diabzi  (MedChemExpress)

Journal: bioRxiv

Article Title: Extracellular cGAMP transmission exacerbates neuroinflammation in aging and multiple sclerosis

doi: 10.64898/2026.09.30.754737

Figure Lengend Snippet: (A) Kinetics of 2′3′- 32 [P]-cGAMP degradation by tissue lysates from the indicated CNS regions of WT mice at 10 weeks of age (n=2 biological replicates). (B, C) mRNA expression levels of Enpp1 in whole brain (B) and spinal cord (C) tissue of WT healthy (n=3 biological replicates) and MOG 35-55 -induced EAE mice at the symptomatic phase (paralysis score ≥ 2) (n=3 biological replicates). (D) Representative thin layer chromatography (TLC) tracking 2′3′- 32 [P]-cGAMP degradation by spinal cord lysate of WT healthy and EAE mice (paralysis score ≥ 2), at the indicated timepoints. (E) Kinetics of 2′3′- 32 [P]-cGAMP degradation by the spinal cord lysate of healthy and EAE mice (n=2 biological replicates). (F) 2′3′-cGAMP levels in healthy (n=3 biological replicates) and EAE (n=4 biological replicates) brain and spinal cord tissue measured by enzyme-linked immunosorbent assay (ELISA). (G) mRNA expression levels of Cxcl10 and Il1b in whole brain and spinal cord tissue of WT healthy (n=3) and MOG 35-55 -induced EAE mice (n=3) mice. (H, I) Expression of Enpp1 in microglia (H), oligodendrocyte precursor cells (OPCs), and committed oligodendrocyte precursors (COPs) (I) across the indicated MS diagnoses compared to healthy donors. Legends: CTRL : control; SPMS : secondary progressive MS; PPMS : primary progressive MS. (EGA: EGAD00001009169). Data are mean ± s.e.m. p -values were calculated using Mann-Whitney t-test. (J) Kinetics of 2′3′- 32 [P]-cGAMP degradation by tissue lysates of the indicated CNS regions of Enpp1 H362A mice at 10 weeks of age (n=2 biological replicates). (K) Paralysis scores for WT, Enpp1 H362A , and Enpp1 H362A Sting gt/gt mice at the indicated times after MOG 35-55 immunization. Number of mice in each group is indicated in the legend. Significance calculated by two-way ANOVA. (I) Paralysis scores in WT mice treated with cGAS inhibitor RU.521 after EAE onset (n= 14 mice for vehicle and n=17 mice for RU.521 treatment). Data was pooled from 2 independent experiments. Significance of the treatment course was calculated by two-way ANOVA and significance of daily score was calculated by two-sided unpaired t-test with Welch’s correction. (M) Experimental scheme to measure mitochondrial DNA leakage in different cell types of the spinal cord during EAE symptomatic phase: spinal cord tissue single cell dissociation, followed by FACS, subcellular fractionation, and qPCR of mitochondrial genes. (N) Cytosolic mtDNA (mt-ND1) in CD11b + CD45 lo microglia, CD11b + CD45 hi macrophages, CD3 + T cells, CD45 − non-immune cells isolated from WT healthy and EAE mice, normalized to total mt-ND1 DNA in the heavy fraction (n=4 biological replicates per group). (O,P) Cytosolic mtDNA (mt-ND1) normalized to total MT-ND1 DNA (O) or total genomic DNA (B2M) (P) in the heavy fraction of MACS-sorted mouse primary spinal cord macrophages (CD11b + ) from WT mice at 10 weeks of age, with or without MOG 35-55 -induced EAE (n=3-4 biological replicates per group). (Q) 2′3′-cGAMP secretion by MACS-sorted mouse primary brain and spinal cord macrophages (CD11b + ) from wild-type mice with or without MOG 35-55 -induced EAE. Cells were cultured for 5 days after sorting and incubated for 48 h in the presence of ENPP1 inhibitor STF-1623. 2′3′-cGAMP levels in the supernatant were measured by enzyme-linked immunosorbent assay (ELISA) (n=5 biological replicates). Data are mean ± s.e.m. p -values were calculated using unless otherwise indicated.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies diluted 1:1,000-1:4,000, including antibodies against tubulin DM1A (Sigma, CP06), STING (CST, #13647), phospho-STING (CST, #72971), phospho-IRF3 (CST, #4947), Flag (CST, #14793), SLC38A2/SNAT2 (Proteintech 25928-1-AP).

