Rnaesy extraction kit

Total RNA from cell lines and tumor samples was extracted with Trizol (Invitrogen) and RNaesy Extraction Kit (QIAGen) as indicated by the manufacturer. cDNA from the different cell lines and tumor samples was obtained from 1 μg of total RNA using random primers and Superscript II system (Life Technologies Inc) as previously described [13] . Gene expression analyses were performed by semiquantitative RT-PCR (sqRT-PCR) and real time RT-PCR (qRT-PCR). For qRT-PCR pre-designed TaqMan probes (GSDMB, GSDMB-1, GSDMB-2, GSDMB-3&4) or SybrGreen PCR reagents (mCherry) (Sigma) were used on an iQ5 iCycler Realtime PCR Detection System (BioRad) using TaqMan “iQ Supermix” or “SYBR Green Supermix” (BioRad), according to the manufacturer's recommendations. GSDMB-3 and -4 were analyzed together as there is no commercially available Taqman probe to discriminate isoform 3. Primers sequences and amplification conditions for sqRT-PCR and for qRT-PCR are indicated in Tables S1 and S2, respectively. All RT-PCRs were performed in triplicates. Relative expression was normalized to β2 microglobulin, β-actin or GAPDH. The comparative threshold cycle (Ct) method was used to calculate the amplification factor as specified by the manufacturer.