PMC

Western blot experiments, recombinant expression of NANOG1 and NANOG2 variants in 293T cells and immunohistological experiments. (A) Western blot experiments using an antiserum against the C-terminal portion of NANOG protein revealed the expression of NANOG1 and NANOG2 protein variants in NTERA-2 cells, and NANOG2 in SEM cells. (B) Cloned Strep-affinity-tagged splice variants of NANOG1 and NANOG2. Predicted molecular weight of all expression constructs is indicated. Recombinant expression of NANOG1 splice variants A and B_b and NANOG2 splice variants D2_c and E in 293T cells were shown. Sizes of protein markers are indicated. (C) Immunohistological analysis of all recombinant NANOG1/2 protein variants expressed in Hela cells. Selected pictures from transfected cells were taken (20×). From left to right: phase contrast, anti-C NANOG antiserum, DAPI counter-staining and a merged picture is shown. All NANOG1/2 protein variants were localized within the nucleus of transfected cells.

Gene expression profiling experiment. (A) The cell line 293 was transfected with episomal pEPI-EGFP vector containing expression cassettes for NANOG1A, NANOG2D2c or NANOG2E. After 10 weeks of culture, GPF-positive cells were sorted and used for RNA isolation. These RNA samples were used for Affymetrix chip experiments, resulting in few deregulated genes shown in the right. White boxed areas: up-regulated genes. Light grey boxed areas: down-regulated genes. Common genes identified in all three experiments using the different NANOG1/2 protein variants are highlighted. (B) Mode of action for the investigated NANOG protein variants. NANOG protein is able to induce transcription of EGR1 (directly or indirectly). EGR1 protein is directly binding to the promoter regions of FOS and p21. P21 protein is necessary for stem cell maintenance, while the FOS transcription factor is necessary for cell proliferation.

NANOG transcription in MLL-rearranged and Teratocarcinoma cell lines. (A) Gene structures of NANOG1 and NANOG2. Primer sets a–c were indicated for both genes. Forward primer of primer set c binds specifically to the 5′-NTR of NANOG1, and thus, enable to monitor transcripts that derive only from the NANOG1 gene. (B) RT–PCR analysis of four different MLL-rearranged cell lines (SEM, RS4;11, NOMO1, KOPN8). The NTERA-2 cell line represents a human embryonic carcinoma cell line and served as positive control for all experiments. M: DNA size marker. N: water control. (C) Validation of transcripts starting in the upstream located SLC2A14 gene. M, DNA size marker. N: water control. (D) Validation of NANOGP8 transcription. Specific oligonucleotides that bind only to the NANOGP8 open reading frame, we were not able to detect NANOGP8 transcripts in the investigated cells lines. M: DNA size marker. N: water control. N-P8, N2, N-P5: cloned NANOGP8, NANOG2 and NANOGP5 cDNAs served as positive controls.

NANOG1/2-specific RT–PCR experiments. (A) The two promoter regions of each NANOG genes are highlighted. Oligonucleotides A–C are indicated. Right upper panel: RT–PCR experiments using the primer combination A/C which allowed to monitor NANOG1- and NANOG2-derived transcripts. Right lower panel: RT–PCR experiments using the primer combination B/C, which allowed to monitor only NANOG1-derived transcripts. As internal control, cDNA from transfected cells were used (NANOG1 B_b and NANOG2 D2_c) and analyzed under identical conditions. N: water control. (B) Tissue cDNA panel. By using the primer combination A/C, a tissue cDNA panel was tested. With the exception of CD34⁺ cells, all other investigated tissue cDNAs remained negative. M: DNA size marker; neg: water control. Middle panel: Screening of t(4;11) patients, healthy volunteers and positive controls. Seven out of ten analyzed t(4;11) patients transcribed the NANOG2 gene. Peripheral blood samples of healthy volunteers were negative. M: DNA size marker; neg: water control. Lower panel: Screening of AML patients with normal karyotype. Out of 20 samples, 16 were positive for NANOG2 transcription, while 5 out of 20 samples were also positive for NANOG1 transcripts. Only 17 out of 20 analyses are shown. M: DNA size marker.

RACE experiments reveal splice variations of NANOG1 and NANOG2 and novel upstream exons. (A) 5′-RACE experiment. NTERA-2 and SEM cDNA was analyzed by using an anchored primer in the final NANOG exon. I–IV: bands cut out from the gel, cloned and sequenced. M: 50-bp ladder; N: water control. (B) Extended gene structure for NANOG1 and NANOG2. Homologous regions between both genes are depicted by grey areas. Repetitive DNA elements are indicated. Potentially transcriptional active ALU-elements are shown in light grey, while promoter-mutated ALU-elements are shown in dark grey. MER: MER-repeat. Arrows: indicate transcription initiation sites. All exons are numbered according to the nomenclature used throughout the manuscript. (C) Splice variants of NANOG1 and NANOG2. All cloned RACE amplimers were sequenced and revealed the additional NANOG1 exons 1a, 1b, 1b*, 2 and NANOG2 exons 1b, 1b*. Open reading frame analyses (marked in grey) of all cloned splice variants revealed putative open reading frames (ORFs) coding for two NANOG1 (splice variants A and B) and three NANOG2 protein (splice variants D, E and F). The number of isolated cDNA clones is indicated. Length of ORFs, predicted molecular weight (MW) and putative protein structures are depicted for all NANOG1/2 mRNA variants.