Techniques: Tissue, Expressing, Thin Layer Chromatography, Enzyme-linked Immunosorbent Assay, Control, MANN-WHITNEY, Single Cell, Cell Fractionation, Isolation, Cell Culture, Incubation, FACS

Journal: bioRxiv

Article Title: Extracellular cGAMP transmission exacerbates neuroinflammation in aging and multiple sclerosis

doi: 10.64898/2026.09.30.754737

Figure Lengend Snippet: (A, B) mRNA expression levels of Cgas (A) and Sting (B) in whole brain tissue of wild-type healthy (n=3 biological replicates) and MOG 35-55 -induced EAE mice (n=3 biological replicates). Data are mean ± s.e.m. p -values were calculated using two-sided unpaired t-test with Welch’s correction (C-E) Mean expression of Enpp1 in excitatory neurons (C), inhibitory neurons (D), and oligodendrocytes (E) in the indicated MS diagnoses. CTRL : control; SPMS : secondary progressive MS; PPMS : primary progressive MS. Significance calculated by Mann-Whitney t-test. (EGA: EGAD00001009169). (F-J) Mean expression of Enpp1 in microglia (F), oligodendrocyte precursor cells (OPCs) and committed oligodendrocyte precursor cells (COPs) (G), excitatory neurons (H), inhibitory neurons (I), and oligodendrocytes (J) in the indicated white matter MS lesions compared to healthy donors. WM : white matter (healthy); NAWM : normal-appearing white matter; AL : active demyelinated lesion; CAL : chronic active demyelinated lesion; CIL : chronic inactive demyelinated lesion; RL : remyelinated lesion. Data are mean ± s.e.m. p -values were calculated using Mann-Whitney t-test. (EGA: EGAD00001009169). (K) Disease incidence in wild-type, Enpp1 H362A and Enpp1 H362A Sting gt/gt mice at the indicated times after MOG 35-55 immunization. Significance calculated by two-way ANOVA.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies diluted 1:1,000-1:4,000, including antibodies against tubulin DM1A (Sigma, CP06), STING (CST, #13647), phospho-STING (CST, #72971), phospho-IRF3 (CST, #4947), Flag (CST, #14793), SLC38A2/SNAT2 (Proteintech 25928-1-AP).

Techniques: Expressing, Tissue, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Extracellular cGAMP transmission exacerbates neuroinflammation in aging and multiple sclerosis

doi: 10.64898/2026.09.30.754737

Figure Lengend Snippet: (A) MACS-sorted CD11b + primary mouse brain microglia from aged wild-type mice were incubated for 48 h in the presence of ENPP1 inhibitor STF-1623. Supernatant was concentrated by solid-phase extraction with aminopropyl columns and 2′3′-cGAMP levels were measured by enzyme-linked immunosorbent assay (ELISA) and normalized to total protein amount in the cell lysates (n=8 biological replicates). (B) Differential gene expression from bulk RNA-seq analysis of young and aged mice forebrains (n=2 biological replicates per group). The volcano plot shows bolded type I IFN and inflammatory from the total list of DEGs (adjusted p-value ≤ 0.05, |[fold change (FC)]| ≥ 1.5). (C) Functional enrichment sorted on adjusted p -value in ascending order. Pathways identified with gene set enrichment analysis (GSEA) using the Hallmark gene set reveal interferon response as the top-enriched pathway in bulk RNA-seq analysis of young and aged mice forebrains (n=2 biological replicates per group). (D,E) Relative transcript levels of type I IFN and interferon-stimulated genes (ISGs) in whole brain tissue (D) and spinal cord tissue (E) of wild-type mice at 10 weeks of age (young) and 22–24 months of age (aged) (n=4–5 mice per group). (F) Kinetics of 2′3′-cGAMP degradation by different CNS regions of Enpp1 H362A mice at 22 months of age (n=2 biological replicates). (G) Strategy to assess the effect of extracellular cGAMP on aging-related neurodegeneration. (H, I) Average latency to fall (H) and average speed at fall (I) across three consecutive trials at the accelerating rotarod test with wild-type (n=6–17 per age group), Enpp1 H362A (n=6–32 per age group), and Enpp1 H362A Sting gt/gt (n=6–15 per age group) (n=3–6 independent experiments performed for different age groups). (J,K,L) Time to turn (TTT), time to descend (TTD), and percentage of failed trials in the pole across three consecutive trials with wild-type (n=8), Enpp1 H362A (n=9 mice per age group), and Enpp1 H362A Sting gt/gt (n=9 mice per age group) mice at 4–6 months of age (n=2 independent experiments). (M) Time spent at the center during open field testing of wild-type (n=10–13 per age group), Enpp1 H362A (n=11–16) and Enpp1 H362A Sting gt (n=9–13) mice at 4 and 22 months of age. Data are mean ± s.e.m. p -values were calculated using two-sided unpaired t-test with Welch’s correction unless otherwise indicated.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies diluted 1:1,000-1:4,000, including antibodies against tubulin DM1A (Sigma, CP06), STING (CST, #13647), phospho-STING (CST, #72971), phospho-IRF3 (CST, #4947), Flag (CST, #14793), SLC38A2/SNAT2 (Proteintech 25928-1-AP).

