Directzol kit

5′UTRs were amplified from cDNA synthesized from MEFs and cloned into the pGL3 luciferase reporter vector (Promega) upstream of the Firefly luciferase open reading frame. MEFs expressing control or Pmpcb sgRNAs were seeded in a 6-well plate and induced with Dox for 24 h. After that, cells were transfected using Lipofectamine LTX (Invitrogen) with 900ng of pGL3-FLuc-Sv40 and 100ng of RLuc. Dox was not removed from the medium to allow 48h induction in total. 24h post-transfection cells were harvested: a fraction was lysed in Trizol for RNA extraction and the rest were lysed in a passive lysis buffer for 20 min. The Rluc/Fluc activity was assessed using the Dual-luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions, using a Glomax microplate luminometer (Promega). RNA extraction was performed using the Zymogen DirectZol kit following manufacturer’s instructions TurboDNA-free kit (Thermofisher) was used to remove transfected DNA completely. cDNA synthesis was performed by the SuperScript III first-strand synthesis kit (Invitrogen) using specific oligos for FLuc, RLuc and actin. qPCR analyses were performed as described previously.

The 5′UTR of Hspa8 was cloned into the pGL3 luciferase reporter vector (Promega) in which the downstream Firefly luciferase open reading frame (ORF) was replaced with the Renilla luciferase (Rluc) ORF. GTML cells expressing either control or eIF4E shRNAs were seeded at a density of 125k cell/well on a 12-well plate. After 24h, cells were transfected with 500 ng of pGL3–5’UTR-RLuc-Sv40 using Lipofectamine 2000 (Invitrogen). 24h post-transfection, cells were harvested: a fraction was lysed in Trizol for RNA extraction and the rest were lysed in a passive lysis buffer for 20 min. The Rluc activity was measured using the Dual-luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions, using a Glomax microplate luminometer (Promega). RNA extraction was performed using the Zymogen DirectZol kit, following manufacturer’s instructions, and the TurboDNA-free kit (Thermofisher) was used to remove transfected DNA completely. cDNA synthesis was performed by the SuperScript III first-strand synthesis kit (Invitrogen) using specific oligos for RLuc and GAPDH. qPCR analyses were performed as described previously, and Rluc activity was normalized to Rluc mRNA expression.