Fig. 2

A Transcription profiles of genes involved in PI3K/Akt pathway measured by qRT-PCR arrays. Undifferentiated and 5-day differentiated LNCaP cells plated on poly-d-lysine coated T75 flasks were treated with and without 100 nM T₃ for 48 h. The total RNA was purified by TRIZOL and the cDNA was synthesized using RT2 first strand kit (QIAGEN, cat#: 330401). For the transcriptome heatmaps assessment, the cDNA was subjected to qRT-PCR array analyses via PI3K-AKT signaling pathway (SAB Target List) H96 (BIO-RAD, cat#: 100-34223). The protocol was described essentially by the manufactures based on the CFX Connect™ Real-Time PCR Detection System (BIO-RAD Cat# 1855200). Amplification was plotted and analyzed in triplicates using the BIO-RAD CFX manager software provided by the manufacturer. For each gene, the brightest red squares indicate at least a fourfold increase over the brightest blue square. A Showed the select genes from PI3K-AKT target list modulated significantly by T₃ treatment and differentiation. Akt, EIF, and mTOR genes regulated by T₃ and differentiation. B CK2 inhibitor TBB disrupts T₃ mediated reduction of viral replication. The viral replication was measured by T₃ removal assays [32] and FLICIT assays as shown in B with modification. TBB (Santa Cruz Bio, cat#: sc-202830) was added at 1 µM for CK2 inhibition. In short, differentiated cells were infected with HSV-1. At 48 hpi, infected cells were treated with (1) T₃, (2) T₃ washout, (3) T₃ plus TBB, or (4) T₃ washout plus TBB. The culture media were collected at 96 hpi and subjected to PLICIT assays. The results showed that infection with 100 nM of T₃ reduced viral replication and hormone washout reversed this reduction. The addition of TBB disrupted the T₃–mediated repression. FLICIT was reported previously [68] and described in the figure. Data in triplicates, two-way ANOVA with Holm-Sidak post hoc analysis suggests that statistically significant differences in fluorescently labeled infected cells exists; a, b, c, d, e p < 0.001. C TBCA reversed T₃-mediated suppression of viral replication in differentiated cells. LNCaP cells were infected under treatment of no T₃, with T₃, 110 nM TBCA (Millipore, cat#: 218710), or T₃ + TBCA followed by plaque assays to measure the release of infectious viruses. No suppression of viral replication was observed for undifferentiated cells under the influence of T₃ and/or TBCA when analyzed by ANOVA (data not shown). T₃ removal assays as described in A were used to investigate the effects of TBCA. At 48 hpi, infected cells were treated with (1) T₃, (2) T₃ washout, (3) T₃ plus TBCA, or (4) T₃ washout plus TBCA. It is shown that TBCA, similar to TBB, reversed the suppression of viral replication by T₃ as measured by viral plaque assay. Data in triplicates were analyzed by Two-way ANOVA with Holm-Sidak post hoc analysis suggests that statistically significant differences in pfu per mL exists; a p < 0.001, b p < 0.046, c p < 0.040