Biolux gaussia luciferase flex assay kit

Gaussia luciferase assays were performed as previously described56 (link). Briefly, at the selected time points, 10 μL of media was collected from the supernatant and stored at −20 °C. Gaussia luciferase activity was measured using the Biolux Gaussia luciferase Flex Assay kit (NEB) in a plate reader (Synergy HT, Biotek) and normalized to the luciferase activity of the secreted cypridina luciferase under the control of a constitutive CMV promoter (pCMV-CLuc) or protein concentration. Details and validation of the constructs are shown in Figure S4.

For transfection experiments, HeLa Ohio cells were cotransfected in 24-well plates with pCMV-GLuc 2- and pRK5-Myc-derived plasmids at a ratio of 1:3 for 24 h. For infection experiments, HeLa Ohio cells stably expressing Gaussia luciferase (HeLa-Gluc) were plated at a density of 0.2 × 10⁶ cells per well in a 24-well plate, and each time point was performed in triplicate. Cells were infected with HRV16 at an MOI of 20 for 1 h at room temperature and then washed in PBS before adding fresh medium and starting a 7-h time course. During the infection time course, culture medium was removed and replaced every 30 min and cells collected every 1 h. As controls, HeLa-Gluc cells either were not infected with virus or were not infected but treated with brefeldin A (BFA) at a final concentration of 5 μg/ml. To determine the Gluc activity in the cells, the wells were washed with PBS and the cells lysed in 250 μl of luciferase cell lysis buffer (B3321; New England BioLabs). Gluc secreted in the media and remaining in the cells was assayed using the BioLux Gaussia luciferase flex assay kit (E3308S; New England BioLabs) according to the manufacturer's instructions. Luminescence was analyzed on a FLUOstar OMEGA plate reader (BMG Labtech) as recommended. Each time point was determined in triplicate, and the experiment was performed independently three times.

Didemnin B (Did B, kindly provided by the Natural Product Branch, NCI, USA) was dissolved in DMSO as a 10 mM stock and stored at − 80 °C. Confluent HEp-2 cells were treated with a range of concentrations from 0 nM to 16 nM. The same volume of DMSO was used as vehicle control. For effect of Did B on translation, HEp-2 cells were transfected with pCMV-Gluc2 plasmid and re-seeded into 24 well plates containing various concentration of Did B as described. The luciferase activity in culture supernatant was measured after 24 and 48 h of treatment using Biolux Gaussia luciferase Flex Assay kit (NEB). Subsequent experiments were conducted with a final concentration of 2.5 nM of Did B. Cell proliferation was monitored using CellTiter 96® Aqueous One Solution Cell Proliferation assay (Promega), as instructed by the manufacturer.

Plasmids for constitutive expression of the TFs were purchased from Addgene (pCMV5-FLAG-FoxO1, pSV Sport SREBP-1c, pEGFP-C1 NFAT3, pcDNA flag PPAR gamma) or Invitrogen (pCMV-Sport6-CREB). 293T/17 cells were seeded in 6-well tissue culture plates, and ∼1 µg of each expression plasmid was cotransfected along with the corresponding reporter plasmid (∼1 µg) using the calcium phosphate transfection. For control experiments, the same amount of pEYFP-N1 plasmid (constitutive expression of yellow fluorescent protein) was transfected. At 48 h post-transfection, supernatants were collected and luciferase activity measured using the BioLux Gaussia Luciferase Flex assay kit (New England Biolabs, Ipswich, MA). Additionally, the PPARγ reporter plasmid was validated using thiazolidinedione (TZD) as the agonist. PPARγ was overexpressed from plasmid (pcDNA flag PPAR gamma) in 3T3-L1 PPARγ reporter cells and 25 µM TZD was used to activate PPARγ for 24 h. The C/EBPβ reporter cell line was validated by up-regulating C/EBPβ with 100 ng/mL oncostatin M (OSM) for 12 h [53] (link). Luciferase activity in the supernatant was determined as described above.

For Gaussia luciferase, the secreted luciferase activity in the cell culture medium was measured using a BioLux^®Gaussia Luciferase Flex Assay Kit (New England Biolabs). Gaussia luciferase activity was quantified using a LumiStar Optima luminescence counter (BMG LabTech, Offenburg, Germany). For the full-length infectious models (HEV-p6), intracellular viral RNA was quantified. RNA was isolated using a Machery-Nucleo Spin RNA II kit (Bioke, Leiden, The Netherlands) and quantified using a NanoDrop ND-1000 spectrophotometer (Wilmington, DE, USA). cDNA was prepared from total RNA using a cDNA Synthesis Kit (Takara Bio Inc, USA). The HEV RNA level was quantified using a SYBR Green–based real-time PCR assay (Applied Biosystems® SYBR® Green PCR Master Mix, Life Technologies, CA, USA) according to the manufacturer’s instructions. The PCR steps consisted of a 10 min holding stage (95 °C) followed by 40 cycles of 15 s at 95 °C, 30 s at 58 °C, and 30 s at 72 °C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene to normalize gene expression. Relative gene expression was normalized to GAPDH using the formula 2^−ΔΔCT (ΔΔCT = ΔCT_sample − ΔCT_control). The HEV primer sequences were as follows: HEV-F, 5’-ATTGGCCAGAAGTTGGTTTTCAC-3’; HEV-R, 5’-CCGTGGCTATAATTGTGGTCT-3’; GAPDH-F, 5’-TGTCCCCACCCCCAATGTATC-3’; GAPDH-R, 5’CTCCGATGCCTGCTTCACTACCTT-3’.

CHO cells carrying the MEEVS‐reporter‐MAC2 were plated at a density of 2 × 10⁵ in 24‐well tissue culture plates and cultured for 48 h. The conditioned media and cells were separately collected and used to measure luciferase activity. Activities of GLuc and CLuc luciferase secreted into the supernatant were measured using a BioLux® Gaussia Luciferase Flex Assay Kit (NEB) and a BioLux® Cypridina Luciferase Assay Kit (NEB), respectively. Twenty microliter of various conditioned cell culture media were transferred to flat solid bottom and opaque‐walled white 96‐well plates (Nunc, Roskilde, Denmark). GLuc and CLuc bioluminescence values were measured immediately after the addition of 50 μL of substrate solution. Expression of ELuc in cells was measured with the Emerald Luc Luciferase Assay Reagent (TOYOBO). Cells were resuspended at a density of 1 × 10⁵ cells per 50 μL with PBS and transferred to flat solid bottom and opaque‐walled white 96‐well plates (Nunc). ELuc bioluminescence values were measured after the addition of 50 μL of assay reagent and incubation for 10 min. An Infinite F500 luminometer (Tecan, Crailsheim, Germany) was used to determine luciferase activity.