Techniques: Incubation, Extraction, Enzyme-linked Immunosorbent Assay, Gene Expression, RNA Sequencing, Analysis, Functional Assay, Tissue

Journal: bioRxiv

Article Title: Extracellular cGAMP transmission exacerbates neuroinflammation in aging and multiple sclerosis

doi: 10.64898/2026.09.30.754737

Figure Lengend Snippet: (A) Representative thin layer chromatography (TLC) tracking 2′3′- 32 [P]-cGAMP degradation by tissue lysates from the indicated CNS regions of Enpp1 H362A mice at 22–24 months of age (n=2 biological replicates). (B,C) Relative transcript levels of the indicated ISGs in whole brain tissue (A) and spinal cord tissue (B) of WT (n=3–4) and Enpp1 H362A (n=4–5) mice at 22–24 months of age. (D,E) mRNA expression levels of Cxcl10 (D) and Il1b (E) at the indicated CNS regions of WT, Enpp1 H362A , and Enpp1 H362A Sting gt/gt mice at 4–6 months of age. (F) Body weights of WT, Enpp1 H362A and Enpp1 H362A Sting gt/gt mice at 22–24 months of age. Only mice weighing between 25–35 g underwent accelerating rotarod and pole testing. (G) Discrimination index during the novel object recognition test (NOR) for WT, Enpp1 H362A and Enpp1 H362A Sting gt/gt mice (n=6 mice per group). Only successfully completed tests are plotted. n.a. stands for not applicable, which we used for mice from the Enpp1 H362A genotype that did not interact with either object. (H) Time to reach 30 s of total exploration during the novel object recognition test (NOR) for WT, Enpp1 H362A , and Enpp1 H362A Sting gt/gt mice at 22–24 months of age. The failure threshold of 600s denotes the total duration of the experiment, and 800s denotes the total time of recording (n=6 mice per genotype). (I-K) Distance moved (I), average velocity (J), and time spent at walls (K) during open field testing of WT (n=13), Enpp1 H362A (n=16) and Enpp1 H362A Sting gt/gt (n=13) mice at 22–24 months of age. (L-N) Distance moved (L), average velocity (M), and time spent at walls (N) during open field testing of WT (n=10), Enpp1 H362A (n=11) and Enpp1 H362A Sting g/gtt (n=9) mice at 22–24 months of age. Data are mean ± s.e.m. p -values were calculated using two-sided unpaired t-test with Welch’s correction unless otherwise indicated.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies diluted 1:1,000-1:4,000, including antibodies against tubulin DM1A (Sigma, CP06), STING (CST, #13647), phospho-STING (CST, #72971), phospho-IRF3 (CST, #4947), Flag (CST, #14793), SLC38A2/SNAT2 (Proteintech 25928-1-AP).

