Extended Data Fig. 5: Inflammatory and hypoxic regulators of HB-EGF in astrocytes.

a, schematic of binding domains, flow cytometric quantification, and representative histograms of membranous HB-EGF (antibody 1) and cytoplasmatic HB-EGF (antibody 2) in astrocytes in response to stimulation with TNF-α and IL-1β over 48 hours. n = 4 per timepoint. b, RT-qPCR analysis of Hbegf expression and quantification of soluble HB-EGF (sHB-EGF) in the supernatant of primary mouse astrocyte in response to stimulation with TNF-α and IL-1β over 48 hours. n = 4 per timepoint. d, schematic of stimulation and supernatant sampling, as well as quantification of sHB-EGF (e) in primary mouse astrocytes stimulated with TNF-α and IL-1β for 8 hours, followed by extensive washing before supernatant sampling. n = 4/6 per timepoint. f, RT-qPCR analysis of Hif1a expression by ACSA2⁺ astrocytes following i.c.v. injection of TNF-α and IL-1β or vehicle. n = 3 per group. g, RT-qPCR analysis of Ldha and Ero1l expression as positive controls for HIF1α related signaling in primary mouse astrocytes stimulated with CoCl₂ over 24 hours. n = 4 per timepoint. h, RT-qPCR analysis of HBEGF expression by human astrocytes under pseudohypoxic conditions (CoCl2). n = 2 per group. i, and Enzyme-linked Immunosorbent Assay (ELISA) of soluble HB-EGF (sHB-EGF) in the supernatant of primary mouse astrocytes under pseudohypoxic conditions (CoCl2). n = 4/16 per group. j, schematic of transcriptional competition between HIF1α and AhR. k, representative scatterplots of HB-EGF expression in primary mouse astrocytes (ACSA2 + GFP+) transduced with a control (Gfap::Scrmbl), AhR (Gfap::Ahr), HIF1α (Gfap::Hif1), or HB-EGF (Gfap::Hbegf) targeting CRISPR/Cas9 vector, quantified by intracellular flow cytometry. n = 3 per group. l, representative histograms depicting HB-EGF staining in astrocytes obtained from Gfap::Scrmbl, Gfap::Ahr and Gfap::Hif1a mice during late-stage EAE. m, RT-qPCR analysis of Hbegf, Ahr, Hif1a, and Ldha in ACSA2+ astrocytes in Gfap::Scrmbl, Gfap::Hif1a, and Gfap::Ahr mice. n = 3 per group. Data shown as mean ± SD. One-way ANOVA with Dunett’s multiple comparisons test (tested against control) in (a, b, c, e, m), unpaired t-test in (f, i), Two-way ANOVA with Sidak’s multiple comparisons test in (g).

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