Fig. 8: Hcy decreases the expression level of Ndufa1 by reducing the expression of the transcription factor Creb1.

A Transcription factor prediction of Ndufa1 was performed by NCBI (https://www.ncbi.nlm.nih.gov/home/download/) and Jaspar (https://jaspar.elixir.no/). The list showed the top 4 candidate genes overall. B RT-qPCR analysis of transcript levels of Creb1, Foxa2, Foxd3, and Gfi1 in the hippocampus of rats treated with or without Hcy. The data were normalized by Gapdh (n = 5 for each group). C Potential locations of Creb1 binding to Ndufa1 predicted by JASPAR. D, E The ChIP-qPCR data of the Ndufa1 potential binding site in the promoter region with Creb1 in the hippocampus of rats (n = 4 for each group, each sample was repeated once). F Hcy reduced the protein levels of Ndufa1 and Creb1, and the overexpression of Creb1 reversed the reduction in HEK293T cells (n = 3 for each group). G Working principle of the dual-luciferase reporter gene. H Overexpression of Creb1 and a dual-luciferase reporter in which Ndufa1 promoter truncations were cloned and fused enhances transcription from the 5′ flanking region of the Ndufa1 gene. I, J p-Creb1 staining in CA1 and CA3 regions of the hippocampus and cortex in Con and Hcy groups. L, M The images of p-Creb1 and Creb1 by western blotting and the relative Creb1 activity were represented by the ratio of p-Creb1/Creb1. K, N–P JC-1 staining, ATP and ROS levels detection in N2a cells with/without Hcy and OE-Creb1 treatment. Q, R N2a cells were individually treated with Hcy (100 μM), DTT (100 μM), or NAC (100 μM) for 24 h. Changes in Ndufa1 protein levels were determined by Western blotting (n = 4). Data were presented as mean ± SEM. Unpaired t-test for (E and L). One-way ANOVA followed by Bonferroni’s post hoc was used for others (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns no significance; n = 6 for each group).