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ERK inhibition attenuates TXNRD1-driven SPP1-high/CXCL9-low remodeling in EGCs (A) CCK-8 viability assay of cultured rat EGCs treated with the indicated concentrations of SCH772984 (0–40 nM) for 24 h; for (B)–(G), cultured EGCs were assigned to Control+DMSO, LV-OE-NC+DMSO, LV-OE-TXNRD1+DMSO, and LV-OE-TXNRD1+SCH772984 groups. SCH772984 was used at 10 nM. (B and C) Representative WB of TXNRD1, p-Raf, Raf, p-MEK, MEK, p-ERK, ERK, and GAPDH (B) and densitometric quantification (C). (D and E) Representative FCM histograms of intracellular <t>ROS</t> (D) and quantification of relative ROS fluorescence intensity (E). (F and G) Representative IF images of CXCL9 and SPP1 in cultured EGCs (F) and quantification of relative fluorescence intensity (G). n = 3 independent biological experiments; each dot represents one independently performed experiment. Quantitative data are presented as mean ± SD with individual data points. One-way ANOVA followed by Tukey’s multiple-comparison test was used. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant. Blots, FCM plots, and micrographs are representative of three independent experiments. Scale bars, 100 μm in (F).
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ERK inhibition attenuates TXNRD1-driven SPP1-high/CXCL9-low remodeling in EGCs (A) CCK-8 viability assay of cultured rat EGCs treated with the indicated concentrations of SCH772984 (0–40 nM) for 24 h; for (B)–(G), cultured EGCs were assigned to Control+DMSO, LV-OE-NC+DMSO, LV-OE-TXNRD1+DMSO, and LV-OE-TXNRD1+SCH772984 groups. SCH772984 was used at 10 nM. (B and C) Representative WB of TXNRD1, p-Raf, Raf, p-MEK, MEK, p-ERK, ERK, and GAPDH (B) and densitometric quantification (C). (D and E) Representative FCM histograms of intracellular <t>ROS</t> (D) and quantification of relative ROS fluorescence intensity (E). (F and G) Representative IF images of CXCL9 and SPP1 in cultured EGCs (F) and quantification of relative fluorescence intensity (G). n = 3 independent biological experiments; each dot represents one independently performed experiment. Quantitative data are presented as mean ± SD with individual data points. One-way ANOVA followed by Tukey’s multiple-comparison test was used. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant. Blots, FCM plots, and micrographs are representative of three independent experiments. Scale bars, 100 μm in (F).
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ERK inhibition attenuates TXNRD1-driven SPP1-high/CXCL9-low remodeling in EGCs (A) CCK-8 viability assay of cultured rat EGCs treated with the indicated concentrations of SCH772984 (0–40 nM) for 24 h; for (B)–(G), cultured EGCs were assigned to Control+DMSO, LV-OE-NC+DMSO, LV-OE-TXNRD1+DMSO, and LV-OE-TXNRD1+SCH772984 groups. SCH772984 was used at 10 nM. (B and C) Representative WB of TXNRD1, p-Raf, Raf, p-MEK, MEK, p-ERK, ERK, and GAPDH (B) and densitometric quantification (C). (D and E) Representative FCM histograms of intracellular <t>ROS</t> (D) and quantification of relative ROS fluorescence intensity (E). (F and G) Representative IF images of CXCL9 and SPP1 in cultured EGCs (F) and quantification of relative fluorescence intensity (G). n = 3 independent biological experiments; each dot represents one independently performed experiment. Quantitative data are presented as mean ± SD with individual data points. One-way ANOVA followed by Tukey’s multiple-comparison test was used. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant. Blots, FCM plots, and micrographs are representative of three independent experiments. Scale bars, 100 μm in (F).
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ERK inhibition attenuates TXNRD1-driven SPP1-high/CXCL9-low remodeling in EGCs (A) CCK-8 viability assay of cultured rat EGCs treated with the indicated concentrations of SCH772984 (0–40 nM) for 24 h; for (B)–(G), cultured EGCs were assigned to Control+DMSO, LV-OE-NC+DMSO, LV-OE-TXNRD1+DMSO, and LV-OE-TXNRD1+SCH772984 groups. SCH772984 was used at 10 nM. (B and C) Representative WB of TXNRD1, p-Raf, Raf, p-MEK, MEK, p-ERK, ERK, and GAPDH (B) and densitometric quantification (C). (D and E) Representative FCM histograms of intracellular <t>ROS</t> (D) and quantification of relative ROS fluorescence intensity (E). (F and G) Representative IF images of CXCL9 and SPP1 in cultured EGCs (F) and quantification of relative fluorescence intensity (G). n = 3 independent biological experiments; each dot represents one independently performed experiment. Quantitative data are presented as mean ± SD with individual data points. One-way ANOVA followed by Tukey’s multiple-comparison test was used. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant. Blots, FCM plots, and micrographs are representative of three independent experiments. Scale bars, 100 μm in (F).
