Fig 2: Whole genome copy number analysis using high resolution SNP-Chips. (A, B) Heat map of DNA copy numbers found in the p and q arms of the chromosomes (horizontal axis) of 56 NSCLC samples from the TCGA data base [top panel, Figure 1A], 9 NSCLC patient samples with EGFR mutations [bottom panel, Figure 1A] and 8 NSCLC cell lines with EGFR mutations [Figure 1B] (vertical axis). DNA copy numbers are indicated by colors (black, blue, green, pink, orange and red are 0, 1, 2, 3, 4 and =5 copies, respectively). Common copy number gain regions are emphasized by red dotted rectangles. Common copy number loss region is emphasized by blue dotted rectangle. (C) At chromosome 8p23.1, a homozygous deletion of SOX7 occurs in the HCC2935 NSCLC cell line. Red dots show raw data. Blue line denotes total gene dosage by CNAG; level 2 indicates diploid (2N) amount of DNA. Sample is mostly hemizygous. Green small vertical bars immediately under the chromosome display heterozygous SNP sites. The bottom lines (Red and Green) denote allele-specific gene dosage (one line indicates gene dosage of the maternal allele, and the other indicates gene dosage of the paternal allele). Sample shows that chromosome 8 is hemizygously deleted except at 8p23.1 where the second allele is also lost in a small region resulting in homozygous deletion of the UNQ9391, RP1L1 and the SOX7 genes.

Fig 3: Methylation analysis of upstream regions of SOX7 gene. (A) Schematic illustration of CpG sites spanning 1,500 bp upstream and 350 bp downstream of the transcription start site of SOX7 transcription. Black arrow denotes the transcription start site (+1). Vertical pink bars denotes the CpG sites. Arrowed bars define the regions subjected to bisulfite sequencing (BS). Bars with circles at their end define the regions subjected to methylation specific PCR (MSP). (B) Bisulfite sequencing of SOX7 gene in NSCLC cell lines. Each circle in each horizontal row represent the analysis of a single clone of bisulfite-treated DNA encompassing either 20 or 35 CpG sites (-678 to -440, left panels; -71 to +251, right panels, respectively). Open and solid circles represent unmethylated and methylated CpG sites, respectively. (C) MPS analysis of upstream region of SOX7 gene in NSCLC cell lines. PCR products in lanes marked “U” and “M” are obtained with unmethylated-specific and methylated-specific primers, respectively. (D) Bisulfite sequencing of SOX7 gene in NSCLC samples and their matched normal lung tissues as described for the NSCLC cell lines in Figure 4B.

Fig 4: Downregulated SOX7 in NSCLC compared to matched normal lung samples. (A) Waterfall graph showing SOX7 mRNA expression in 62 paired human NSCLCs compared to normal lung tissue from the same patient. SOX7 mRNA expression was normalized to β-actin mRNA. (B) Statistical analysis of SOX7 mRNA expression in 62 paired human NSCLCs and normal lung tissues. Delta threshold cycle value (DCt) was calculated from the given threshold (Ct) value by the formula DCt = (Ct SOX7 – Ct β-actin) in each sample. P value was calculated with Paired T-test. (C) Relationship between significant SOX7 mRNA levels in the NSCLC samples and clinicopathological features of the patients and their NSCLC. Statistical differences were observed in histological differences (p=0.0222). The p values were calculated with Mann–Whitney T-test.

Fig 5: IaI maintains pluripotency and differentiation capacity in long-term culture.Immunofluorescence staining of H207 hES cell line for (a) stem cell markers Oct4 (green), Sox2 (green), Nanog (red) and SSEA-4 (green) after 16 passages in E8:IaI, and for the three germ layers after 4 weeks of differentiation through embryoid body (EB) formation: mesoderm with alpha smooth muscle actin (SMA, green), ectoderm with Nestin (red) and ß-III-tubulin (green) and endoderm with alpha-fetoprotein (AFP, green) and PDGF-receptor (red), and (b) after endoderm directed differentiation for endoderm markers Sox7 (red), Sox17 (green) and HNF3ß (green) and stem cell markers Oct4 (green) and Nanog (red), as well as negative control with secondary antibodies. All samples were co-stained with DAPI (Blue) for nuclei detection. Scale bars show 100 µm, white boxes show close-up images.