- (Number 3D, thin arrows). Magnified number insets have been provided to MTEP hydrochloride show enlarged views of the corresponding images. IF dual-staining HNmMM cells samples for the melanoma marker Melan-A and induced pluripotent stem cell markers showing manifestation of NANOG (A, reddish), SOX 2 (B, reddish) SOX2 (C, reddish), KLF4 (C, reddish), and c-MYC (D, reddish).