96 well plate luminometer

Luciferase reporter assays were performed as previously described [51 (link)]. Briefly, cells (2 × 10⁵) were seeded into 24-well plates and then transiently transfected with TOP FLASH or FOP FLASH and Renilla pRL-TK plasmids. (cat: E2231; Promega) using Lipofectamine 3000 Reagent (cat: 11668019, Thermo Fisher Scientific). After 48 h, relative luciferase activity was measured using a dual-luciferase reporter assay detection kit (cat: E1980; Promega). Briefly, the culture medium was discarded, and the cells were washed with PBS and lysed with passive lysis buffer (Promega). The cell lysates were transferred to a 96-well plate luminometer (Corning, New York, USA) and analyzed using a microplate reader (Bioteck, Winooski, VT, USA). Calculated the ratio of firefly luciferase luminescence (TOP FLASH or FOP FLASH) to Renilla luciferase luminescence for each sample, and then normalized the ratio of experimental sample wells to the ratio of control sample wells

NSCLC cells were seeded into 24-well plates (3 × 10⁴ cells/well in triplicate) and cultured at 37 °C under 5% CO₂ for 24 h. Cells were then transiently transfected with TOP flash or FOP flash and Renilla pRL-TK plasmids using Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. After 48 h, luciferase activity was determined using a dual-luciferase reporter assay (Promega, Madison, WI, USA). Briefly, the culture medium was discarded, and the cells were washed with PBS and lysed with passive lysis buffer (Promega). The cell lysates were transferred to a 96-well plate luminometer (Corning, New York, USA) and analyzed using a microplate reader (Bioteck, Winooski, VT, USA, #Synergy H/MD).

A total of 3,000 human Sertoli cells were seeded onto 96-well plates (Corning, United States). After 24 h of culture, Lipofectamine 3000 transfection agent (Sigma, United States) was employed to transfect miR-100-3p mimics or the mimics control to these cells. Forty-eight hours later, human Sertoli cells were transfected with 500 ng plasmids containing the binding sequence in 3′ untranslated regions (UTRs) of SGK3, firefly luciferase (reporter), or the renilla luciferase (internal control) (Genecreate, China) using the Lipofectamine 3000 reagent (Sigma, United States). Forty-eight hours after transfection, human Sertoli cells were lysed, and luciferase activity was determined using the 96-well plate luminometer (Corning, United States). The results were normalized to cells transfected with miRNA mimics control.