Journal: JCI Insight

Article Title: An IKBKE variant conferring functional cGAS/STING pathway deficiency and susceptibility to recurrent HSV-2 meningitis

doi: 10.1172/jci.insight.173066

Figure Lengend Snippet: ( A – H ) iPSC derived microglia ( A – D ) and neurons ( E – H ) were transfected with IKBKE or control siRNA. ( A and E ) The efficiency of transfection was examined by measuring IKBKE mRNA levels. Cells were infected with HSV-2 for 6 hours, and IFNA2 , IFNB1 , and TNFA mRNA levels were quantified. ( I – L ) iPSC-derived microglia were transfected with siRNA targeting cGAS ( I and J ) or TLR3 ( K and L ). Microglia were infected with HSV-2, and IFNB1 mRNA was quantified ( J and L ). Unpaired t test was used for statistical analysis. ( M ) Graphical illustration of experimental setup for microglia-neuron crosstalk experiments. Created with BioRender.com. ( N ) iPSC-derived neurons were treated with Human Type 1 IFN Neutralizing Antibody Mixture or control IgG (both 1:100) 30 minutes before addition of supernatants from HSV-2–infected microglia or treatment with IFN-β (10 ng/mL). Six hours later, the medium was removed, and the neurons were infected with HSV-2. Supernatants were collected after 16 hours for plaque assay ( O ). Microglia subjected to IKBKE or control knockdown with siRNA were infected with HSV-2. The cells were washed, and the medium was replaced after 1 hour. Supernatants were collected from the microglia after 24 hours and added to the neurons. Six hours later, the medium was removed, and the neurons were infected with HSV-2. Supernatants were collected 16 hours later for plaque assay. Data presented are pooled from 2 independently performed experiments. ( P ) Cells were treated as in panel N , and culture supernatants were analyzed for cell viability. Data presented are from 1 of 2 experiments performed. ( N – P ) Groups were compared with Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. Error bars represent SEM ( A – H ) and SD ( N – P ), and *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: To neutralize type I IFN bioactivity in supernatants from microglia, neurons were cultured with Human Type 1 IFN Neutralizing Antibody Mixture (PBL) 30 minutes before addition of culture supernatants from microglia (1:100).

Techniques: Derivative Assay, Transfection, Control, Infection, Plaque Assay, Knockdown

Journal: Cell reports

Article Title: CytoMAP: A Spatial Analysis Toolbox Reveals Features of Myeloid Cell Organization in Lymphoid Tissues

doi: 10.1016/j.celrep.2020.107523

Figure Lengend Snippet:

Article Snippet: Mix-n-Stain CF Dye Antibody Labeling Kits , Biotium , Cat# 92433-92339.

Techniques: Recombinant, Electron Microscopy, Antibody Labeling, Adjuvant, Software, Plasmid Preparation

Journal: AIDS (London, England)

Article Title: Activation of the signal transducer and activator of transcription 1 signaling pathway in thymocytes from HIV-1-infected human thymus

doi: 10.1097/01.aids.0000060415.18106.1a

Figure Lengend Snippet: (a) Quantitative measurement of differential gene expression. RNA from six different yeast-specific genes was mixed with the HIV or mock-infected RNA samples in the microarray assay. RNA from each yeast gene was added at a known ratio (3, 10 or 25) in the Cy3 (HIV) or Cy5 (mock) sample (Cy3/Cy5 added). The six yeast genes were arrayed on the slides in quadruplets. Positive values indicate threefold, 10-fold or 25-fold more RNA were used in the Cy3 (HIV) sample. Negative values (−3, −10 or −25) indicate that threefold, 10-fold or 25-fold more RNA were included in the Cy5 (mock) sample. An equal amount of RNA in Cy3 and Cy5 samples is indicated as 1 (+1 or −1). The X axis shows the added ratios and the Y axis indicates Cy3/Cy5 readout data from the assay. Error bars indicate standard deviations. (b) RNA sample and reaction control. RNA samples from HIVJD (Cy3 labeled) or mock (Cy5 labeled) infected severe combined immunodeficient (SCID)-hu Thy/Liv mice were analysed using the microarray assay. Relative expression levels of 23 000 Mr highly basic protein (HBP), ribosomal subunit S9 (S9 rRNA) and α-tubulin were quantified in quadruplets. Three sets of human genes known to express in multiple tissues (complex target 1–3) were also included as controls. Positive values (Cy3 signal/Cy5 signal) indicate more RNA in the Cy3 (HIV) sample, thus induction of expression by HIV. Negative values (Cy5 signal/Cy3 signal) indicate more RNA in the Cy5 (mock) sample, thus suppressed expression by HIV. Error bars indicate standard deviations from the quadruplet arrays. The relative expression of three T cell markers (TCRβ, CD8 and Thy-1) from the microarray assay was also shown. Comparison of HIV (JD or NL4-3) and mock infected samples from different donor tissues showed similar results. (c) Alteration of gene expression in SCID-hu Thy/Liv mice after HIV-1 infection. A small number (< 0.2%) of genes were induced (> threefold) in HIV-1-infected Thy/Liv organs over mock infected samples. The majority of genes were in the +2.5 to −2.5 range (99.5%). Error bars indicate standard deviations from three experiments (two with JD and one with NL4-3) with different donor tissues.

Article Snippet: Monoclonal antibodies reactive with human CD4 and CD8 cells were purchased from Becton Dickinson (San Jose, CA, USA).

Techniques: Gene Expression, Infection, Microarray, Control, Labeling, Expressing, Comparison