A cDNA encoding the monomeric derivative of DsRed fluorescent protein was cloned into the piggybac cDNA expression vector with the PTS1 (serine-lysine-leucine) sequence72 (link) using the In-Fusion HD Cloning Kit (TAKARA Bio Inc., Shiga, Japan).
The LC3-GFP cDNA was amplified from the pEGFP-LC3 plasmid (Addgene #21073) using the Tks Gflex DNA polymerase (TAKARA Bio Inc.) and cloned into the piggybac cDNA expression vector. PTS1-DsRed and pEGFP-LC3 was introduced using a CUY21 electroporator (NEPA Gene, Tokyo, Japan). Fluorescent images were captured using a BZ-X710 all-in-one fluorescent microscope (KEYENCE Corporation, Osaka, Japan) and analyzed integrated values of fluorescent brightness for each channel by an image analysis software BZ-X Analyzer Ver 1.3 (KEYENCE Corporation). To determine whether each fluorescent protein localized to peroxisomes and autophagosomes, a chemical activator of peroxisome proliferation, Wy1464330 (link) and that of autophagy, Rapamycin was used73 (link). DsRed/GFP double-positive dots were counted as peroxisomes processed by autophagy (pexophagy).
The LC3-GFP cDNA was amplified from the pEGFP-LC3 plasmid (Addgene #21073) using the Tks Gflex DNA polymerase (TAKARA Bio Inc.) and cloned into the piggybac cDNA expression vector. PTS1-DsRed and pEGFP-LC3 was introduced using a CUY21 electroporator (NEPA Gene, Tokyo, Japan). Fluorescent images were captured using a BZ-X710 all-in-one fluorescent microscope (KEYENCE Corporation, Osaka, Japan) and analyzed integrated values of fluorescent brightness for each channel by an image analysis software BZ-X Analyzer Ver 1.3 (KEYENCE Corporation). To determine whether each fluorescent protein localized to peroxisomes and autophagosomes, a chemical activator of peroxisome proliferation, Wy1464330 (link) and that of autophagy, Rapamycin was used73 (link). DsRed/GFP double-positive dots were counted as peroxisomes processed by autophagy (pexophagy).
Free full text: Click here