Techniques: Thin Layer Chromatography, Tissue, Expressing

Journal: bioRxiv

Article Title: Extracellular cGAMP transmission exacerbates neuroinflammation in aging and multiple sclerosis

doi: 10.64898/2026.09.30.754737

Figure Lengend Snippet: (A) Schematic of scRNA-seq analysis of brain regions of WT, Enpp1 H362A and Enpp1 H362A Sting gt/gt mice at 4–6 months of age. (B-E) UMAP plots of the annotated clusters of single-cell transcriptomes from wild-type, Enpp1 H362A and Enpp1 H362A Sting gt/gt mice at 4–6 months of age. Cell types of pooled brain regions of each genotype (B), different brain regions (C), different genotypes (D), and Leiden clusters (E). (F) Hallmark Pathway Rescue Score ( -NES_STING x NES_ENPP1 ) for major cell types of pooled brain regions for each indicated genotype. NES_ENPP1 refers to Hallmark Pathway functional enrichment of Enpp1 H362A versus wild-type and NES_STING refers to Enpp1 H362A Sting gt/gt versus Enpp1 H362A . (G) Volcano plot showing differentially expressed genes (DEGs) between Enpp1 H362A versus WTmicroglia at 6 months of age. Genes with FDR values < 0.05 and |log 2 FC| > 0.25 were called as DEGs for each comparison, and highlighted if rescued by Enpp1 H362A Sting gt/gt . (H,I) Western blot (n=3 biological replicates) for phosphorylated IRF3 (pIRF3), STING, and tubulin as a loading control from MACS-sorted CD11b + primary mouse brain microglia from WT adult mice following incubation with the indicated concentration of cyclic dinucleotides (CDNs). One representative Western blot shown in (H) and quantification of three blots are shown in (I). Data are mean ± s.e.m. p -values were calculated using two-sided unpaired t-test with Welch’s correction. (J,K) Western blot (n=2 biological replicates) for pIRF3 and tubulin as loading control from MACS-sorted CD11b + primary mouse brain microglia from WT and Lrrc8c KO adult mice following incubation with or without 100 µM 2′3′-cGAMP. One representative Western blot shown in (J) and quantification of multiple blots are shown in ( K). Data are mean ± s.e.m. p -values were calculated using two-sided unpaired t-test with Welch’s correction. (L,M) Expression of Hallmark IFN-γ response signature in microglia binned by expression of Lrrc8a . Hallmark IFN-γ response module score in microglia from wild-type (L) and Enpp1 H362A (M) mice at 4–6 months of age, all four brain regions pooled. Microglia were binned by log-normalized Lrrc8a expression into Zero + Low (≤ 0.5) and Med + High (> 0.5). Inner box plots show the median and interquartile range, with whiskers at 1.5× IQR. P-values were calculated from two-sided Mann–Whitney U tests.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies diluted 1:1,000-1:4,000, including antibodies against tubulin DM1A (Sigma, CP06), STING (CST, #13647), phospho-STING (CST, #72971), phospho-IRF3 (CST, #4947), Flag (CST, #14793), SLC38A2/SNAT2 (Proteintech 25928-1-AP).

Techniques: Analysis, Single Cell, Functional Assay, Comparison, Western Blot, Control, Incubation, Concentration Assay, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Extracellular cGAMP transmission exacerbates neuroinflammation in aging and multiple sclerosis

doi: 10.64898/2026.09.30.754737

Figure Lengend Snippet: (A,B) Bubble heatmaps showing expression of selected marker genes for cluster annotation (A) and cell type assignment (B). Dot size indicates the fraction of expressing cells, colored based on average expression levels. (C) Pie plot of cell type compositions of pooled brain regions of WT, Enpp1 H362A and Enpp1 H362A Sting gt/gt mice at 4–6 months of age. (D) Pie plot of cell type compositions across the indicated brain regions of WT, Enpp1 H362A and Enpp1 H362A Sting gt/gt mice at 4–6 months of age. (E) Enpp1 expression levels and percent expressing-cell types in different brain regions of wild-type mice at 4–6 months of age. (F) Summary of rescued DEGs in different cell types of 4–6 months old mice (pct_threshold of expressing cells = 0.1). (G) Fraction of interferon-responsive microglia (IRM) by region in wild-type, Enpp1 H362A and Enpp1 H362A Sting gt/gt mice at 4–6 months of age. (H) Expression of Hallmark IFN-γ response signature in microglia binned by expression of Lrrc8c . Hallmark IFN-γ response module score in microglia from wild-type, Enpp1 H362A ,and Enpp1 H362A Sting gt/gt mice at 4–6 months of age, all four brain regions pooled. Microglia were binned by log-normalized Lrrc8c expression. Inner box plots show the median and interquartile range, with whiskers at 1.5× IQR. P-values were calculated from two-sided Mann–Whitney U tests.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies diluted 1:1,000-1:4,000, including antibodies against tubulin DM1A (Sigma, CP06), STING (CST, #13647), phospho-STING (CST, #72971), phospho-IRF3 (CST, #4947), Flag (CST, #14793), SLC38A2/SNAT2 (Proteintech 25928-1-AP).

