Glomax multi detection system photometer

The transfection assay was performed with the Dual-Luciferase Reporter Assay System (Promega) in the GloMax-Multi Detection system Photometer (Promega). MiR145-5p mimic, miR145-5p inhibitor, control mimic and inhibitor were synthesized by Gene RIB Bio (Guangzhou, China). 24 h before transfection, cells were seeded into 24-well plates at 1×10⁵ cells/well. MiR145-5p mimic (50 nM) or miR145-5p inhibitor (100 nM) were transfected into the PMVECs using Lipofectamine 3000 (Invitrogen). For luciferase assays, each wild-type or deletion vector was transfected into the cells at 500 ng, together with 50 ng/well of pRL-TK (Promega). 24 h after transfection, luciferase activities were measured with a VARIOSKAN FLASH (Thermo Fisher Scientific). The experiment was performed three times independently, and the average expression levels of the target genes are presented as the mean±s.e.m.

The BmNs (corresponding to BmN-SWU1) cell line derived from the ovary of B. mori (stored in our laboratory) was cultured at 27 °C in IPL-41 medium (AppliChem, Gatersleben, Germany) containing a 10% FBS (Gibco, Grand Island, NE, USA). Transfection of overexpression and luciferase reporter vectors was performed using the X-treme GENE HP DNA transfection reagent (Roche Applied Science, Basel, Switzerland). Luciferase activity was measured using commercially available kits (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The vector p-IE1-Rlucp (stored in our laboratory) was used to drive the expression of Renilla luciferase via the B. mori nuclear polyhedrosis virus IE1 promoter as an internal control. The vector ratio (w/w) between p-VgRP-2.5k and p-IE1-Rlucp was 1:0.1. The ratio (w/w) between the reporter vector and overexpression vectors was 1:1. The measurement of luciferase activity was performed with the Dual Luciferase Reporter Assay kit (Promega, Madison, WI, USA), using the GloMax-Multi Detection System Photometer (Promega, Madison, WI, USA).

One day posttransfection, cells apoptosis was induced by 2.5‐μg/ml actinomycin D for 24 hr. Then, GFP‐positive and mCherry‐positive cells (i.e., transfected cells and infected cells) and GFP‐negative and mCherry‐negative cells (i.e., nontransfected cells and noninfected cells) were separately sorted by flow cytometry on a MoFlo Astrios EQ (Beckman Coulter) high‐speed cell sorter and distributed into 96‐well plates (10⁴ or 4 × 10⁴ cells/well). Caspase 3/7 activity was measured using the caspase‐Glo assay kit (Promega). Briefly, the caspase‐Glo reagent was added to each well (100 μl), and the plate was mixed gently and then incubated at room temperature for 1.5 hr. The luminescence of each sample was measured in a Glomax multidetection system photometer (Promega). The experiments were performed in triplicate.