Screening applications.Supplies and methodsReagentsAll fluorescently labeled oligonucleotides have been HPLC-purified and obtained from IBA-GmBh (Germany) and IDT (Coralville, IA, USA). Unlabeled oligonucleotides were bought from IDT (Coralville, IA, USA). The peptide nucleic acids (PNA) oligomer, P was synthesized employing standard strong phase Fmoc chemistry on Nova Syn TGA resin (Novabiochem, Germany) making use of analytical grade reagents (Applied Biosystems, USA), purified by reverse phase HPLC (Shimadzu, Japan) as previously reported and stored at 0 until additional use (Prakash et al., 2016). Bovine serum albumin (66 kDalton), nigericin, valinomycin, monensin, chloride ionophore I, Isopropyl b-D-1-thiogalactopyranoside (IPTG), amitriptyline hydrochloride, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and conduritol b epoxide (CBE) were obtained from Sigma (USA). LysoTracker Deep Red, TMR-Dextran (ten kDa) and Oregon Green 488 maleimide was obtained from Molecular Probes, 760173-05-5 Purity & Documentation Invitrogen (USA). Lysosomal enzyme kits namely lysosomal sulfatase assay kit was bought from Marker Gene (USA); Magic Red Cathepsin L assay kit from Immunochemistry Technologies. Gly-Phe b-naphthylamide was purchased from Santa Cruz Biotechnology (USA). All other reagents have been purchased from Sigma-Aldrich (USA) unless otherwise specified. BSA was maleylated as outlined by a previously published protocol (Haberland and Fogelman, 1985). Trizol was purchased from Invitrogen (U.S.A.).Sample preparationAll oligonucleotides were ethanol precipitated and quantified by their UV absorbance. For I-switch (I4cLYA488/A647) sample preparation, five mM of I4 and I40 were mixed in equimolar ratios in 20 mM potassium phosphate m-PEG7-thiol Autophagy buffer, pH five.5 containing 100 mM KCl. The resulting remedy was heated to 90 for five min, cooled to the space temperature at 5 /15 mins and equilibrated at four overnight. Samples have been diluted and made use of within 7 days of annealing. A sample of Clensor was similarly prepared using HPLC purified oligonucleotides and PNA oligomer at a final concentration of 10 mM by mixing D1, D2 and P (see Table S1 for sequence data) in equimolar ratios in ten mM sodium phosphate buffer, pH 7.two and annealed as described above. For ImLy, Oregon Green maleimide was first conjugated to the thiol labeled oligonucleotide (Hermanson, 2008). Briefly, to 10 mM thiol labelled oligonucleotide in HEPES pH 7.4, 500 mM of TCEP (tris-carboxyethylphosphine) was added to minimize the disulfide bonds. Injections have been performed, inside the dorsal side in the pseudocoelom, just opposite for the vulva, of one-day old wild form hermaphrodites applying an Olympus IX53 Easy Inverted Microscope (Olympus Corporation from the Americas, Center Valley, PA) equipped with 40X, 0.6 NA objective, and microinjection setup (Narishige, Japan). Injected worms were mounted on two.0 agarose pad and anesthetized applying 40 mM sodium azide in M9 buffer. In all situations labeling was checked soon after 1 hr incubation at 22 .Colocalization experimentsI4cLYA647 or ClensorA647 sample was diluted to 100 nM utilizing 1X Medium 1 and injected in ten arIs37 [pmyo-3::ssGFP] hermaphrodites as described previously by our lab (Surana et al., 2011). Imaging and quantification of the number of coelomocytes labeled, soon after 1 hr of incubation, was carried out on the Leica TCS SP5 II STED laser scanning confocal microscope (Leica Microsystems, Inc., Buffalo Grove, IL) using an Argon ion laser for 488 nm excitation and He-Ne laser for 633 excitation having a set of dic.