Journal: bioRxiv
Article Title: Toxicity of extracellular cGAMP and its analogs to T cells is due to SLC7A1-mediated import
doi: 10.1101/2024.09.21.614248
Figure Lengend Snippet: (A and B) Activated human primary CD3 + T cells from 2 healthy donors were electroporated with Cas9 protein and non-targeting (scramble) or SLC7A1-targeting sgRNA. Cells were treated with 50 µM cGAMP for 2 h three days later, and signaling was assessed by Western blot (n = 3 biological replicates). Indel score was determined by ICE (Synthego). One representative Western blot from Donor 2 is shown in (A) and quantification of multiple blots from 2 donors are shown in (B). Significance calculated by unpaired, two-tailed t-test. Data are shown as the mean +/- SD. *p < 0.05 (C and D) Activated mouse CD4+ and CD8+ T cells were electroporated with Cas9 protein and Rosa26-targeting or SLC7A1-targeting sgRNA. Cells were treated with 50 µM cGAMP for 3 h three days later, and signaling was assessed by Western blot (n = 4 - 6 biological replicates). One representative Western blot from each group of CD4+ and CD8+ T cells are shown in (C) and quantification of multiple blots from the two groups are shown in (D). Significance calculated by unpaired, two-tailed t-test. Data are shown as the mean +/- SD. *p < 0.05, ****p<0.0001 (E and F) Rosa26- and SLC7A1-knocked out CD4+ and CD8+ mouse T cells from (C and D) were electroporated with 100 nM of cGAMP for 2 h, and signaling was assessed by Western blot (n = 3 biological replicates). One representative Western blot from each group of CD4+ and CD8+ T cells are shown in (E) and quantification of multiple blots from the two groups are shown in (F). Significance calculated by unpaired, two-tailed t-test. Data are shown as the mean +/- SD. *p < 0.05. (G) Schematic of diazirine probe crosslinking assay. Purified FLAG-SLC7A1 proteins were incubated with a 3’3’-cGAMP probe containing a diazirine motif, and then UV irradiated at 365 nm. Crosslinked proteins were then conjugated with a R110 fluorescence tag containing an alkyne handle (R110-N 3 ) via click chemistry. Proteins were visualized were visualized on SDS-PAGE gel and R110 fluorescence and Flag signal was assessed by Western Blot. (H) Purified FLAG-SLC7A1 protein was incubated with 500 µM of 3’3’-cGAMP diazirine probe, and with or without 2’3’- or 3’3’-cGAMP (lower concentration: 500 µM, higher concentration: 2mM), and irradiated at 365 nm for 20 min. Crosslinked protein signals were assayed by Western Blot. Flag blot is shown in (Supplementary Figure 3B) and R110 fluorescence blot is shown in (H).
Article Snippet: To a crosslinked reaction mixture, 0.1 mM of R110 azide (MedChemExpress) was added into a pre-mixed complex of 1 mM of CuSO 4 and 5 mM BTTAA (Sigma-Aldrich), which were mixed with 5 mM of sodium ascorbate to a total volume of 15 μL.
Techniques: Western Blot, Two Tailed Test, Purification, Incubation, Irradiation, Fluorescence, SDS Page, Concentration Assay
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