Fig. 2

Functional characterization of NHIP transcript levels in response to neuronal hypoxia. In a–e RT-qPCR assays, NHIP levels were normalized to GAPDH with at least three independent experiments per condition. A NHIP levels in human tissues, including adult brain, fetal brain, placenta, and testis. B NHIP levels in placenta samples from the discovery group (ASD n = 17, TD n = 11). ASD samples show significantly lower NHIP levels than TD samples (Mann-Whitney-Wilcoxon, p value = 0.009). C NHIP levels in human cell lines, HEK293T, IMR90, LUHMES, and SH-SY5Y. In LUHMES cells, NHIP levels were significantly higher in differentiated neurons compared to undifferentiated neurons (Mann-Whitney-Wilcoxon, p value = 0.034). D Differentiated LUHMES cells are more sensitive to hypoxia than undifferentiated LUHMES cells. Formation of reactive oxygen species (ROS) was measured in differentiated and undifferentiated LUHMES cells treated with 100 nM CoCl₂, a hypoxia mimetic, or vehicle (mock) (Mann-Whitney-Wilcoxon, p value = 0.0001). E NHIP levels increase in response to hypoxia, specifically in differentiated neurons. Differentiated or undifferentiated LUHMES cells were treated with 100 nM CoCl₂. In differentiated LUHMES cells, CoCl₂ treatment significantly increased NHIP levels (Mann-Whitney-Wilcoxon, p value = 0.009). F NHIP overexpression in HEK293T cells resulted in a faster doubling time than vector control cells, indicating increased cell proliferation. (Hour 142, Mann-Whitney-Wilcoxon, p value = 0.045, NHIP overexpression cells n = 3, control cells n = 3, effect size = 3.10; Hour 166, Mann-Whitney-Wilcoxon, p value = 0.0009, NHIP overexpression cells n = 3, control cells n = 3, effect size = 7.80). G Vector design of NHIP peptide-eGFP (dotted line represents excised ATG of EGFP) and combined phase and fluorescent microscopy. Green, eGFP linked to NHIP peptide; red, mCherry, transfection positive control. Scale bars, 100 μm. H Immunofluorescent staining of human frontal cortex, showing nuclear localization with anti-NHIP, but not pre-immune control. Blue, DAPI nuclear counterstain; red, anti-NHIP staining. Scale bars, 100 μm. Data are mean ± SEM