Efficient Human Cytomegalovirus Replication in Primary Endothelial Cells Is SOCS3 Dependent

Abstract

<b><i>Background:</i></b> In immunocompromised patients, human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality. Suppressor of cytokine signaling (SOCS) proteins are very potent negative regulators of the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. We hypothesized that HCMV exploits SOCS1 and/or SOCS3 to its advantage. <b><i>Methods:</i></b> All experiments were carried out with primary human lung-derived microvascular endothelial cells (HMVEC). SOCS1 and SOCS3 were silenced by transfecting the cells with siRNA. HCMV was propagated and titered on human lung-derived fibroblasts MRC5. Real-time PCR and Western blot were used to detect mRNA and protein levels, respectively. <b><i>Results:</i></b> The data presented show that an efficient replication of HCMV in HMVEC is dependent on SOCS3 protein. Time course analysis revealed an increase in SOCS3 protein levels in infected cells. Silencing of SOCS3 (siSOCS3) resulted in inhibition of viral immediate early, early, and late antigen production. Consistently, HCMV titers produced by siSOCS3 cultures were significantly decreased when compared to control transfected cultures (siCNTRs). STAT1 and STAT2 phosphorylation was increased in siSOCS3-infected cells when compared to siCNTR-treated cells. <b><i>Conclusion:</i></b> These findings indicate the implication of SOCS3 in the mechanism of HCMV-mediated control of cellular immune responses.

The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina

“…The specimens were observed under a laser confocal microscope (LSM700 or 900, Carl Zeiss). The primary antibodies used in this study were guinea pig anti-Rax (1:500) ( 36 ), goat anti-Lhx2 (1:1000, Santa Cruz, sc-19342), rabbit anti-Rhodopsin (1:2,000, LSL, LB-5597), goat anti-S-opsin (1:100, Santa Cruz, sc-14363), rabbit anti-M-opsin (1:500, Millipore, AB5405), rabbit anti-Chx10 (1:500) ( 97 ), mouse anti-Pax6 (1:250, DSHB), rabbit anti-Sox9 (1:500, Millipore, AB5535), rabbit anti-Calbindin (1:1000, Calbiochem, PC253L), mouse anti-S100β (1:2,500, Sigma, S-2532), guinea pig anti-Rbpms (1:1000, Millipore, ABN1376), mouse anti-GFAP (1:500, Sigma, G3893), mouse anti-Socs3 (1:100, Abcam, SO1) ( 98 , 99 ), rabbit anti-Vimentin (1:500, Abcam EPR3776), and rabbit anti-GS (1:500, Sigma, G2781). Cy3-conjugated (1:500, Jackson ImmunoResearch Laboratories), Alexa Fluor 488–conjugated (1:500, Sigma), and DyLight 649–conjugated (1:500, Jackson) secondary antibodies were used.…”