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ERK inhibition attenuates TXNRD1-driven SPP1-high/CXCL9-low remodeling in EGCs (A) CCK-8 viability assay of cultured rat EGCs treated with the indicated concentrations of SCH772984 (0–40 nM) for 24 h; for (B)–(G), cultured EGCs were assigned to Control+DMSO, LV-OE-NC+DMSO, LV-OE-TXNRD1+DMSO, and LV-OE-TXNRD1+SCH772984 groups. SCH772984 was used at 10 nM. (B and C) Representative WB of TXNRD1, p-Raf, Raf, p-MEK, MEK, p-ERK, ERK, and GAPDH (B) and densitometric quantification (C). (D and E) Representative FCM histograms of intracellular <t>ROS</t> (D) and quantification of relative ROS fluorescence intensity (E). (F and G) Representative IF images of CXCL9 and SPP1 in cultured EGCs (F) and quantification of relative fluorescence intensity (G). n = 3 independent biological experiments; each dot represents one independently performed experiment. Quantitative data are presented as mean ± SD with individual data points. One-way ANOVA followed by Tukey’s multiple-comparison test was used. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant. Blots, FCM plots, and micrographs are representative of three independent experiments. Scale bars, 100 μm in (F).
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ERK inhibition attenuates TXNRD1-driven SPP1-high/CXCL9-low remodeling in EGCs (A) CCK-8 viability assay of cultured rat EGCs treated with the indicated concentrations of SCH772984 (0–40 nM) for 24 h; for (B)–(G), cultured EGCs were assigned to Control+DMSO, LV-OE-NC+DMSO, LV-OE-TXNRD1+DMSO, and LV-OE-TXNRD1+SCH772984 groups. SCH772984 was used at 10 nM. (B and C) Representative WB of TXNRD1, p-Raf, Raf, p-MEK, MEK, p-ERK, ERK, and GAPDH (B) and densitometric quantification (C). (D and E) Representative FCM histograms of intracellular <t>ROS</t> (D) and quantification of relative ROS fluorescence intensity (E). (F and G) Representative IF images of CXCL9 and SPP1 in cultured EGCs (F) and quantification of relative fluorescence intensity (G). n = 3 independent biological experiments; each dot represents one independently performed experiment. Quantitative data are presented as mean ± SD with individual data points. One-way ANOVA followed by Tukey’s multiple-comparison test was used. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant. Blots, FCM plots, and micrographs are representative of three independent experiments. Scale bars, 100 μm in (F).
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ERK inhibition attenuates TXNRD1-driven SPP1-high/CXCL9-low remodeling in EGCs (A) CCK-8 viability assay of cultured rat EGCs treated with the indicated concentrations of SCH772984 (0–40 nM) for 24 h; for (B)–(G), cultured EGCs were assigned to Control+DMSO, LV-OE-NC+DMSO, LV-OE-TXNRD1+DMSO, and LV-OE-TXNRD1+SCH772984 groups. SCH772984 was used at 10 nM. (B and C) Representative WB of TXNRD1, p-Raf, Raf, p-MEK, MEK, p-ERK, ERK, and GAPDH (B) and densitometric quantification (C). (D and E) Representative FCM histograms of intracellular <t>ROS</t> (D) and quantification of relative ROS fluorescence intensity (E). (F and G) Representative IF images of CXCL9 and SPP1 in cultured EGCs (F) and quantification of relative fluorescence intensity (G). n = 3 independent biological experiments; each dot represents one independently performed experiment. Quantitative data are presented as mean ± SD with individual data points. One-way ANOVA followed by Tukey’s multiple-comparison test was used. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant. Blots, FCM plots, and micrographs are representative of three independent experiments. Scale bars, 100 μm in (F).
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ERK inhibition attenuates TXNRD1-driven SPP1-high/CXCL9-low remodeling in EGCs (A) CCK-8 viability assay of cultured rat EGCs treated with the indicated concentrations of SCH772984 (0–40 nM) for 24 h; for (B)–(G), cultured EGCs were assigned to Control+DMSO, LV-OE-NC+DMSO, LV-OE-TXNRD1+DMSO, and LV-OE-TXNRD1+SCH772984 groups. SCH772984 was used at 10 nM. (B and C) Representative WB of TXNRD1, p-Raf, Raf, p-MEK, MEK, p-ERK, ERK, and GAPDH (B) and densitometric quantification (C). (D and E) Representative FCM histograms of intracellular ROS (D) and quantification of relative ROS fluorescence intensity (E). (F and G) Representative IF images of CXCL9 and SPP1 in cultured EGCs (F) and quantification of relative fluorescence intensity (G). n = 3 independent biological experiments; each dot represents one independently performed experiment. Quantitative data are presented as mean ± SD with individual data points. One-way ANOVA followed by Tukey’s multiple-comparison test was used. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant. Blots, FCM plots, and micrographs are representative of three independent experiments. Scale bars, 100 μm in (F).

