Alamarblue cytotoxicity assay

On-TARGET plus siRNA SMARTpools targeting genes encoding for prohibitin (PHB), peripherin (PRPH), phosphatidylethanolamine binding protein 1 (PEBP1), enolase-1 (ENO1), stomatin-like protein 2 (STOML2), protein disulfide isomerase family A member 3 (PDIA3), DEP domain containing MTOR-interacting protein (DEPTOR) and non-targeting siRNA control (NTC) were purchased from Dharmacon (GE Life Sciences). The siRNA SMARTpool sequences are shown in S3 Table. Briefly, various concentrations of siRNAs were prepared using DharmaFECT Cell Culture Reagent in a total volume of 50 μL (DCCR) (Dharmacon, GE Life Sciences) and incubated for 5 minutes at RT. DharmaFECT 1 Transfection Reagent (1 μL) (Dharmacon, GE Life Sciences) was then added to the siRNA mixture and topped up to final volume of 100 μL with DCCR. After 30 minutes incubation with transfection reagent at RT, NSC-34 cells (2.5 × 10⁵ cells/ 400 μL) were seeded onto 24 wells plate and reverse transfected with the siRNA constructs. At 48 hour post-transfection (h.p.t.), cellular viability was assessed using alamarBlue cytotoxicity assay (Invitrogen) and the cells were subjected to EV71 infection at M.O.I. 10. Culture supernatant and cell lysate were harvested at 48 h.p.i. for viral titer determination and Western blot analysis. Gene silencing of PHB was also performed in RD and SK-N-SH cells following the same procedure.

BHK21, HeLa and HSMM cells were seeded on 96-well plates at densities of 12,500, 15,000 and 20,000 cells per well, respectively. After overnight incubation, cell culture media was replaced with media containing proteasome inhibitors or vehicle control at various concentrations and incubated for 24h (BHK21 and HSMM) or 48h (HeLa) at 37°C. Cell viability was then determined using the alamarBlue® cytotoxicity assay (Invitrogen) as previously described [69 (link)].