Uvsolo ts

RNA from 1 × 10⁶ cultured and pooled PCSCs was isolated using the NucleoSpin^® RNA Plus kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s guidelines. The quality and concentration of isolated RNAs were assessed via nanodrop ultraviolet spectrophotometry. For each sample, 1 µg of RNA was transcribed into cDNA according to the protocol of Thermo Fisher Scientific. Analyses of PCSC gene expression profiles were performed with the taq-S PCR Kit (New England Biolabs, Frankfurt am Main, Germany) according to the manufacturer’s guidelines. The specific primer sequences used for characterization are listed in Table 3 below.
Amplified RT-PCR transcripts were loaded on agarose gel (Sigma-Aldrich) with 0.001% ethidium bromide (Carl Roth GmbH), run at 100 V, and imaged with a trans-illuminator (UVsolo TS; Biometra, Göttingen, Germany).

For the analysis of mRNA expression, RNA was isolated using the NucleoSpin RNA Kit (Macherey Nagel, Düren, Germany) according to the manufacturer’s guidelines. cDNA synthesis was performed using 1 µg RNA and the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Bremen, Germany). The quantification of RNA was performed using the Primers listed below (Table 2) and the Platinum SYBR Green qPCR Super-Mix UDG (Invitrogen, Thermo Fisher Scientific, Bremen, Germany) according to the manufacturer’s guidelines and assayed with a Rotor Gene 600 (Qiagen, Hilden, Germany). Reverse transcriptase PCR was performed using the Taq DNA Polymerase with ThermoPol^® Buffer (New England Biolabs, Frankfurt am Main, Germany) and the primers listed below according to the manufacturer’s guidelines. Amplified RT-PCR products were separated on an 2% agarose gel (Sigma-Aldrich, Munich, Germany) with 0.001% ethidium bromide (Carl Roth GmbH, Karlsruhe, Germany) run at 100 V, and were imaged with a trans-illuminator (UVsolo TS; Biometra, Göttingen, Germany).

1 × 10⁶ cells from U251-MG cell lines were pooled and resuspended in 300 µl lysis-buffer with SDS as well as 200 ng/ml Proteinase K and shook for 4 h at 55 °C. After a heat shock with 95 °C for 5 min, the genomic PCR was performed with GoTaq DNA polymerase (Promega Corporation, Mannheim, Germany) according to manufacturer’s guidelines. Sequences for used PLEKHG5 primer pair:PLEKHG5

(fwd 5′-TTGTCCTTATGACGCCCTAGC-3′;

rev 5′-CACTGCACTCCCTGTCTCAAAGAA-3′).

Amplified DNA was loaded on a 2% agarose gel with 0.001% EtBr (Carl Roth GmbH, Karlsruhe, Germany) and ran for 30 min by 100 V, followed by imaging with a trans-illuminator (UVsolo TS, Biometra).