Prl tk e2241

HEK293T and CHO cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) (SH30022.01B, HyClone, Logan, USA) with 10% fetal bovine serum (FBS) (P30-330250, PAN-Biotech, Aidenbach, Germany) in 12/48-well plates and Lipofectamine^TM 2000 (11668027, Invitrogen) was used for transfection according to the routine protocol. For luciferase assays, cells per well was transfected with 0.4 μg recombinant constructs and 10 ng pRL-TK (E2241, Promega). Then luciferase activities were measured by a dual-luciferase reporter assay system (Promega, Madison, WI, USA) and a Modulus Single Tube Multimode Reader (Turner Biosystems, Sunnyvale, CA, USA) according to the manufacturer’s protocol. The experiments were repeated at least 3 times, and the results were expressed as the means ± SD.

HEK293T (3142C0001000001715) and COS7 (3142C0001000000088) cells were obtained from China Center for Type Culture Collection and cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) (SH30022.01B, HyClone, Logan, USA) with 10% fetal bovine serum (P30-330250, PAN-Biotech, Aidenbach, Germany) in 6/48-well plates and LipofectamineTM 2000 (11668027, Invitrogen, CarIsbad, USA) was used for transfection according to the routine protocol. For luciferase assays, cells per well was transfected with 0.5 μg recombinant constructs and 1 ng pRL-TK (E2241, Promega Madison, USA). Then luciferase activities were measured by a dual-luciferase reporter assay system (Promega) and a Modulus Single Tube Multimode Reader (Turner Biosystems, Sunnyvale, CA, USA) according to the manufacturer’s protocol. The experiments were repeated at least 3 times, and the results were expressed as the mean ± SEM.

The target gene of miR-136-5p was predicted using the web-based bioinformatic resource (starBase). Dual-luciferase reporter gene assay was conducted in order to verify whether IL-6 and CRP were direct target genes of miR-136-5p. The pMIR-reporter plasmid (MLCC13738, Miaolingbio Inc., Wuhan, Hubei, China) was introduced using the endonuclease sites SpeI and Hind III. The Mut site of complementary sequence was designed on the IL-6-Wt and CRP-Wt. IL-6-Mut or CRP-Mut plasmid was constructed with promoter-Renilla luciferase reporter plasmid (PRL-TK; E2241, Promega Corporation, Madison, WI, USA) used as the internal reference. miR-136-5p mimic and miR-136-5p negative control (NC) were co-treated with luciferase reporter plasmid and then were transferred into human embryonic kidney (HEK)-293T cells (CRL1415, Shanghai Xin Yu Biotech Co., Ltd, Shanghai, China). Finally, the fluorescence density was detected using a fluorescence detector (Glomax20/20, Promega Corporation, Madison, WI, USA) [39 (link)].

Genomic DNA from GM12282 and GM11931 was extracted using the Quick-DNA Miniprep kit (Zymo Research) and used as a template to amplify the DNA sequence of 490 bp (REF) or 485 bp (ALT) centered on rs143348853, respectively. The PCR products were inserted using the BamHI and SpeI sites in the STARR-seq luciferase vector (#99297, Addgene). We found that there is a second genetic variant that differs between the two LCLs in the region of interest (rs11867847); in order to modify this polymorphism, the REF template was amplified in two pieces using a second pair of primers (site-directed mutagenesis). See Supplementary Table 3 for the list of primers. 500,000 MEC1 cells were washed once with calcium and magnesium-free PBS and nucleofected with 1 μg of the luciferase plasmids (ALT, REF, or empty vector) and 1 μg of a plasmid expressing Renilla Luciferase (pRL-TK E2241, Promega) using the Neon Transfection System 10 μl kit (Thermo Scientific) with the following reagents and parameters: R and E buffers, 2 pulses, 1100 V and 30 ms. Cells were immediately cultured with IMDM GlutaMAX with 10% FBS without antibiotics. 24 h later, luciferase expression was measured using the Dual-Luciferase Reporter Assay System (Promega). A total of 4 biological replicates were obtained.

To obtain reporter plasmid 3xPSMA4-ARE-Luc, three copies of the ARE sequence (TGACTCTGCA) from the promoter region of the human PSMA4 gene were inserted into the pGL4.37 Vector (Promega, Madison, WI, USA) using XhoI and SacI restriction enzymes, as previously described [5 (link)]. Co-reporter plasmid encoding renilla luciferase pRL-TK (E2241) was purchased from Promega (Madison, WI, USA). Constructs pCMV-dR8.2 (Addgene, #8455) and pCMV-VSV-G (Addgene, #8454) required for lentiviral transduction were a kind gift from Bob Weinberg (MIT, Boston, MA, USA). Plasmid Ub^G76V-GFP (Addgene, #11941) was a kind gift from Nico Dantuma, and construct pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP (Addgene, #71237) was a kind gift from Charles Gersbach.