Inhibition of protein kinase R (PKR) activity attenuated HBx-siRNA-induced innate immune responses.

<p>(A) HepG2.2.15 cells were treated with or without PKR inhibitor C16, and then transfection experiments were performed for 24 h. Levels of IFN-α and IFN-β mRNA were analyzed by real-time PCR and presented relative to mock transfection (left). The levels of IFN-α in supernatants were examined by ELISA (right). (B) The experiment was performed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027931#pone-0027931-g004" target="_blank">Fig. 4A</a>. Levels of IFN-stimulated gene (ISG)15 and ISG56 mRNA were analyzed by real-time PCR and presented relative to mock transfection. (C) p-Stat1 expression was detected by flow cytometry when siRNA was transfected for 4 h. (D) The experiment was performed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027931#pone-0027931-g004" target="_blank">Fig. 4A</a>, mRNA levels of TNF-α and IL-6 were analyzed by quantitative real-time PCR and were presented relative to mock transfection. (E) siPKR were transfected into cells, then PKR protein expression was assayed by Western Blot (top). siRNA4 and siRNA targeting PKR were cotransfected into HepG2.2.15 cells for 24 h, then mRNA levels of IFN-α and IFN-β were analyzed and presented relative to mock transfection (bottom). Data are expressed as the mean ± SD from at least three separate experiments. *<i>p</i><0.05 versus siRNA4-treated group.</p

HBx-siRNA induced the expression of type I interferon (IFN) and inflammatory cytokines in HepG2.2.15 cells.

<p>(A) HepG2.2.15 cells were transfected with HBx-siRNAs; 24 h later, the mRNA levels of IFN-α, IFN-β, ISG15 and ISG56 were measured by real-time PCR. The mRNA level in each sample was expressed as a fold change of the RNA level in lipofectamine (LF)-treated cells. The levels of mRNA are presented relative to mock control. (B) Cells were transfected with HBx-siRNAs for 24 h. Levels of TNF-α, IL-6, iNOS, and IL-8 mRNA were analyzed by real-time PCR and presented relative to those of LF-treated cells. (C) HepG2.2.15 cells were incubated with phycoerythrin (PE)-labelled anti-p-Stat1 antibody after HBx-siRNA transfection for 4 h, and were then analyzed by flow cytometry. p-Stat1 expression was also detected when IFN receptor was neutralized. (D) Levels of CXCL2, CCL4, and CXCL10 mRNA were analyzed by real-time PCR and presented relative to those of LF-treated cells. Data are expressed as the mean ± SD from at least three separate experiments. *<i>p</i><0.05 versus LF-treated group.</p

Sequences of primers specific for human genes used for real-time PCR analysis.

<p>Sequences of primers specific for human genes used for real-time PCR analysis.</p

Inhibition of protein kinase R (PKR) activity attenuated HBx-siRNA-induced hepatitis B virus (HBV) inhibition.

<p>HepG2.2.15 cells were treated with or without C16, and then transfection experiments were performed at a concentration of 150 nM. Levels of HBx RNA mRNA <b>(</b>A) and HBsAg proteins in supernatants (C) were examined as described previously. Levels were expressed as percentages of corresponding levels in scramble siRNA-treated cells. HBV DNA in supernatants was detected by real-time PCR (B). Immunostaining for HBcAg were visualized under a microscope (D). siRNA4 and siPKR were cotransfected into HepG2.2.15 cells, then levels of HBx mRNA were analyzed by quantitative real-time PCR and presented relative to mock transfection (E). Data are expressed as the mean ± SD from at least three independent experiments. *<i>p</i><0.05 versus corresponding solvent-treated group.</p

HBx-siRNA promoted the expression of PKR and the activation of PKR signaling pathway.

<p>(A<b>)</b> Real-time PCR was used to analyze the mRNA expression of PKR after HBx-siRNAs treatment for 24 h. The mRNA level in each sample was expressed as fold change compared with RNA level in LF-treated cells. Data are expressed as the mean ± SD from at least three separate experiments. *<i>p</i><0.05 versus LF-treated group. (B) Western blot analysis of the activation of the protein kinase R (PKR) and PKR substrate, translation initiation factor 2 (eIF-2)α, from HepG2.2.15 cells. Cells were transfected with siRNA4 (150 nM) at different time points (0, 30, 60, 90, 120, and 180 min) using lipofectamine. The panel used an antibody specific to whole PKR or eIF2-α that demonstrates equal loading of total protein. Histogram showed the relative expression of p-PKR or p-eIF2-α after normalization to whole PKR or eIF2-α (right). (C) Western blot analysis of the activation of the PKR and eIF2-α by siRNA4 at different time points (0, 4, 6, 8, 12, and 24 h). Histogram showed the relative expression of p-PKR or p-eIF2-α (bottom). (D) Western blot analysis of the activation of the PKR by scramble siRNA at different time points (0, 4, 6, 8, 12, and 24 h). Histogram showed the relative expression of p-PKR (bottom).</p

Protein kinase R (PKR) over-expression enhanced type I interferon (IFN) production and HBx-siRNA-mediated hepatitis B virus (HBV) inhibition.

