Victor x2 multilabel reader

For the BCAR4 luciferase reporter assay, pRL-TK-BCAR4 or pRL-TK-BCAR4 mutant vectors and pGL3 control (Promega, Madison, WI) were transfected into HEK-293T cells along with miR-644a mimic or miR-644a inhibitor using ExFect® Transfection reagent (Vazyme) according to the instructions of the manufacturer. For miR-644a target gene CCR7 luciferase reporter assay, pRL-TK-CCR7-3΄UTR or pRL-TK- CCR7-3΄UTR mutant vectors and pGL3 control (Promega) were transfected into HEK-293T cells along with miR-644a mimic or miR-644a inhibitor using ExFect® Transfection reagent (Vazyme). The luciferase activity was measured 48 h after transfection using the Dual-Glo luciferase reporter assay system (Promega) according to the guidelines of the manufacturer. Luminescence values were recorded by a Victor X2 multilabel reader (PerkinElmer).

The in vitro cytotoxicity of the VIP126 and SN38 payloads was investigated in parental NCI-H1975 and in transfected NCI-H1975-P-gp and NCI-H1975-BCRP NSCLC cells. Cells (6 × 10³ cells/well) were plated in RPMI-1640 medium supplemented with 2% fetal calf serum (FCS) minimal media (MM) in 96-well plates and allowed to adhere at 37 °C/5% CO₂ for 24 h. Then, the test compounds VIP126 and SN38 were added at concentrations of 1000–250–62.5–15–4–1–0.25 nM in MM (no elastase, final dimethyl sulfoxide (DMSO) concentration of 0.1%) in triplicates. The cells were incubated at 37 °C/5% CO₂ for 72 h. Then, Alamar Blue (#DAL1100, Invitrogen, Waltham, MA, USA) was added at a 1:1 ratio, the cells were incubated at 37 °C/5% CO₂ for 4 h, and fluorescence (530 nm/590 nm) was measured with a VICTOR X2 Multilabel Reader (PerkinElmer, Waltham, MA, USA). The fluorescent signal was directly proportional to the number of viable cells. The half-maximal growth inhibition (IC₅₀) was determined from the dose–response curves using the GraphPad Prism software.

The wild-type or mutant forms of HOTAIR were cloned into pRL-TK vectors, and the pGL3 vector (Promega) was used as a control. These constructs were co-transfected into HEK-293T cells with miR-19a-3p mimic or miR-19a-3p inhibitor using ExFect® Transfection reagent (Vazyme). After 48 hours of transfection, the luciferase activity was measured using the Dual-Glo luciferase reporter assay system (Promega). The luciferase activity values were quantified using a Victor X2 multilabel reader (PerkinElmer).