Journal: bioRxiv

Article Title: The cGAS-STING pathway regulates microglial chemotaxis in genome instability

doi: 10.1101/2023.08.25.554654

Figure Lengend Snippet: (A) Representative immunoblot of STING pathway activation in ATM KO C20 cells (clones #1 and #2). Loading control: Vinculin. Quantification of phosphorylated proteins is relative to respective total proteins and normalised to WT. (B) Relative mRNA levels of IFNB1 and IFIT2 in WT and ATM KO C20 cells treated with 1 µM STING agonist diABZI, or DMSO as control, for 5 h. C q values normalised to IPO8 and DMSO-treated WT cells. Mean log 2 fold change ± SD (n=3). Two-way ANOVA with Tukey’s post-hoc comparison test. (C) Representative immunoblot of HMC3 cells treated with 10 nM ATM inhibitor AZD1390 (ATMi), or DMSO as control, for 1, 3, 6 or 9 days. Loading control: Vinculin. NS: nonspecific. Quantification of phosphorylated proteins is relative to respective total proteins and normalised to Day 1 DMSO control. (D) Representative images of IRF3 localisation in HMC3 cells stimulated for 2 h with 1 µM diABZI, or DMSO as control, or with 10 nM ATMi or DMSO for 6 days (diABZI and ATMi stimulation are part of separate experiments). Red: IRF3, cyan: DNA (DAPI). 20x magnification, scale bar = 25 µm. Yellow arrows indicate cells with nuclear IRF3. (E) Percentage of cells with nuclear translocation of IRF3 treated as in (D). Mean ± SD (n=3). Unpaired, two-tailed Student’s t -tests. (F) Relative IFNB1 mRNA levels in HMC3 cells treated with ATMi or DMSO for 6 days followed by 1 µM diABZI, or DMSO as a control, for 5 h. C q values normalised to RPS13 and DMSO-treated cells. Mean log 2 fold change ± SD (n=3). Two-way ANOVA with Tukey’s post-hoc comparison test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns = not significant.

Article Snippet: To induce ATM activation, cells were treated with 1 μM camptothecin (CPT; Cayman Chemical Company) for 1 h. The STING agonist diABZi (Selleck Chemicals) was used at 1 μM.

Techniques: Western Blot, Activation Assay, Clone Assay, Control, Comparison, Translocation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: The cGAS-STING pathway regulates microglial chemotaxis in genome instability

doi: 10.1101/2023.08.25.554654

Figure Lengend Snippet: (A) Relative mRNA levels of CCL5 and CXCL10 in WT and ATM KO C20 cells and upon treatment with 1 µM diABZI, or DMSO as control, for 5 h. C q values normalised to IPO8 and DMSO-treated WT cells. Mean log 2 fold change ± SD (n=3). Two-way ANOVA with Tukey’s post-hoc comparison test. (B) Schematic of experimental workflow. WT and STING1 KO HMC3 cells were treated with 10 nM ATMi, or DMSO as a control, for 6 days. mRNA levels were measured by RT-qPCR. Secretion was measured 24 h after media change to low FBS supplemented with 1 µM diABZI, or DMSO as a control. (C) Relative mRNA levels of CCL5 and CXCL10 from cells treated as in (B). C q values normalised to RPS13 and DMSO-treated WT cells. Mean log 2 fold change ± SD (n=3). Two-way ANOVA with Tukey’s post-hoc comparison test. (D) CXCL10 concentration in cell culture supernatants. Mean ± SD (n=3). Dashed line indicates lower limit of detection (LLOD) = 0.12 pg/mL. Nd: not detected. One-way ANOVA with Tukey’s post-hoc comparison test in WT cells. (E) Representative immunoblot of cerebellar homogenates from individuals with A-T and matched individuals with no known diseases of the CNS as controls (Ctrl). Loading control: GAPDH. NS = nonspecific. (F) Relative mRNA levels of STING1 , CGAS , CCL5, and CXCL10 as in (E). C q values normalised to RPS13 . Mean 2 -δCT ± SD shown (n=3). Paired t -test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns = not significant.

Article Snippet: To induce ATM activation, cells were treated with 1 μM camptothecin (CPT; Cayman Chemical Company) for 1 h. The STING agonist diABZi (Selleck Chemicals) was used at 1 μM.

Techniques: Control, Comparison, Quantitative RT-PCR, Concentration Assay, Cell Culture, Western Blot