*4.11. Real Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)*

Total RNA was isolated from the HCT116, HT-29, DU145, and PC-3 cells, 24 h after incubation with the respective GSH-NS B or GSH-NS D IC50. Briefly, the cells were collected in RNA Cell Protection Reagent (Qiagen, Milano, Italy) and stored at −80 ◦C. Total RNA was obtained by the RNeasy Plus Mini Kit (Qiagen Milano, Italy). Total RNA concentration (µg/mL) was determined using the fluorometer Qubit (Invitrogen) and the Quant-IT RNA Assay Kit (Invitrogen). Calibration was carried out by applying a two points standard curve, according to the manufacturer's instructions. RNA sample integrity was determined by the Total RNA 6000 Nano Kit (Agilent Technologies, Milano, Italy) using the Agilent 2100 Bioanalyzer (Agilent Technologies, Milano, Italy).

Real-time RT-PCR analysis was carried out using 1 µg of total RNA, which was reverse transcribed in a 20 µL cDNA reaction volume using the QuantiTect Reverse Transcription Kit (Qiagen, Milano, Italy). Each 10 µL real-time RT-PCR reaction was obtained using 12.5 ng of cDNA, according to the manufacturer's instructions. Quantitative RT-PCR was performed by the SsoFast EvaGreen (Bio-Rad, Milan, Italy) and the QuantiTect Primer Assay (Qiagen, Milano, Italy) was used as the gene-specific primer pair for the studied gene panel (Table 4).



The transcript of the reference gene 18S ribosomal RNA (*RRN18S*) was used to normalize mRNA data and real-time RT-PCR was performed by the MiniOpticon Real Time PCR system (Bio-Rad, Milan, Italy). The PCR protocol conditions were as follows: a HotStarTaq DNA polymerase activation step at +95 ◦C for 30 s, followed by 40 cycles at +95 ◦C for 5 s and +55 ◦C for 10 s. All runs were performed on at least three independent cDNA preparations per sample and all samples were run in duplicate. At least two non-template controls were included in each PCR run. Quantification data analyses were performed by the Bio-Rad CFX Manager software version 1.6 (Bio-Rad, Milan, Italy), according to the manufacturer's instructions. These analyses were performed in compliance with MIQE guidelines (Minimum Information for Publication of Quantitative Real-time PCR Experiments) [63].
