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Fig. 2 | Cell & Bioscience

Fig. 2

From: New insights on thyroid hormone mediated regulation of herpesvirus infections

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

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