Nmdi14

NMDI14 (Sigma) stock solution was made in DMSO as per manufacturer’s instructions. AB wild-type zebrafish embryos were dechorionated on agarose-coated 10 cm plates. At 3 hpf, NMDI14 was added to a final concentration of 4.8 μM. At 24 hpf, embryos (20/treatment) were rinsed with fresh fish water and added to 500 μl of Trizol (Thermo Fisher Scientific) for RNA preparation.

For the Western blotting experiments and the IHC, the following antibodies were used: rabbit polyclonal anti-GABARAPL1 (D5R9Y, Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-UPF1 (D15G6, Cell Signaling), rabbit polyclonal antibody anti-SMG1 (Q25, Cell Signaling), rabbit polyclonal anti-b-actin (A5060, Sigma Aldrich, St. Louis, MI, USA), monoclonal anti-Vimentin (#7902917, Ventana Medical Systems, Oro Valley, AZ, USA), secondary goat polyclonal anti-rabbit HRP (BI2407, Abliance, Compiègne, France) and anti-mouse HRP (BI2413C, Abliance).
For the cellular treatments, cycloheximide (01810, Sigma, St. Louis, MI, USA), G418 (11811023, ThermoFisher, Santa Cruz, CA, USA) and NMDi14 (SML1538, Sigma-Aldrich) were used.
The pGFP-GABARAP vector cloning was previously reported in [18 (link)]. The pGFP-GABARAP + 3′UTR-GABARAPL1 was synthesized by amplifying the 3′UTR of GABARAPL1 added with SacII and BamHI restriction sites via PCR using the following primers: forward—5′-TAAGCACCGCGGGTGGTTGGAAGCCCAGCAG and reverse—ATTTTATTAACGAATGGAATTTTTAGCCTAGGATTCGT. The amplified sequence was then cloned downstream GABARAP CDS in a pGFP-GABARAP plasmid using the SacII and BamHI sites.
The siRNAs used in the transfection were purchased from Eurogentec (Seraing, Belgium). The sequences are listed in Table 1.

RPE1-hTERT human retinal pigment epithelial cell lines were cultured in DMEM/F12 medium (11504436, Fisher Scientific) supplemented with 1% penicillin/streptomycin, 10% FCS and 2 mM l-glutamine. HCT116 human colorectal carcinoma cell lines were grown in McCoy’s 5A medium (26600080, Thermo Fisher Scientific) supplemented with 1% penicillin/streptomycin, 10% FCS and 2 mM l-glutamine. U2OS human bone osteosarcoma cell lines were grown in DMEM medium (41965062, Thermo Fisher Scientific) supplemented with 1% penicillin/streptomycin, 10% FCS and 2 mM l-glutamine. Mirin (M9948), ML216 (SML0661-5MG), and NMDI14 (SML1538) was purchased from Sigma. Cell counting were performed using a NucleoCounter NC-3000™ system (Chemometec). Cell cycle analyses were performed according to a two-step cell cycle protocol on a NucleoCounter NC-3000™ system (Chemometec).

Human HeLa cells from ATCC were grown at 37 °C with 5% CO2 in DMEM (Gibco, LifeTechnologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 2 µg/mL penicillin/streptomycin (Sigma-Aldrich, St Louis, MO, USA). Drosophila S2 cells were grown in Schneider’s medium (Invitrogen, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 50U/mL penicillin and 50 µg/mL streptomycin (Invitrogen) at 25 °C. Transfection of HeLa or S2 cells with plasmid vectors was performed respectively with JetPrime reagent (Polyplus transfection, Illkirch, France) and FuGene HD transfection Reagent (Promega, Madison, WI, USA) according to the manufacturer specifications. Cycloheximide, Actinomycin D and NMDI14 were purchased from Sigma.