Pgl3 control plasmid

SINV DNA templates for transcription were generated by XhoI linearization of pToto1101 (Rice et al., 1987 (link)). SINV RNA was transcribed in vitro by Sp6 RNA polymerase (New England Biolabs) in the presence of the cap analog [m7G(5’)ppp(5’)G] (New England Biolabs). Fluc DNA templates for transcription were amplified from the pGL3-Control plasmid (Promega). Fluc RNA was transcribed in vitro using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen). Biotin-labeled RNAs were generated by adding 10mM biotin-16-UTP (Roche Life Science) to in vitro transcription reactions. JEV replicon DNA templates for transcription were generated by XhoI linearization and transcribed in vitro using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen). Transcribed RNAs were purified using the Quick-RNA Miniprep Kit (Zymo Research) and biotinylation was confirmed by streptavidin dot blot (Chan et al., 2020 (link)).

Intronic artificial miRNAs directed against PLN were cloned into the pSM155 vector50 (link), kindly provided my Michael A. Frohman (Stony Brook University, NY), using asymmetric BsmBI restriction sites (Supplementary Fig. 7). For amiRNA candidates screening, a reporter construct was obtained by inserting the full-length human PLN complementary DNA (cDNA; amplified by PCR from human heart cDNA) downstream of the luciferase coding sequence of the pGL3-control plasmid (Promega). For the PLN-R14del reporter vector, a pair of complementary primers encoding mutant PLN was annealed and inserted in the same position of pGL3-control vector. HEK-293T cells were co-transfected with both pSM155-amiRNA and reporter vectors in a 5:1 ratio using calcium phosphate transfection, and luciferase activity from cell lysates was measured 24 h post transfection using Luciferase assay reagent (Promega). Transfection efficiency was normalized by measuring the green fluorescent protein fluorescence from pSM155 vectors. PSM155 vectors containing a scrambled miRNA or a luciferase-directed miRNA were used as negative and positive controls, respectively.

The human pri-miR-31 sequence containing miR-31 pre-miRNA and its flanking sequences on both sides was amplified from genomic DNA using the primers listed in Supplementary Table 1, and was subsequently cloned into pcDNA3.0 vector and lentivirus shuttle vector plenti6 (Invitrogen). For knockdown of miR-31 expression, tough decoy (TuD) RNA against miR-31 (5′-CGggatccGACGGCGCTAGGATCATC AACAGCTATGCCAGATCTCATCTTGCCTCAAGTATTCTGGTCACAGAATACAACAGCTATGCCAGATCTCATCTTGCCTCAAGATGATCCTAGCGCCGTCTTTTTTctcgagCGG-3′) were designed according to reference [25 (link)]. The TuD sequence was cloned into lentiviral shuttle vector plenti6-U6 vector.
For miR-31 sensor vector, the 3′-UTR of ISL1 gene including potential miR-31 binding sites was amplified from cDNAs prepared from Hep-12 cells, and the corresponding mutant 3′-UTRs were obtained by overlap-extension PCR. These amplification products were subsequently cloned into the downstream of firefly luciferase gene of the pGL3-control plasmid (Promega, Madison, WI).
Lentiviral constructs were transfected with the ViraPower Packaging Mix (Invitrogen) into 293FT cells to generate lentivirus. Cells infected with virus are selected by 5 μg/ml blasticidin (Invitrogen). The pool of antibiotic-resistance cells were used for subsequent assay.

3′-Untranslated region (3′-UTR) reporter plasmids for miR-200c were constructed via insertion of miR-200c seed sequence into the XbaI restriction site 3′ to luciferase gene in the pGL3-control plasmid (Promega, Madison, WI, USA). Briefly, HEK-293 cells were seeded in 24 well cell culture clusters (Corning Incorporated; Corning, NY, USA). When reached 70% confluences, the cells were co-transfected with hsa-miR-200c/control miRNA and wild/mutated 3’-UTR of BMI-1, E2F3. After 48 h of transfection, the cells were harvested for firefly and Renilla luciferase activity assay. The Renilla luciferase activities were used to normalize the transfection efficiency.

The pGL3 luciferase reporter construct containing 500 bp 5′-flanking sequence of human PKCβ promoter was kindly provided by Dr. Alan P. Fields (Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida). Promoter activity was assayed by transfection of 1 μg pGL3-PKCβ promoter construct into HEK293 cells stably expressing stabilin-1 or empty vector clones using FuGene HD transfection reagent (Roche) as described by manufacturer. In parallel, HEK293 cells were transfected with 1 μg pGL3 basic plasmid or 1 μg pGL3 control plasmid (both from Promega). All cells were co-transfected with 200 ng of pRL-SV40 vector expressing Renilla luciferase (Promega) as an internal control for transfection efficiency. Forty eight hours after transfection, firefly and Renilla luciferase activity was measured using Dual-Luciferase Reporter Assay System from Promega according to manufacturer instructions. Luciferase activity was measured using luminometer Tecan Infinite 200. Results are expressed as firefly luciferase activity normalized to Renilla luciferase and firefly luciferase activity of cells transfected with pGL3 control plasmid.