Inducible Expression of GFP-RPL3 Fusion

ORFs encoding GFP alone, or GFP fused to human RPL3, either WT or R246A/R249A-mutated (RPL3 fused to N-terminus of GFP) were cloned into the pcDNA5/FRT/TO plasmid (Thermo Fisher Scientific) under the control of a doxycycline (Dox)-inducible promoter, similarly as described in ref. (32 (link)). HEK293-derived, Flp-In™ T-REx™ 293 cells (Thermo Fisher Scientific) were co-transfected with pOG44 and pcDNA5/FRT/TO-derived plasmids, according to manufacturer's instructions, and selected with 200 μg/ml Hygromycin B (Thermo Fisher Scientific). The pool of surviving cells was maintained in high-glucose DMEM + GlutaMAX™ medium (Gibco) supplemented with 10% FBS and P/S. To test ORF expression, cells were left untreated or induced with 1 μg/ml Dox for 24 h and then fixed in cold acetone for 10 min. Cells were visualized by fluorescence microscopy after incubation with 1 μg/ml Hoechst 33258 (Sigma-Aldrich) to stain the nuclei (λ_ex = 405 nm), similarly as described in the above section.