Techniques: Expressing, Marker, MANN-WHITNEY

Journal: bioRxiv

Article Title: Extracellular cGAMP transmission exacerbates neuroinflammation in aging and multiple sclerosis

doi: 10.64898/2026.09.30.754737

Figure Lengend Snippet: (A) Volcano plot showing Enpp1 effect ( Enpp1 H362A versus wild-type) DEGs in astrocytes at 4–6 months of age. Genes with FDR values < 0.05 and |log 2 FC| > 0.25 were called as differentially expressed genes for each comparison. (B) Bubble plot showing STING-dependent DEGs in astrocytes by comparing Enpp1 effect ( Enpp1 H362A versus wild-type) and Sting gt/gt effect ( Enpp1 H362A Sting gt versus Enpp1 H362A ). (C,D) STING signaling output as measured by pIRF3 levels in MACS-sorted ACSA-2 + primary mouse brain astrocytes from WT and Lrrc8c KO adult mice with or without treatment with 100 µM 2′3′-cGAMP and/or 20 µM DCPIB. One representative Western blot shown in (C) and quantification of n = 2 biological replicates is shown in (D). (E,F) STING signaling output as measured by pIRF3 levels in primary human brain astrocytes with or without treatment with 100 µM 2′3′-cGAMP and/or 20 µM DCPIB One representative Western blot is shown in (E) and quantification of n = 4 biological replicates is shown in (F). (G,H) STING signaling output as measured by pIRF3 levels in MACS-sorted ACSA-2 + primary mouse brain astrocytes from WT adult mice with or without treatment with 100 µM 2′3′-cGAMP and/or 1-2 mM L-glutamate. One representative Western blot is shown in (G) and quantification of n = 2 biological replicates is shown in (H). (I) Intracellular 2′3′-cGAMP concentrations in human primary astrocytes after treatment with or without 10 µM 2′3′-cGAMP and/or 1-2 mM L-glutamate, measured by enzyme-linked immunosorbent assay (ELISA) (n = 5–6 biological replicates). (J) Intracellular 2′3′-cGAMP concentrations in HEK 293 LRRC8A KO cells expressing system xc-(SLC7A11 and SLC3A2) after treatment with 10 µM 2′3′-cGAMP, measured by enzyme-linked immunosorbent assay (ELISA) (n = 5–6 biological replicates). (K) Intracellular 2′3′-cGAMP concentrations in HEK 293 LRRC8A KO cells expressing system xc-(SLC7A11 and SLC3A2) after treatment with or without 10 µM 2′3′-cGAMP and/or erastin (50 µM) and/or L-cystine (1 mM) and/or L-glutamate (1-2 mM), measured by enzyme-linked immunosorbent assay (ELISA) (n = 5–6 biological replicates). (L) STING signaling output as measured by pIRF3 levels in MACS-sorted ACSA-2 + primary mouse brain astrocytes from WT adult mice treated with or without 100 µM 2′3′-cGAMP and/or 20 µM WAY-213613. One representative Western blot is shown out of n = 2 biological replicates. (M,N) STING signaling output as measured by pIRF3 levels in U937 SLC19A1 KO LRRC8A KO tet-h SLC1A2 -Flag cells treated with or without 2 µg/ml doxycycline (Dox) for 72 h, and treated with or without 50 µM 2′3′-cGAMP and/or 1 µM WAY-213613 for 1h. . One representative Western blot is shown in (M) and quantification of n = 2 biological replicates is shown in (N). (O,P) STING signaling output as measured by pIRF3 levels in U937 SLC19A1 KO LRRC8A KO tet-h SLC1A2 -Flag cells treated with or without 2 µg/ml doxycycline (Dox) for 72 h, and electroporated with 100 nM 2’3’-cGAMP.One representative Western Blot is shown in (O), and quantification of n = 2 biological replicates is shown in (P). (Q) STING signaling output as measured by pIRF3 levels in U937 SLC19A1 KO LRRC8A KO tet-h SLC1A2 -Flag cells treated with or without 2 µg/ml doxycycline (Dox) for 72 h, and treated with 50 µM 2′3′-cGAMP and/or the indicated concentrations of L-glutamate.(n = 2 biological replicates). One representative Western blot out of n = 2 biological replicates is shown.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies diluted 1:1,000-1:4,000, including antibodies against tubulin DM1A (Sigma, CP06), STING (CST, #13647), phospho-STING (CST, #72971), phospho-IRF3 (CST, #4947), Flag (CST, #14793), SLC38A2/SNAT2 (Proteintech 25928-1-AP).

