Cell culture lysis reagent

Mouse brain and lung tissues and mouse whole blood samples were collected from a control (mice that were not injected with tumour cells) and tumour-inoculated mice. The extracted tissues were then cut into ~25 mg pieces and homogenised in a cell culture lysis reagent (Promega). The tissue lysates were maintained at 4 °C for 30 mins with agitation and either transferred directly to an opaque 96-well plate or “spiked” with a titration of U87MG-Fluc or MU20-Fluc cells before being added to the opaque 96-well plate. Luciferase readings were performed using a luciferase assay kit (Promega) and a luminometer (Promega). The luciferase activity was presented as relative luciferase units (R.L.U).

hDPSCs were plated at a concentration of 5.0 × 10⁴ cells/well in a 24-well plate and were lysed 24 h after transfection using 100 μL of lysis buffer (Cell Culture Lysis Reagent; Promega). Luciferase activity was measured using a luciferase assay substrate (Luciferase Assay System; Promega) and a luminometer (AB-2200; ATTO, Tokyo, Japan).

ROS were determined using 2’,7’-dichlorofluorescin diacetate (DCFDA, Abcam, Cambridge, UK) according to the supplier’s protocol with slight modifications [34 (link)]. Briefly, SK-OV-3 cells were seeded in 96-well plates as mentioned above. The next day, cells were incubated with CUR-NP for 4 h. Tert-butyl hydroperoxide (TBHP, 50 µM) was used as positive control. Cells were subsequently washed using PBS containing Ca²⁺ and Mg²⁺ (pH 7.4) and supplemented with fresh medium. Irradiation was carried out at 457 nm with a radiation fluence of 8.6 J/cm². The cells were washed again with PBS and incubated with culture medium (IMDM without phenol red) containing 25 µM DCFDA for 1 h. Cell culture lysis reagent (Promega, Mannheim, Germany) was used to lyse the cell and fluorescence was recorded using a FLUOstar Optima plate reader (λ_ex 480 nm/λ_em 520 nm).

BeWo or JEG-3 cells were treated with scrambled siRNA (sicon) or siRNA against the 3′-untranslated region (UTR) of p21 (sip21) or mixed siRNAs against the coding region of p21 (sip21 #2). After 24 h, the syncytin-2 (2 µg) promoter plasmid [43 (link)] was transfected with FuGENE^® HD transfection reagent (Promega GmbH, Walldorf, Germany) for 48 h. Cells were harvested with cell culture lysis reagent from Promega GmbH (Walldorf, Germany; #E1531) and the assays were performed with the luciferase assay system from Promega GmbH (Walldorf, Germany; #E1501).

A total of 8,300 TZM-bl cells were seeded per well in a 96-well plate. At a confluence of ~40%, the cells were infected with 100 µl of cell-free supernatant of infected CD4⁺ T cells obtained 7 days postinfection in the presence of DEAE-dextran (final concentration, 40 µg/ml). Forty-eight hours later, the cells were lysed with Cell Culture Lysis Reagent (catalog no. E153A; Promega), lysates were frozen at −80°C for 2 h, and relative light units (RLU) were determined using the luciferase assay system (Promega). The RLUs obtained were normalized to the capsid antigen p24 levels to obtain RLUs per picogram of p24 capsid antigen. Each measurement was performed in duplicate.

The Huh7/Rep-Feo cell line harboring the subgenomic replicon RNA of the N strain (genotype 1b) was seeded at 2 × 10⁴ cells per well in a 48-well plate and incubated at 37 °C for 24 h. A serial dilution of Compounds 2 and 3 was added to each well of the plate. The treated cells were harvested 72 h post-treatment and lysed in cell culture lysis reagent (Promega, Madison, WI, USA). Luciferase activity in the harvested cells was estimated with a luciferase assay system (Promega). The resulting luminescence was detected by a Luminescencer-JNR AB-2100 (ATTO, Tokyo, Japan). The anti-HCV activity was calculated as the intensity of the luminescence relative to that of the control (in the absence of Compounds 2 and 3 but in the presence of DMSO).

HEK 293 cells seeded into a 100-mm dish were transfected with 4.5 μg pNF-κB-Luc or pAP-1-Luc cis-reporter plasmids (Agilent Technologies, Inc., Santa Clara, CA, USA) for 6 h at 37 °C. The gWIZ-green fluorescent protein (GFP) plasmid was used as an internal control for normalization. Transfected cells were split into 12-well plates and further transfected with or without FLAG-tagged DUSP5 expressing plasmids using OmicsFect. After 32 h of transfection, cells were treated PMA for additional 16 h. Cells were lysed with Cell Culture Lysis Reagent (Promega, Madison, WI) and then extracted samples were analyzed with Luciferase Assay Reagent (Promega). Luminescence from the product was measured with a multiwall plate reader (Synergy H1: Luminometer filter). Relative fold induction of luciferase activity was determined and normalized to GFP. All luciferase assays were repeated at last three times.