Fig 2: Co-expressions of TMEM98, GPR64 and FAT4 in the CD44+/CD24- BCC subset. BCCs, stably transfected with Oct4-GFP, were co-labeled with CD44-APC and CD24-PE and the population of CD44 + /CD24- (Top panels) further studied for the expression of TMEM98, GPR64 and FAT4 (middle panels). CD44+/CD24- populations were further stratified based on Oct4 expression (Oct4hi, Oct4med and Oct4low) and then studied for TMEM98, GPR64 and FAT4 (lower panels). The ? Geometic (Geo) mean fluorescence intensity (MFI) for TMEM98, GPR64 and FAT4 are shown in each panel. The Geo mean was calculated as the change from the actual MFI – the MFI of the isotype.

Fig 3: Expression of selected genes in BCC subsets. (A) Real time PCR for GPR64, FAT4, TMEM98, ALOX5 AP, DUSP4, RAP1A and FN1. The RNA was isolated from Oct4hi, Octmed and Oct4low subsets from MDA-MB-231 stably transfected with pEGFP1-Oct3/4. The results are shown as relative to ß-actin expression with mean ± SD for four independent experiments. (B) Western blots for GPR64, TMEM98, FAT4, RAP1A and FN1 using whole cell extracts expression from Oct4hi, Octmed and Oct4low, isolated as for ‘A’. The results represent four different independent experiments.

Fig 4: Characterization of BCC subsets. ALDH1 activity was assessed in positive control with K562 cells (A) and experimentally, with heterogeneous MDA-MB-231 cells (B). The presence of Oct4-dsRED-SB in MBA-MB-231 cells was determined by Bix treatment in flow cytometry (C). Different subsets of Oct4-dsRED-SB-transfected MDA-MB-231 cells (D) were assessed for ALDH1 (E). Similar sorting of three MDA-MB-231 subsets were analyzed for telomere length using the TeloTAGG kit (F). MDA-MB-231, stably transfected with Oct4-GFP (left panel) was labeled with PE-anti-TMEM-98, -GPR64 and -FAT4 and analyzed by flow cytometry based on the relative GFP levels (n = 5; G and H). Further assessment for TMEM-98, GPR64 and FAT4 within Oct4hi BCCs was based on size. Oct-4-GFP transfected MDA-MB-231 cells were gated based on GFP expression (I, top second panel) followed by gating of the 5% of cells with the highest GFP (I, R3, top third panel-Oct4hi). R3 was divided based on side (SSC) and forward scatter (FSC) (R4-R6) and then analyzed for TMEM98, GPR64 and FAT4 (I, lower panels). The results represent four independent experiments.