Pcr primers

At humane endpoint, mice were sacrificed with an overdose of ketamine and immediately perfused with ice-cold Ringer’s solution (Sigma-Aldrich, 96724-100TAB). The brain was extracted, and a piece of tumor was immediately snap-frozen in liquid nitrogen for storage at −80 °C. Alternatively, cultured cells were harvested from plates using TRIzol (ThermoFisher, 15596026). RNA was isolated from the frozen tumor pieces or cells with the RNeasy Lipid Tissue Mini Kit (Qiagen, 74804) according to the manufacturer’s instructions. RNA quantity was assessed with a NanoDrop 2000 spectrometer, while quality was confirmed via electrophoresis of samples in a 1% bleach gel as previously described^{99 (link)}. RNA was used to generate cDNA with a First Strand Superscript III cDNA synthesis kit (ThermoFisher, 18080051) according to the manufacturer’s instructions and with equal amounts of starting RNA. Quantitative PCR was performed with the validated BioRad PCR primers (Supplementary Table 2, except Cxcl1 and Cxcl2 whose sequence were obtained from ref. ^{100 (link)}) using SsoAdvanced Universal green Supermix (BioRad, 1725271). Fold changes in gene expression were determined relative to a defined control group using the 2^-ΔΔCt method or by z score, with β-actin or HPRT used as housekeeping genes.

sox1a mutant lines were generated by CRISPR/Cas9 targeted genome editing relying on non-homologous end joining repair mechanism, as described in detailed protocols provided by Auer et al. (2014b) (link), Gagnon et al. (2014) (link), and Talbot and Amacher (2014) (link). Optimal target sites were selected using the CHOPCHOP web tool (Montague et al., 2014 (link)). cas9 mRNA was transcribed from a plasmid provided by Jao et al. (2013) (link) using Ambion mMESSAGE mMACHINE Kit. Guide RNAs were generated by PCR and transcribed using HiScribe T7 High-Yield RNA Synthesis Kit (NEB). 110–140 pg of guide RNA and 170 pg of cas9 mRNA per embryo was injected at one-cell stage into the cell. Mutants were screened by high-resolution melt analysis (HRMA) (Dahlem et al., 2012 (link)) using Biorad Precision Melt Supermix and confirmed by Sanger sequencing. The mutated sequences are shown in Figure 2—figure supplement 1. Mutants were genotyped for all further experiments by allelic discrimination via KASP chemistry using PCR primers designed by the manufacturer (LGC Genomics) and the CFX Connect Real-Time PCR Detection system (BIO-RAD) for detection and analysis.

Cells exposed to the tested fluoroquinolones at concentrations of LC₁₀, LC₅₀, and LC₉₀ were analyzed for the expression of selected genes (BAX, BCL2, TOP2A, TOP2B, and CDKN1A). Total RNA from tested cells was purified using a RNeasy Mini Kit (Qiagen, Germany). The cell pellet was disrupted with an appropriate volume of lysis buffer and processed according to the manufacturer’s protocol. The obtained material was subjected to qualitative and quantitative analysis using the NanoDrop™ Lite Spectrophotometer (Thermo Fisher Scientific, USA). Reverse transcription reactions for complementary DNA synthesis were performed using the Transcriptor First Strand cDNA Synthesis Kit with the Anchored-oligo(dT)₁₈ Primer (Roche, Switzerland) according to the manufacturer’s recommendations. Gene expression was evaluated by quantitative polymerase chain reaction (qPCR) using LightCycler^® 480 SYBR Green I Master (Roche, Switzerland) and PCR primers (Bio-Rad, USA) for selected genes with SDHA and TBP as the reference genes. The obtained gene expression results were compared to control cells and subjected to statistical analysis.