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: Frontiers in Oncology

Article Title: Use of phage display biopanning as a tool to design CAR-T cells against glioma stem cells

doi: 10.3389/fonc.2023.1124272

Figure Lengend Snippet: Dual peptide CAR-T cell design. (A) Diagram of the CAR construct designs. The E-28t28z and E-8t28z constructs use CD28 and CD8 as the hinge/transmembrane domains respectively, while both constructs employed CD28 for the co-stimulatory domain. The E-28t28z-tCD34 and E-8t28z-tCD34 constructs are identical to the E-28t28z and E-8t28z constructs but feature co-expression of truncated CD34 as an extracellular marker. (B) Diagram of a CAR-T cell with the different dual peptide-based constructs as the antigen-recognition domain. (C) CD34+ expression analysis by flow cytometry allows for a built-in quantification of transduction efficiency. (D) ELISA results displaying IFN-gamma secretion from CAR- or mock-transduced (GFP) T cells co-cultured overnight against T387 GSCs or cell-free media, **** = p < 0.0001. Values shown in the bar chart represent the average of two independent experiments. (E) CD3ζ western blotting with GAPDH as a housekeeping gene, used for additional support to confirm the presence or lack of the CAR following transduction. LTR, Long terminal repeat; S, signal peptide; P2A, Porcine teschovirus-1 2A self-cleaving peptide; tCD34, Truncated CD34.

Article Snippet: Membranes were incubated with 0.2 μg/mL mouse anti-human CD3ζ (Santa Cruz Biotechnology, SC-166275, Dallas, TX) and 0.04 μg/mL rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, SC-25778, Dallas, TX).

Techniques: Construct, Expressing, Marker, Flow Cytometry, Transduction, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot