Journal: iScience

Article Title: Depletion of HIV reservoir by activation of ISR signaling in resting CD4 + T cells

doi: 10.1016/j.isci.2022.105743

Figure Lengend Snippet: ISR/ATF4 activation by HA15 activates anti-viral IFIT1/cell death signaling in primary CD4 + T cell model of latency The primary CD4 + T cell model of latency was treated with or without 20 μM HA15 for 48 h. Cells were collected 12 h, 1 day, and 2 days after the treatment. (A) Total RNA was isolated, and RT-qPCR was performed to analyze the expression of IFN-I/IFIT signaling genes. p<0.05; ∗∗∗, p<0.001; analyzed with One-way ANOVA (n = 3). (B–D) Primary CD4 + T cell model are treated with DMSO or HA15 (20 μM) for 4 days. Cells are collected and prepared for HIV RNA FISH-Flow to determine the percentage of IFIT1+ cells The percentage of IFIT+/HIV RNA+ cells and cell death (% of cleaved PARP1+) was determined by HIV RNA FISH-Flow ∗, p<0.05; ∗∗, p<0.01; ∗∗∗, p<0.001; ∗∗∗∗, p<0.0001; analyzed with two-tailed t -test (n = 3–6).

Article Snippet: IFIT1 (D2X9Z) Rabbit mAb (Pacific BlueTM Conjugate) , Cell Signaling Technology , Cat#96740.

Techniques: Activation Assay, Isolation, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: iScience

Article Title: Depletion of HIV reservoir by activation of ISR signaling in resting CD4 + T cells

doi: 10.1016/j.isci.2022.105743

Figure Lengend Snippet:

Article Snippet: IFIT1 (D2X9Z) Rabbit mAb (Pacific BlueTM Conjugate) , Cell Signaling Technology , Cat#96740.

Techniques: Recombinant, Staining, Reverse Transcription, Cell Isolation, Software

Journal: Frontiers in Immunology

Article Title: Extracellular Vesicles From KSHV-Infected Cells Stimulate Antiviral Immune Response Through Mitochondrial DNA

doi: 10.3389/fimmu.2019.00876

Figure Lengend Snippet: List of primers used for PCR.

Article Snippet: MA), mouse monoclonal anti-KSHV ORF65 , rabbit polyclonal anti-GAPDH (Cusabio, Houston, TX), rabbit polyclonal anti-calnexin (Bioss Antibodies Inc.), mouse monoclonal anti-HDAC1 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-mtTFA (Santa Cruz Biotechnology), rabbit polyclonal anti-MX1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT44L (Bioss Antibodies Inc.) and mouse monoclonal anti-β-actin antibodies (Sigma, St. Louis, MO) were used as primary antibodies.

Techniques: Amplification

Journal: Frontiers in Immunology

Article Title: Extracellular Vesicles From KSHV-Infected Cells Stimulate Antiviral Immune Response Through Mitochondrial DNA

doi: 10.3389/fimmu.2019.00876

Figure Lengend Snippet: EV was an essential factor in the induction of ISGs by the supernatant from KSHV-infected cells. (A) KSHV EVs isolated by centrifugal filtration induced IFIT1 expression. The supernatant from KSHV-infected cells was separated by centrifugal filter device with a cut-off of 100 kDa. High molecular weight (HMW) proteins (the retained materials by a filter) and low molecular weight (LMW) proteins (the flow-through) was applied to HUVECs, followed by analyzing mRNA expression by RT-qPCR. (B) Induction of ISGs was correlated with the amount of EVs. The same volume of EVs was isolated from the same amount of the supernatant from mock- or KSHV-infected cells. Then, each EV was applied to HUVECs, followed by analyzing mRNA expression by RT-qPCR. ΔCT indicated the normalized CT value of IFIT1 with reference gene, β-actin. (C) Knockdown of Rab27b suppressed the induction of ISGs by KSHV EVs. The expression of Rab27b was suppressed by shRNA in HUVECs. After KSHV infection, mock EVs or KSHV EVs were isolated from the Rab27b-suppressed HUVECs. Each prepared EV was applied to uninfected HUVECs, and mRNA expressions for ISGs were analyzed. The grouping of blots cropped from different gels and full-length blots are included in a . Data are shown as the mean ± SD, n = 6, ** p < 0.01.

Article Snippet: MA), mouse monoclonal anti-KSHV ORF65 , rabbit polyclonal anti-GAPDH (Cusabio, Houston, TX), rabbit polyclonal anti-calnexin (Bioss Antibodies Inc.), mouse monoclonal anti-HDAC1 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-mtTFA (Santa Cruz Biotechnology), rabbit polyclonal anti-MX1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT44L (Bioss Antibodies Inc.) and mouse monoclonal anti-β-actin antibodies (Sigma, St. Louis, MO) were used as primary antibodies.

Techniques: Infection, Isolation, Filtration, Expressing, Molecular Weight, Quantitative RT-PCR, shRNA

Journal: Frontiers in Immunology

Article Title: Extracellular Vesicles From KSHV-Infected Cells Stimulate Antiviral Immune Response Through Mitochondrial DNA

doi: 10.3389/fimmu.2019.00876

Figure Lengend Snippet: Induction of ISGs by KSHV EVs is associated with mtDNA. (A) mRNA expression of IFIT1 in HUVECs treated with DNase I or RNase-treated EVs. (B) Quantification of mtDNA in mock EVs vs. KSHV EVs. Genomic DNA was isolated from the same number of EVs, and mtDNA-related genes were analyzed by qPCR. (C) Western blotting for the cytoplasmic fraction from KSHV-infected HUVECs. Cytoplasmic fraction was extracted by digitonin, and its purity was analyzed by western blot analysis. WCE: whole cell extract, Pel: pellet after extraction of the cytoplasmic fraction, Cyt: cytoplasmic fraction. (D) Quantification for genomic and mitochondrial DNA in the cytoplasmic fraction from mock- vs. KSHV-infected HUVECs. (E,F) Induction of IFIT1 in cGAS or STING-suppressed HUVECs by KSHV EVs. The expression of cGAS (E) or STING (F) was suppressed by shRNAs. Mock EVs or KSHV EVs were treated with each indicated knockdown cell, and the induction in IFIT1 expression was analyzed by RT-qPCR. Data are shown as the mean ± SD, n = 6, ns, not significant, * p < 0.05, and ** p < 0.01.The grouping of blots cropped from different gels and full-length blots are included in a .

Article Snippet: MA), mouse monoclonal anti-KSHV ORF65 , rabbit polyclonal anti-GAPDH (Cusabio, Houston, TX), rabbit polyclonal anti-calnexin (Bioss Antibodies Inc.), mouse monoclonal anti-HDAC1 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-mtTFA (Santa Cruz Biotechnology), rabbit polyclonal anti-MX1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT44L (Bioss Antibodies Inc.) and mouse monoclonal anti-β-actin antibodies (Sigma, St. Louis, MO) were used as primary antibodies.

Techniques: Expressing, Isolation, Western Blot, Infection, Quantitative RT-PCR