<p>HepG2.2.15 cells were transfected with 2 µg of full-length PKR plasmid or the control plasmid. 24 h later, the cells were transfected with HBx-siRNA (150 nM). (A) Over-expression of PKR at the transcriptional level. (B, C) Levels of IFN-α and IFN-β were detected by real-time PCR at 24 h. (D, E) mRNA expression levels for HBx and HBs/p were detected by real-time RT-PCR at 24 h. (F) HepG2.2.15 cells were transfected with 1 µg of hPKR-K296R or the control plasmid. Then cells were collected and 6-His was assayed by FACS (top). When transfected with hPKR-K296R for 24 h, the cells were transfected with HBx-siRNA4 (150 nM). Another 24 h later, levels of IFN-α and IFN-β were detected. The levels of all examined parameters were expressed as percentages of the corresponding parameter in LF-treated control-vector transfected cells (bottom). Data are expressed as the mean ± SD from at least three independent experiments. *<i>p<</i>0.05 versus corresponding control-vector transfected cells.</p

Highly Sensitive Dual-Mode Optical Thermometry of Er³⁺/Yb³⁺ Codoped Lead-Free Double Perovskite Microcrystal

In this Letter, erbium (Er3+) and ytterbium (Yb3+) codoped perovskite Cs2Ag0.6Na0.4In0.9Bi0.1Cl6 microcrystal (MC) is synthesized and demonstrated systematically to the most prospective optical temperature sensing materials. A dual-mode thermometry based on fluorescence intensity ratio and fluorescence lifetime provides a self-reference and highly sensitive temperature measurement under dual wavelength excitation at a temperature from 300 to 470 K. Combined with the white-light emission derived from self-trapped excitons (STEs), the characteristic emission peak of Er3+ ions can be observed under 405 nm laser excitation. The fluorescence intensity ratio (FIR) between perovskite and Er3+ is used as temperature-dependent probe signal, of which maximum value for relative and absolute sensitivities reaches to 1.40% K–1 and 8.20 × 10–2 K–1. Moreover, Er3+ luminescence becomes stronger with the feeding Yb3+ increasing under 980 nm laser excitation. The energy transfer of Er3+ and Yb3+ is revealed by power-dependent photoluminescence (PL) spectroscopy, and the involved upconversion mechanism pertains to the two-photon excitation process. The results reveal that the Er3+/Yb3+ codoped lead-free double perovskite MC is a good candidate for a thermometric material for the novel dual-mode design

Sequences of chemically synthesized HBx-siRNAs.

<p>Sequences of chemically synthesized HBx-siRNAs.</p

HBx-siRNA inhibits hepatitis B virus (HBV) expression in HepG2.2.15 cells.

<p>(A) Scramble siRNA or HBx-siRNA (siRNA1, siRNA2, siRNA3, siRNA4) (100 nM) was transfected into HepG2.2.15 cells using lipofectamine 2000 (LF). LF-treated group was used as a control. The cells were harvested, and RNA was extracted at 24 h after transfection. Expression of HBx mRNA were detected by real-time PCR (top). The level of each sample is expressed as the percentage of the RNA level in scramble siRNA-treated cells. The levels of HBx protein were detected by western blot at 48 h after transfection (bottom). (B) HepG2.2.15 cells were transfected for 48 h, and then cells were fixed, immunostained for HBcAg and visualized under a microscope. Representative fields from each cell group are shown (original magnification ×100). (C, D) The supernatants were harvested after 48 h, and the dose-dependent reductions in HBsAg (C) and HBeAg (D) were detected with ELISA. Data showing the silencing effect are representative of three independent experiments. Data are expressed as the mean ± SD from at least three separate experiments. *<i>p</i><0.05 versus the LF-treated group.</p

Highly Sensitive Dual-Mode Optical Thermometry of Er³⁺/Yb³⁺ Codoped Lead-Free Double Perovskite Microcrystal

In this Letter, erbium (Er3+) and ytterbium (Yb3+) codoped perovskite Cs2Ag0.6Na0.4In0.9Bi0.1Cl6 microcrystal (MC) is synthesized and demonstrated systematically to the most prospective optical temperature sensing materials. A dual-mode thermometry based on fluorescence intensity ratio and fluorescence lifetime provides a self-reference and highly sensitive temperature measurement under dual wavelength excitation at a temperature from 300 to 470 K. Combined with the white-light emission derived from self-trapped excitons (STEs), the characteristic emission peak of Er3+ ions can be observed under 405 nm laser excitation. The fluorescence intensity ratio (FIR) between perovskite and Er3+ is used as temperature-dependent probe signal, of which maximum value for relative and absolute sensitivities reaches to 1.40% K–1 and 8.20 × 10–2 K–1. Moreover, Er3+ luminescence becomes stronger with the feeding Yb3+ increasing under 980 nm laser excitation. The energy transfer of Er3+ and Yb3+ is revealed by power-dependent photoluminescence (PL) spectroscopy, and the involved upconversion mechanism pertains to the two-photon excitation process. The results reveal that the Er3+/Yb3+ codoped lead-free double perovskite MC is a good candidate for a thermometric material for the novel dual-mode design