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Fig. 3 | Virology Journal

Fig. 3

From: Herpes simplex virus type 2 infection triggers AP-1 transcription activity through TLR4 signaling in genital epithelial cells

Fig. 3

HSV-2 triggered AP-1 transcriptional activation via TLR4-MyD88/TRIF pathway in genital epithelial cells. a HSV-2 infection induced AP-1 activation. HEC-1-A cells were co-transfected with pAP-1-luc and pRL-TK. After 24 h, cells were mock-treated or treated with acyclovir (5 μg/ml), and then infected with HSV-2 or UV-treated HSV-2. RLUs were determined as described. b HSV-2 could induce c-Jun phosphorylation. HEC-1-A cells were infected with HSV-2 (moi = 1), and at indicated time point, cells were harvested and lysed. c-Jun and phosphorylated c-Jun were determined via western blot. c TLR4 specific siRNA knockdown efficiency determination. HEC-1-A cells were transfected with TLR4-specific siRNA oligonucleotide, and the efficiency of TLR4 knockdown was evaluated after 24 h via real-time PCR. d Knockdown of TLR4 could attenuate HSV-2-induced AP-1-driven transcription activity and phosphorylation of c-Jun. For AP-1 activity assay, HEC-1-A cells were co-tranfected with TLR4-specific siRNA oligonucleotide or negative control (NC), pAP-1-luc and pRL-TK plasmids. Cells were mock-infected or infected with HSV-2 (moi = 1) 24 h post-transfection. RLUs were determined as described. For p-c-Jun detection, HEC-1-A cells were transfected with TLR4-specific siRNA oligonucleotide or NC. And after 24 h, cells were mock-infected or infected with HSV-2 (moi = 1). c-Jun and phosphorylated c-Jun were determined via western blot 24 h p.i. ef Knockdown of TLR4 could impede HSV-2 virions production. HEC-1-A cells transfected with TLR4 siRNA or NC siRNA. After HSV-2 infection for 24 h, cells were freezing and thawing, and the released virions were titrated in a Vero-ICP10P luciferase reporter system (e). Or HSV-2 gD expression was evaluated via In-cell Western as described (f). g MyD88 and TRIF specific siRNAs knockdown efficiency determination. The approach was the same as that described in (c). hi Knockdown of MyD88 or TRIF expression could attenuate HSV-2-induced AP-1-driven transcription activity and phosphorylation of c-Jun (h), and impede HSV-2 replication (i). The approach was the same as that described in (d and f). All experiments were performed three times, and the representative results were shown

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