HIF1 signaling in cultured myenteric neurons negatively regulates CCL2 expression. (A) Confocal images of colonic muscularis tissue cross-sections from water- or 1×DSS- (3.5%) treated WT mice at week 1, stained with HIF1α (hypoxic cells) and Hu (neuronal somas) antibodies, and DAPI (nuclei). Scale bars, 25 μm. (B)Uchl1 and s100b expression by qPCR in primary adult myenteric neuronal cultures as compared to total colonic muscularis tissue and sorted hematopoietic CD45⁺ cells as a negative control, normalized to Actb (n = 3 per group, n—independent cell culture or independent cell sorting). (C) Confocal images of primary adult myenteric neuronal cultures derived from colonic muscularis of Actl6b^CreR26-STOP^fl/WTtdTomato (Cre⁺) mice at steady state, stained with tdTomato (Actl6b:tdTomato⁺ cells), PGP9.5 (whole neurons), s100β (enteric glia), and Hu (neuronal somas) antibodies, and DAPI (nuclei). Scale bars, 100 μm. (D)Vegfa expression by qPCR in cultured adult colonic myenteric neurons from WT mice, treated with CoCl₂, HIF1α inhibitor PX-478, or their combinations, normalized to Actb (n = 3 per group, n—replica wells in the same cell culture). (E)Ccl2 expression by qPCR in cultured adult colonic myenteric neurons as compared to adult colonic muscularis tissue, normalized to Actb (n = 5 per group, n—independent cell culture or mice). (F) Complementary to Fig. 6 F: Ccl2 expression by qPCR in cultured adult colonic myenteric neurons treated with CoCl₂, LPS, or their combination, normalized to Actb (n = 6 per group, n—independent cell culture). (G)Ccl2 expression by qPCR in cultured SB or colonic myenteric neurons treated with CoCl₂, IL-1β, or their combination, normalized to Actb (n = 4–6 per group, n—replica wells in the same cell culture). (H)Actb mRNA expression (Cq, Quantification cycle) by qPCR in cultured adult colonic myenteric neurons treated with CoCl₂, LPS, IL-1β, or their combination (n = 3, n—replica wells in the same cell culture). (I)Il1a expression by qPCR in cultured adult colonic myenteric neurons treated with CoCl₂, LPS, or their combination, normalized to Actb (left, n = 3, n—replica wells in the same cell culture; right, n = 6, n—independent cell culture). (J)Csf1 expression by qPCR in cultured adult colonic myenteric neurons treated with CoCl₂, LPS, or their combination, normalized to Actb (left, n = 3, n—replica wells in the same cell culture; right, n = 5, n—independent cell culture). (K)Vegfa expression by qPCR in cultured adult colonic myenteric neurons treated with CoCl₂, LPS, or their combination, normalized to Actb (left, n = 3, n—replica wells in the same cell culture; right, n = 6, n—independent cell culture). (L)Actb mRNA expression (Cq) by qPCR in cultured colonic myenteric neurons treated with increasing concentrations of PX-478 as indicated (n = 6, n—replica wells in the same cell culture). All graphs show the mean ± SEM. Data are representative of at least two independent experiments with similar results (A–L) or as indicated. Statistical analyses: unpaired Student’s t test (D, E, and, G–L), paired t test (F), and two-way ANOVA for multiple comparisons (B), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. SB, small bowel.