Lipofectamine kit

PVT1 siRNA (si-PVT1) and siRNA negative control (si-NC) were synthesized by Shenggong Bioengineering Co., Ltd. (Shanghai, China) (Table 1). MiR-93-5p mimics, miR-93-5p inhibitor, mimics negative control (mimics-NC), and inhibitor negative control (INC) were purchased from Jima Pharmaceutical Technology Co., Ltd. (Shanghai, China). Cells were transfected with the above agents by using a Lipofectamine kit (Invitrogen). Cells without transfection were considered as the control. The transfection efficiency was identified by qRT-PCR. After the transfection for 48 h, cells were incubated in DMEM containing 5.55 mmol/l (normal physiological environment, NG group) or 30 mmol/l d-Glucose (diabetic physiological environment, HG group) for another 48 h. Cells were collected for the following assay.

Luciferase reporter constructs of AP-1-Luc or GAS/ISRE-Luc were used in the determination of transcriptional activity of AP-1 or IRF3 (Promega, Madison, WI, USA), and those of TNF-α (−1260/+60)-Luc, iNOS (−1592/+183)-Luc or IFN-β (−1814/+11)-Luc for the promoter activity of TNF-α, iNOS or IFN-β gene40 (link)41 (link). Cells were transfected with each reporter construct in combination with Renilla control vector using a lipofectamine kit (Invitrogen, Carlsbad, CA, USA). Cell extracts were subjected to dual-luciferase assay (Promega, Madison, WI, USA). Firefly luciferase activity as the reporter was normalized to Renilla activity as a reference for transfection efficiency.

B16F0 cells were transfected with plasmid construct encoding firefly luciferase reporter fused to the promoter region of MITF-M (MITF-M-Luc) or that of TYR (TYR-Luc) along with Renilla control vector using lipofectamine kit (Invitrogen, 11668), and incubated for 48 h. The cells harboring each reporter plasmid were stimulated with 100 nM α-MSH in the presence of BI2B for 20 h, and harvested for dual-luciferase assays (Promega, E1910). The promoter activity of MITF-M or TYR was measured by Firefly luciferase activity, and normalized by Renilla luciferase activity, indicating the transfection efficiency.

The luciferase constructs containing the 2-kb promoter of MMP-9 gene were previously described (11 (link)). The putative SOX5-binding site from −1,300 to −900 was deleted by using the Quick Change II site-directed mutagenesis kit (Stratagene, Cedar Creek, TX, USA) as previously described (11 (link)). MH7A cells were seeded on 24-well plates at 2 × 10⁵ cells/well and transfected with Ad-SOX5 or Ad-EGFP for 48 h. After overexpression of SOX5, 1 µg of full-length promoter/luciferase fusion plasmid DNA or the plasmid that deletion of SOX5-binding site was transfected into MH7A by Lipofect-AMINE kit (Invitrogen), respectively. 100 ng of pRL-SV40 control vector (Renilla luciferase) was co-transfected as an internal control for transfection efficiency. Luciferase activity in cell lysates was measured after 24 h with a dual luciferase reporter assay (Promega).

B16F0 cells were transfected with luciferase reporter construct, MITF-M-Luc or TYR-Luc, using lipofectamine kit (Invitrogen, 11668). Renilla control vector was co-transfected as a reference of transfection efficiency. Lysates of transfected cells were subjected to dual luciferase assays (Promega, E1910). Firefly luciferase activity, indicating the promoter activity of MITF-M or TYR, was normalized to Renilla luciferase activity.

B16-F0 cells were transfected with the reporter construct, Tyro (−2236/+59)-Luc or MITF-M (−2200/+95), in combination with the Renilla control vector using a lipofectamine kit (Invitrogen, Madison, WA, USA). The cell extract was subjected to a dual luciferase assay with a premixed kit (Promega, Carlsbad, CA, USA). Firefly luciferase activity, reporting the promoter activity of the MITF-M or Tyro gene, was normalized to the Renilla activity as a reference of transfection efficiency.