Pgvb2

The luciferase reporter plasmids pRORE1x3-wt, pRORE2x3-wt, pRORE1x3-mt, and pRORE2x3-mt containing triplet repeats of NCEH1-ROREs were constructed. Synthetic oligonucleotides corresponding to sense and antisense ROREs, as used for EMSA (Supplementary Table S1), were phosphorylated with T4 DNA polynucleotide kinase (Takara Bio, Shiga, Japan), mixed, and annealed. Each resulting double-stranded oligonucleotide was cloned into the SmaI site of the reporter vector PGVP2 (Nippon Gene, Tokyo, Japan) containing the SV40 promoter. Moreover, the human NCEH1 promoter [pNCEH1(− 1689/+ 128)], from − 1689 to + 128 relative to the TSS, was amplified by PCR and inserted as a KpnI/MluI-fragment into the promoterless luciferase expression vector PGVB2 (Nippon Gene). The wild-type and mutant promoter sequences for RORE2 [pNCEH1(− 140/+ 128)-wt and -mt] were generated by PCR using the pNCEH1(− 1689/+ 128) plasmid as a template. Briefly, the primer sets Promoter-RORE2-FW-KpnI, Promoter-RORE2mt-FW-KpnI, and Promoter-RV-MluI were synthesized to incorporate the desired mutation (Supplementary Table S1), and a continuous fragment was produced. This fragment was then cloned as a KpnI/MluI-fragment into PGVB2. All cloned plasmids were purified using a Qiagen Plasmid Mini Kit (Qiagen, Valencia, CA, USA). Inserts were confirmed by sequencing using PGVB2-FW and PGVB2-RV primers.

The human PEPCK promoter, from -1305 to +51 relative to the transcriptional start site, was amplified by PCR and inserted into the luciferase expression vector PGVB2 (Nippon Gene, Tokyo, Japan). This plasmid was then used in linker-scanning mutagenesis [27 (link)] to generate the deletion plasmids dC1 (deletion of C1 from -1305 to -323), dC2C3 (deletion of C2 and C3 from -323 to -251), dC4 (deletion of C4 from -251 to -203), and dC5 (deletion of C5 from -78 to -43). The mutated promoter ROREmt was generated by PCR using the wild type sequence as template. Briefly, the primers ROREmt-FW and ROREmt-RV were synthesized to incorporate the desired mutation (Fig 1 and S1 Table). These primers were then used in PCR reactions with PGVB2-FW and PGVB2-RV, respectively, to generate overlapping fragments that contain the mutation. The resulting fragments were then used in a final PCR reaction with PGVB2-FW and PGVB2-RV to generate a continuous mutated fragment, which was then cloned as a MulI/HindIII-fragment into PGVB2. The mutated promoter C3mt was generated in a similar manner using the primers C3mt-FW and C3mt-RV to incorporate mutations in the C3 binding site (Fig 1 and S1 Table). Plasmids were purified using QIAGEN Plasmid Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol.

Luciferase assay was carried out as previously described [28 (link),32 (link),34 (link),70 (link)]. Briefly, LuM1 or MMP3 KO cells were seeded at 2 × 10⁴ cells per well in a 96-well plate and cultured for 24 h in RPMI 1640 medium with 10% FBS. The human CCN2/CTGF promoter-driven firefly luciferase reporter plasmid (pTS589) [32 (link)] and mutant plasmids including ⊿TRENDIC [33 (link)], ⊿TRENDIC/⊿BCE [28 (link),33 (link)], pDS3 (202-bp promoter), and pDS4 (88-bp promoter) were described previously [32 (link)]. Any one of these reporter plasmids (100 ng/well) or the promoterless vector PGV-B2 (Nippon Gene, Tokyo, Japan) was cotransfected with a Renilla luciferase control vector phRL-TK (Promega, Madison, WI) at 20 ng/well using Lipofectamine 3000 transfection reagent (ThermoFisher). Media were replaced with RPMI 1640 without FBS and EVs were added to the media at concentrations of 0, 5 or 10 µg/mL and cells were incubated for 24 h. Cells were lysed and luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).