KLF8 and Ki67 expression in non-CNS tumors.

<p>KLF8 (A, C, E) and Ki67 (B, D, F) protein expression was analyzed in some additional tissue samples of breast cancer metastasis (A, B), meningioma (C, D) and non-neoplastic brain (E, F). All tissue samples analyzed confirmed ubiquitous expression of this transcription molecule, irrespective of the assumed proliferation rate.</p

PCR and WB of KLF8 after shRNA knockdown in U87-MG.

<p>(A) U87-MG cells transfected with either scrambled (scr) or KLF8-shRNA (kd) were cultivated for up to 3 days (day 0 = day of seeding). Cells were harvested every 24 hrs and RNA was isolated, transcribed into cDNA and amplified by qPCR; data were normalized relative to levels of the house keeping gene TBP. Semi-quantitative qPCR displayed a clear knock-down in KLF8 expression already 48 hrs after transfection (day 0). Expression levels decreased to about 10% in KLF8-shRNA treated cells compared to cells treated with scrambled shRNA on day 2 after seeding. (B) Subsequent Western Blot analysis of the nuclear fraction of KLF8-kd U87-MG cells on day 4 after seeding revealed that KLF8 protein was still detectable in all transfected U87-MG cells but only to a small extent in the KLF8-knockdown cells indicating that shRNA-knockdown was successful in these transfected cells in concordance with the qPCR results (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030429#pone-0030429-g005" target="_blank">Figure 5A</a>).</p

Western blot and densitometric analysis of KLF8 Western blots in gliomas.

<p>Western blot was performed for samples from gliomas of different WHO grades (GBM °IV, LGG °II) as well as non-neoplastic brain (NNB) (A). Densitometric analysis of the Western blot revealed no significant difference in expression of the transcription factor KLF8 in GBM compared to LGG and non-neoplastic brain samples. Protein expression was normalized to the house-keeping gene β-actin (set as 1.0, B).</p

Quantitative PCR.

<p>Samples of low-grade gliomas LGG and GBM (n = 10 each) were analyzed for KLF8-mRNA regulation. Compared to non-neoplastic brain (set as 100%), qPCR did not show any significant difference in the amount of KLF8-mRNA in LGG (97.1%) and GBM (99.3%).</p