Techniques: Comparison, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

Journal: bioRxiv

Article Title: Extracellular cGAMP transmission exacerbates neuroinflammation in aging and multiple sclerosis

doi: 10.64898/2026.09.30.754737

Figure Lengend Snippet: (A) STING signaling output as measured by pIRF3 levels in HEK 293 LRRC8A KO cells expressing system xc-(SLC7A11 and SLC3A2), xCT (SLC7A11 alone), or 4F2hc (SLC3A2 alone) after treatment with or without 100 µM 2′3′-cGAMP. One representative Western blot is shown out of n = 2 biological replicates (B) STING signaling output as measured by pIRF3 levels in U937 SLC19A1 KO LRRC8A KO tet-h SLC1A2 -Flag cells treated with or without 2 µg/ml doxycycline (Dox) for 72 h, then treated with or without 50 µM 2’3’-cGAMPand/or erastin (10 µM) or WAY-213613 (20 µM).

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies diluted 1:1,000-1:4,000, including antibodies against tubulin DM1A (Sigma, CP06), STING (CST, #13647), phospho-STING (CST, #72971), phospho-IRF3 (CST, #4947), Flag (CST, #14793), SLC38A2/SNAT2 (Proteintech 25928-1-AP).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Extracellular cGAMP transmission exacerbates neuroinflammation in aging and multiple sclerosis

