FIGURE

Fig 8

ID
ZDB-FIG-210304-13
Publication
Lu et al., 2021 - A novel role of Zebrafish TMEM33 in negative regulation of interferon production by two distinct mechanisms
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Fig 8

(A and B) Overexpression of TMEM33 restores MAVS-mediated decrease of viral titer. EPC cells seeded in 24-well plates overnight were transfected with 0.25 ?g of MAVS-Myc and 0.25 ?g 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 were seeded in 6-well plates overnight and then transfected with 2 ?g of MAVS-Myc and 2 ?g of TMEM33-HA 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 ?-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 anti-N, anti-P, anti-Myc, anti-HA, and anti-?-actin Abs, respectively. (E) TMEM33 enhances the SVCV-induced K48-linked ubiquitination of MAVS. EPC cells were transfected with 5 ?g MAVS-Myc, 5 ?g TMEM33-HA or empty vector, and 1 ?g Ub-HA, Ub-K48O-HA or Ub-K63O-HA. At 24 h post-transfection, the cells were uninfected or infected with SVCV (MOI = 1) for 18 h, then the cells were treated with MG132 for 6 h. Cell lysates were IP with anti-Myc-affinity gel. Then the immunoprecipitates and WCLs were analyzed by IB with the indicated Abs. (F-J) Overexpression of TMEM33 inhibits the expression of ifn (F), vig1 (G), isg15-1 (H), irf7 (I), and rig-i (J) induced by MAVS. EPC cells seeded in 6-well plates overnight were transfected with 2 ?g of TMEM33-HA or empty vector together with 2 ?g of MAVS-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 ?-actin gene and were represented as fold induction relative to the transcriptional level in the control cells, which was set to 1. (K and L) TMEM33-?TM1 and TMEM33-?TM2 have no effect on MAVS-mediated suppression of viral genes transcription and protein expression. EPC cells were seeded in 6-well plates overnight and transfected with the indicated plasmids (2 ?g 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.

Expression Data

Expression Detail
Antibody Labeling
Phenotype Data

Phenotype Detail
Acknowledgments
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