Fig 2

(A and B) Overexpression of TMEM33 suppresses poly I:C/SVCV-induced IFNφ1pro/ISRE-Luc activation and displays a dose-dependent manner. EPC cells were seeded in 24-well plates and transfected the next day with 250 ng IFNφ1pro-Luc (A) or ISRE-Luc (B) and 25 ng pRL-TK, plus pcDNA3.1-TMEM33 (250 ng or 200/400 ng) or pcDNA3.1(+) (control vector). At 24 h post-transfection, cells were untreated (null) or transfected with poly I:C (1 μg/ml) or treated with SVCV (MOI = 1). Luciferase activities were monitored at 24 h after stimulation. The promoter activity is presented as relative light units (RLU) normalized to Renilla luciferase activity. (C-G) Overexpression of TMEM33 inhibits the expression of ifn (C), vig1 (D), isg15-1 (E), irf7 (F), and rig-i (G) induced by poly I:C in EPC cells. EPC cells seeded in 6-well plates overnight were transfected with 2 μg TMEM33-Myc or empty vector and transfected with poly I:C at 24 h post-transfection. At 24 h after stimulation, total RNAs were extracted to examine the mRNA levels of cellular ifn, vig1, isg15-1, irf7, and rig-i. (H) Effects of TMEM33 RNAi on the expression of endogenous TMEM33. EPC cells were seeded in 6-well plates overnight and transfected with 100 nM si-TMEM33#1, si-TMEM33#2, or si-NC (negative control). At 24 h post-transfection, the cells were transfected with poly I:C or treated with SVCV (MOI = 1). At 24 h post-stimulation, total RNAs were extracted to examine the transcriptional levels of TMEM33. (I and J) Effects of TMEM33 RNAi on the poly I:C/SVCV-induced epc ifn and epc vig1 transcription. EPC cells were seeded in 6-well plates and transfected with 100 nM si-NC or si-TMEM33#2. At 24 h post-transfection, cells were untreated or transfected with poly I:C or treated with SVCV for 24 h before qPCR analysis was performed. The relative transcriptional levels were normalized to the transcription of β-actin and represented as fold induction relative to the transcriptional level in the control cells, which was set to 1. Data were expressed as mean ± SEM, n = 3. Asterisks indicate significant differences from control (*, p < 0.05).