doi: 10.64898/2026.09.30.754737

Figure Lengend Snippet: (A) Quantification of intracellular 2′3′-cGAMP concentrations in primary mouse cortical and striatal neurons following 15-minute treatment with 10 µM 2′3′-cGAMP by enzyme-linked immunosorbent assay (ELISA) and normalized to total protein amount in the cell lysates (n=21 biological replicates). (B) Bulk RNA sequencing volcano plot of primary mouse cortical and striatal neuron cultures after 24 h treatment with 10 µM 2′3′-cGAMP and 1 µM ENPP1 inhibitor STF-1623. Genes with FDR values < 0.05 and |log 2 FC| > 1 were considered differentially expressed after 2′3′-cGAMP treatment. (C) Cxcl10 transcript levels determined by RT-qPCR of primary mouse cortical and striatal neuron cultures after pre-treatment with glutaminase inhibitor telaglenastat (CB-839, 10µM), followed by L-Gln (2 mM) or erastin (50 µM) for 15 min, then 2′3′-cGAMP for 6h (n=3 technical replicates per condition, replicated across 3 independent experiments). (D) Cxcl10 transcript levels determined by RT-qPCR of primary mouse cortical and striatal neuron cultures after pre-treatment with erastin (50 µM), DCPIB (20 µM), or both for 15 min, followed by 2′3′-cGAMP for 6h (n=3 technical replicates per condition). (E) Cxcl10 transcript levels determined by RT-qPCR of primary mouse cortical and striatal neuron cultures after pre-treatment with erastin (50 µM), DCPIB (20 µM), or both for 15 min, followed by 2′3′-cGAMP for 30 min. All compounds were washed out and cells were incubated in compound-free media for 5h (n=3 technical replicates per condition). (F) Intracellular 2′3′-cGAMP concentration in 293T tet-h SLC38A2 -Flag cells as measured by ELISA following incubation with or without 2 µg/ml doxycycline (Dox) for 18 h and treatment with or without 10 µM 2′3′-cGAMP for 15 min in the presence of ENPP1 inhibitor STF-1623. Measured values are normalized to total protein amount in the cell lysates (n = 6 biological replicates). (G) Western blot assessing STING signaling through pIRF3 levels and SLC38A2 expression in U937 Slc19a1 KO tet-h SLC38A2 -Flag cells following incubation with or without 2 µg/ml doxycycline (Dox), the indicated concentrations of L-Glutamine, and 50 µM 2′3′-cGAMP . One representative Western blot of n = 2 biological replicates is shown. (H) Western blot assessing STING signaling through pIRF3 levels in U937 Slc19a1 KO tet-h SLC38A2 -Flag cells following incubation with or without 2 µg/ml Dox, 50 µM 2′3′-cGAMP, the indicated concentrations of L-Glutamine, and the glutaminase inhibitor telaglenastat (GLSi).. One representative Western blot of n = 2 biological replicates is shown. (I) Western blot assessing pIRF3 levels in U937 Slc19a1 KO tet-h SLC38A2 -Flag cells following incubation with 2 µg/ml doxycycline (Dox) and cGAMP, with or without erastin (50µM). One representative Western blot of n = 2 biological replicates is shown. (J) Western blot to measure pIRF3 in U937 Slc19a1 KO tet-h SLC38A2 -Flag cells following incubation with or without Dox and cGAMP, plus the indicated concentrations of L-cystine. One representative Western blot of n = 2 biological replicates is shown. (K) Heatmap of the Wilcoxon rank-sum z-score for a 15-gene ISG panel; all panel genes are shown regardless of expression level. Left, Enpp1 H362A vs. WT; right, Enpp1 H362A Sting gt/gt vs. Enpp1 H362A . Positive (red) z indicates higher expression in the first-named genotype. *: FDR < 0.05, Benjamini–Hochberg correction across all genes in each comparison. Region × neuron-type combinations with fewer than 30 cells per genotype were not tested and are not shown. Data are mean ± s.e.m. p -values were calculated using two-sided unpaired Student’s t-tests unless otherwise indicated.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies diluted 1:1,000-1:4,000, including antibodies against tubulin DM1A (Sigma, CP06), STING (CST, #13647), phospho-STING (CST, #72971), phospho-IRF3 (CST, #4947), Flag (CST, #14793), SLC38A2/SNAT2 (Proteintech 25928-1-AP).

Techniques: Enzyme-linked Immunosorbent Assay, Bulk RNA Sequencing, Quantitative RT-PCR, Incubation, Concentration Assay, Western Blot, Expressing, Comparison, RNA Sequencing

Journal: bioRxiv

Article Title: Extracellular cGAMP transmission exacerbates neuroinflammation in aging and multiple sclerosis

doi: 10.64898/2026.09.30.754737

Figure Lengend Snippet: (A) Functional enrichment sorted on adjusted p -value in ascending order. Pathways identified with gene set enrichment analysis (GSEA) using the Hallmark gene set reveal interferon response as the top-enriched pathway in bulk RNA-seq analysis of primary mouse cortical and striatal neurons treated with 10 µM 2′3′-cGAMP and 1 µM ENPP1 inhibitor STF-1623 after 24h. (B) Per-gene ISG responses in inhibitory and excitatory neurons by brain region. Heatmap of the Wilcoxon rank-sum z-score for the 40 ISGs with the largest absolute z-score in any column, selected from a curated 124-gene ISG list (118 detected). Left, Enpp1 H362A vs. WT; right, Enpp1 H362A Sting gt/gt vs. Enpp1 H362A . Positive (red) z indicates higher expression in the first-named genotype. * FDR < 0.05, Benjamini–Hochberg across all genes in each comparison. Region × neuron-type combinations with fewer than 30 cells per genotype were not tested and are not shown (inhibitory midbrain; excitatory striatum, left).

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies diluted 1:1,000-1:4,000, including antibodies against tubulin DM1A (Sigma, CP06), STING (CST, #13647), phospho-STING (CST, #72971), phospho-IRF3 (CST, #4947), Flag (CST, #14793), SLC38A2/SNAT2 (Proteintech 25928-1-AP).

Techniques: Functional Assay, Analysis, RNA Sequencing, Expressing, Comparison