Pgl3 based vector

Wild-type (WT) or MUT HAGLR binding to miR-182-5p was subcloned into the pGL3 vector. BMSCs were co-transfected with miR-182-5p (RiboBio, Guangzhou, China) and 10 μg of pur-WT-HAGLR or pur-MUT-HAGLR. WT or MUT Hoxa10 and miR-182-5p were subcloned into a pGL3-based vector (Promega). miR-182-5p (RiboBio) was co-transfected with 10 μg pluco-WT-Hoxa10 or pluco-MUT-Hoxa10. After transfection of 48 h, a test of luciferase activity was done via the dual luciferase detection system (Promega Corporation, Fitchburg, WI, USA) [38 (link)].

Expression plasmids of FLAG-tagged Homo sapiens SCAMP5, SCAMP1, and TFEB were generated by PCR using human cDNA library and subcloned into the pcDNA3+based vector (Invitrogen). EGFP-α-synuclein-WT and EGFP-α-synuclein-A53T were generated by subcloning into the EGFP-C1 vector and mutagenesis. Tandem Fluorescent-Tagged LC3 (tf-LC3 or mRFP-GFP-LC3) was kindly provided by Dr. Yoshimori [23 (link)]. Luciferase reporter constructs pGL3-pSCAMP5 was generated by PCR of the predicted human SCAMP5 promoter (~3400bp upstream of SCAMP5 mRNA) using human genomic DNA library and cloned into pGL3-based vector (Promega). Lentivirus Lv-pSYN-Flag-SCAMP5 was generated by subcloning of SCAMP5 into the lv-fhSYN-eNpHR3.0-E (minus 2a-GFP) vector, and the lentivirus was generated in HEK-293T cells with Pspax+pMD2.G coating plasmids. The human SCAMP5 and TFEB siRNA constructs were synthesized at Genepharma with the following sequences: SCAMP5-542(sense, 5’ CCCAUUUACAAGGCCUUCATT; antisense, 5’UGAAGGCCUUGUAAAUGGGTT); SCAMP5-771(sense, 5’ CCCUCAGCAUGGUUCAUAATT; antisense, UUAUGAACCAUGCUGAGGGTT); SCAMP5-1101(sense, 5’ GGACCAGAGUUAUAUAUAUTT; antisense, AUAUAUAUAACUCUGGUCCTT); TFEB-870(sense, AGACGAAGGUUCAACAUCATT; antisense, UGAUGUUGAACCUUCGUCUTT); TFEB-981(sense, UACAUCCGGAGGAUGCAGATT; antisense, UCUGCAUCCUCCGGAUGUATT).