Proteasome-mediated degradation plays a role in STING reduction.

<p>(A) Time course assessment of STING mRNA expression after MRC-5 cells were transfected with pcDNA3.1 (0.05μg). (B) Western blotting assessment of STING after MRC-5 cells were transfected with pcDNA3.1 (0.05μg) with treatment of Bortezomib (20 nM) or DMSO for 18h. (C)Western blotting assessment of immunoprecipitated STING with anti-ubiquitin antibody.</p

STING degradation occurs in HDCs but not in HEK293.

<p>(A) Western blotting assessment of STING and RIG-I in MRC-5, 2BS, KMB17 and hUC-MSC cells after transfection with 0.5 μg of pcDNA3.1 for 24 h. (B) MRC-5 and HEK293 cells were transfected with 0.5 μg of pcDNA3.1 for 24 hours. The indicated molecules were assessed by western blotting. (C, D) qRT-PCR assessment of mRNA levels (C) and ELISA assessment of protein levels (D) of IL-6 in MRC-5 and HEK293 cells after transfection of pcDNA3.1. (E) ULK1 S556 phosphorylation was assessed by western blotting in MRC-5 and HEK293 cells. Asterisk (*), P <0.05.</p

Neutralization ICs of sera collected at various times after infection against the infecting virus

Each panel represents neutralization of macaques infected with the same virus: a) SIVMneCL8; b) SIVMne35wkSU; c) SIVMne170; d) SIVMne027. For each panel, the x axis shows the time when sera samples were collected. The y-axis shows the IC– the reciprocal dilution of sera required to inhibit infection by 50%. Neutralization ICs measured by the TZM-bl cells are shown in solid lines. Neutralization ICs measured in sMAGI cells are shown in dotted lines.<p><b>Copyright information:</b></p><p>Taken from "Heavily glycosylated, highly fit SIVMne variants continue to diversify and undergo selection after transmission to a new host and they elicit early antibody dependent cellular responses but delayed neutralizing antibody responses"</p><p>http://www.virologyj.com/content/5/1/90</p><p>Virology Journal 2008;5():90-90.</p><p>Published online 4 Aug 2008</p><p>PMCID:PMC2518139.</p><p></p

RIG-I or IL-6 promotes STING degradation by activating ULK1 in MRC-5 cells.

<p>(A) Western blotting assessment of the levels of ULK1 S556 phosphorylation triggered by dsDNA. (B) Western blotting assessment of the ULK1 S556 phosphorylation in MRC-5 cells with RIG-I knockdown or IL-6R antibody treatment.</p

RIG-I is involved in regulating the DNA-induced STING degradation.

<p>(A) Western blotting analysis of RIG-I expression after MRC-5 cells were transfected with pcDNA3.1. (B) Time course assessment of RIG-I mRNA after MRC-5 cells were transfected with pcDNA3.1 (0.05μg) for the indicated time points. (C-G) Western blotting shows a reverse correlation between STING and RIG-I under different conditions: dose (C), time (D, E), DNA types (F), and Bortezomib treatment (G). (H) Silencing RIG-I partially reverses the STING degradation. (I) qRT-PCR assessment of RIG-I expression after STING knockdown in MRC-5 cells. Asterisk (*), P <0.05.</p

The plasmid DNA induces STING protein reduction in MRC-5 cells.

<p>(A) After 48 h transfection with different concentrations of pcDNA3.1, the MRC-5 cell lysates were analyzed by immunoblotting with anti-STING. (B, C) Time course assessment of STING expression induced by DNA. (D) Western blotting assessment of STING after 48 h transfection of MRC-5 cells with equal amount of circular plasmid pcDNA3.1, plasmid vector PGL-3, liner plasmid pcDNA3.1, 2 kb PCR DNA fragment amplified from pcDNA3.1, genomic DNA derived from MRC-5 cells, DNA virus (rTv-Fluc and HSV-2).</p

STING serves as a major mediator for the plasmid DNA-induced IFN-β response.

<p>(A) Analysis of the dose effect on IFN-β production induced by DNA transfection. MRC-5 cells were transfected with pcDNA3.1 with indicated amounts for 6 hours. IFN-β mRNA was analyzed by qRT-PCR. (B) Time course assessment of IFN-β induction by pcDNA3.1 (0.05μg). (C, D) After 24 h transfection of MRC-5 cells with pcDNA3.1, cells were infected with rTV-Fluc (C) or Sendai virus (D) for another 24 hours before a luciferase report assay for rTV-Fluc (C) and qRT-PCR for Sendai virus (D). (E) Western blotting showing STING knockdown by siRNA after 72 h transfection in MRC-5 cells. (F) qRT-PCR assessment of IFN-β after STING was knockdown in MRC-5 cells that were transfected with pcDNA3.1(0.2μg). (G) A luciferase report assay showing rTV-Fluc virus replication after STING knockdown. Data are presented as mean ± SD (n = 3). Asterisk (*), P <0.05.</p

IL-6 and RIG-I contribute additively to the DNA-induced STING degradation.

<p>(A) qRT-PCR assessment of expression of indicated interleukins after MRC-5 cells were transfected with pcDNA3.1 (0.05 μg) for 9 hours. (B) ELISA analysis of IL-6 protein levels in the supernatants of cells at different time points. (C) qRT-PCR analysis of IL-6 mRNA at different time points. (D) Western blotting assessment of STING expression after MRC-5 cells were treated with IL-6R antibody with pcDNA3.1 transfection. (E) Western blotting assessment of STING expression after application of IL-6R antibody and siRIG-I. (F) qRT-PCR (up) and ELISA analysis (down) of IL-6 expression after STING knockdown. Asterisk (*), P <0.05.</p

Presentation_1.PDF

<p>Using 5′ rapid amplification of cDNA ends, Illumina MiSeq, and basic flow cytometry, we systematically analyzed the expressed B cell receptor (BCR) repertoire in 14 healthy adult PBMCs, 5 HIV-1+ adult PBMCs, 5 cord blood samples, and 3 HIS-CD4/B mice, examining the full-length variable region of μ, γ, α, κ, and λ chains for V-gene usage, somatic hypermutation (SHM), and CDR3 length. Adding to the known repertoire of healthy adults, Illumina MiSeq consistently detected small fractions of reads with high mutation frequencies including hypermutated μ reads, and reads with long CDR3s. Additionally, the less studied IgA repertoire displayed similar characteristics to that of IgG. Compared to healthy adults, the five HIV-1 chronically infected adults displayed elevated mutation frequencies for all μ, γ, α, κ, and λ chains examined and slightly longer CDR3 lengths for γ, α, and λ. To evaluate the reconstituted human BCR sequences in a humanized mouse model, we analyzed cord blood and HIS-CD4/B mice, which all lacked the typical SHM seen in the adult reference. Furthermore, MiSeq revealed identical unmutated IgM sequences derived from separate cell aliquots, thus for the first time demonstrating rare clonal members of unmutated IgM B cells by sequencing.</p

KEGG pathway enrichment analysis of differentially expressed genes between S28 and S6.

<p>Enrichment factor is calculated as followed: </p><p></p> Gene hits is the number of hits in the selected pathway; Gene_pathway is the number of genes in the selected pathway of KEGG background; Hits_total represents the number of total hits in all pathways; Gene _total is the number of total genes in all pathways of KEGG background.<p></p