Siatf6

Predesigned siRNAs (100 pmol) were transfected into MRC5 cells with Lipofectamine 3000 reagent (Cat#L3000015, Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. We used the following siRNAs: siRNA negative control (Cat#sc-37007, Santa Cruz), sip62 (Cat#1144479, Bioneer, Daejeon, Korea), siNrf2 (Cat#sc-37030, Santa Cruz), siATF6 (Cat#sc-37699, Santa Cruz), siHSF1 (Cat#sc-35611, Santa Cruz), siHIF1 (Cat#sc-35561, Santa Cruz), and siRTN4 (Cat#sc-43974, Santa Cruz).

Total RNA from human term trophoblast previously transfected with siRNA against IRE1α, ATF6 and PERK were used [17 (link)]. Briefly, VCT cells were transfected with 16.6 nM siATF6 (SantaCruz Biotechnology, Labforce, Muttenz, Switzerland), 16,6 nM of siIRE1α (SantaCruz Biotechnology, Labforce, Muttenz, Switzerland) and 16,6 nM of siPERK (SantaCruz Biotechnology, Labforce, Muttenz, Switzerland) or 50 nM control siRNA (SantaCruz Biotechnology, Labforce, Muttenz, Switzerland) using Interferin transfection reagent (Polyplus transfection SA, Illkirch-Graffenstaden, France) and following the manufacturer’s protocol [17 (link)]. RNA extracted from trophoblast cells incubated with LPV or DMSO control with or without IRE1α inhibitor (STF-083010) was also analyzed. An amount of 500 ng of total RNA were reversed transcript with SuperScript^® III Reverse Transcriptase Kit (Invitrogen^TM, Carlsbad, CA, USA). The qPCR was performed using cDNA diluted 1/5 in RNase-DNase free water using the the Takyon^TM ROX SYBR^® MasterMix blue dTTP (Eurogentec, Kaneka, Liège, Belgium). Data were normalized using SDHA, 18S and HPRT as endogenous controls. Used primers are described in Table 2.