Journal: iScience

Article Title: TXNRD1-driven enteric glial remodeling promotes neuronal dysfunction in Hirschsprung disease

doi: 10.1016/j.isci.2026.117636

Figure Lengend Snippet: ERK inhibition attenuates TXNRD1-driven SPP1-high/CXCL9-low remodeling in EGCs (A) CCK-8 viability assay of cultured rat EGCs treated with the indicated concentrations of SCH772984 (0–40 nM) for 24 h; for (B)–(G), cultured EGCs were assigned to Control+DMSO, LV-OE-NC+DMSO, LV-OE-TXNRD1+DMSO, and LV-OE-TXNRD1+SCH772984 groups. SCH772984 was used at 10 nM. (B and C) Representative WB of TXNRD1, p-Raf, Raf, p-MEK, MEK, p-ERK, ERK, and GAPDH (B) and densitometric quantification (C). (D and E) Representative FCM histograms of intracellular ROS (D) and quantification of relative ROS fluorescence intensity (E). (F and G) Representative IF images of CXCL9 and SPP1 in cultured EGCs (F) and quantification of relative fluorescence intensity (G). n = 3 independent biological experiments; each dot represents one independently performed experiment. Quantitative data are presented as mean ± SD with individual data points. One-way ANOVA followed by Tukey’s multiple-comparison test was used. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant. Blots, FCM plots, and micrographs are representative of three independent experiments. Scale bars, 100 μm in (F).

Article Snippet: ROS assay kit , MedChemExpress , HY-K0320.

Techniques: Inhibition, CCK-8 Assay, Viability Assay, Cell Culture, Control, Fluorescence, Comparison

NAC suppresses TXNRD1-induced ROS accumulation, ERK activation, and SPP1-high/CXCL9-low remodeling in EGCs Cultured rat EGCs were assigned to Control+DMSO, LV-OE-NC+DMSO, LV-OE-TXNRD1+DMSO, LV-OE-NC+NAC, and LV-OE-TXNRD1+NAC groups. NAC was used at 1. (A and B) Representative WB of p-ERK, ERK, and tubulin (A) and quantification of the p-ERK/ERK ratio (B). (C and D) Representative FCM histograms of intracellular ROS (C) and quantification of relative ROS fluorescence intensity (D). (E) TrxR activity in EGC lysates. (F and G) Representative IF images of CXCL9 and SPP1 in cultured EGCs (F) and quantification of relative fluorescence intensity (G). n = 3 independent biological experiments; each dot represents one independently performed experiment. Quantitative data are presented as mean ± SD with individual data points. One-way ANOVA followed by Tukey’s multiple-comparison test was used. ∗ p < 0.05, ∗∗∗ p < 0.001; ns, not significant. Blots, FCM plots, and micrographs are representative of three independent experiments. Scale bars, 100 μm in (F).

Journal: iScience

Article Title: TXNRD1-driven enteric glial remodeling promotes neuronal dysfunction in Hirschsprung disease

doi: 10.1016/j.isci.2026.117636

Figure Lengend Snippet: NAC suppresses TXNRD1-induced ROS accumulation, ERK activation, and SPP1-high/CXCL9-low remodeling in EGCs Cultured rat EGCs were assigned to Control+DMSO, LV-OE-NC+DMSO, LV-OE-TXNRD1+DMSO, LV-OE-NC+NAC, and LV-OE-TXNRD1+NAC groups. NAC was used at 1. (A and B) Representative WB of p-ERK, ERK, and tubulin (A) and quantification of the p-ERK/ERK ratio (B). (C and D) Representative FCM histograms of intracellular ROS (C) and quantification of relative ROS fluorescence intensity (D). (E) TrxR activity in EGC lysates. (F and G) Representative IF images of CXCL9 and SPP1 in cultured EGCs (F) and quantification of relative fluorescence intensity (G). n = 3 independent biological experiments; each dot represents one independently performed experiment. Quantitative data are presented as mean ± SD with individual data points. One-way ANOVA followed by Tukey’s multiple-comparison test was used. ∗ p < 0.05, ∗∗∗ p < 0.001; ns, not significant. Blots, FCM plots, and micrographs are representative of three independent experiments. Scale bars, 100 μm in (F).

Article Snippet: ROS assay kit , MedChemExpress , HY-K0320.

Techniques: Activation Assay, Cell Culture, Control, Fluorescence, Activity Assay, Comparison

Journal: iScience

Article Title: TXNRD1-driven enteric glial remodeling promotes neuronal dysfunction in Hirschsprung disease

doi: 10.1016/j.isci.2026.117636

Figure Lengend Snippet:

Article Snippet: ROS assay kit , MedChemExpress , HY-K0320.

Techniques: Model Organisms, Recombinant, CCK-8 Assay, Apoptosis Assay, ROS Assay, Calcium Assay, Enzyme-linked Immunosorbent Assay, Generated, Software, Genotyping