Fig 10

Fig 10

(A and B) Overexpression of TMEM33 increases TBK1-mediated decline of viral titer. EPC cells seeded in 24-well plates overnight were transfected with 0.25 μg of TBK1-Myc and 0.25 μg of TMEM33-HA or empty vector. At 24 h post-transfection, cells were infected with SVCV (MOI = 0.001) for 48 h. Then, cells were fixed with 4% PFA and stained with 1% crystal violet (A). Culture supernatants from the cells infected with SVCV were collected, and the viral titer was measured according to the method of Reed and Muench (B). (C) EPC cells seeded in 6-well plates overnight were transfected with 2 μg of TMEM33-HA or empty vector together with 2 μg of TBK1-Myc or empty vector. At 24 h post-transfection, cells were infected with SVCV (MOI = 1). After 24 h-infection, total RNAs were extracted to examine the mRNA levels of cellular g, l, m, n, and p. The relative transcriptional levels were normalized to the transcriptional level of the β-actin gene and were 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 values (*, p < 0.05). (D) The same samples were prepared similarly as described above for panel C. The lysates were detected by IB with the indicated Abs. (E-I) Overexpression of TMEM33 blocks the expression of ifn (E), vig1 (F), isg15-1 (G), irf7 (H), and rig-i (I) induced by TBK1. EPC cells seeded in 6-well plates overnight were transfected with 2 μg of TMEM33-HA or empty vector together with 2 μg of TBK1-Myc or empty vector. At 24 h after transfection, total RNAs were extracted to examine the mRNA levels of cellular ifn, vig1, isg15-1, irf7, and rig-i. The relative transcriptional levels were normalized to the transcriptional level of the β-actin gene and were represented as fold induction relative to the transcriptional level in the control cells, which was set to 1. (J and K) TMEM33-ΔTM1 and TMEM33-ΔTM2 have a little impact on TBK1-mediated inhibition of viral genes transcription and protein expression. EPC cells were seeded in 6-well plates overnight and transfected with the indicated plasmids (2 μg each) for 24 h. At 24 h post-transfection, cells were infected with SVCV (MOI = 1) for 24 h. qPCR and immunoblot analysis were performed similarly as in C and D. All experiments were repeated for at least three times with similar results.