Journal: Redox Biology
Article Title: p300-mediated histone H3K18 lactylation promotes mitochondrial ROS accumulation via mitophagy inhibition to potentiate dopamine agonists efficacy in prolactinomas
doi: 10.1016/j.redox.2026.104077
Figure Lengend Snippet: WASH1 inhibits mitophagy by binding to the ubiquitin-associated (UBA) domain of p62 in pituitary tumor cells. (A) MMQ and AtT-20 cells in the vector group, OE-p300 group, OE-p300 with Washc1 knockdown group 1 (sh1+OE), and OE-p300 with Washc1 knockdown group 2 (sh2+OE) were treated with BRC (10 μM) for 48 h, followed by WB analysis to detect the expression of LC3BI/II, p62, translocase of outer mitochondrial membrane 20 (TOM20), translocase of inner mitochondrial membrane 23 (TIM23), and cytochrome c oxidase subunit IV (COX IV) (n = 3). (B) WASH1-Flag protein was overexpressed in AtT-20 cells with Washc1 knockout, and immunoprecipitation-mass spectrometry (IP-MS) was used to analyze potential interacting proteins of WASH1. (C) Mouse WASH1 protein and the UBA domain of p62 were subjected to molecular docking using the HDOCK server ( http://hdock.phys.hust.edu.cn/ ). Appropriate prediction models were selected, and docking results were analyzed using PyMOL (Version 3.1). (D) In MMQ and AtT-20 cells with p300 overexpression and Washc1 knockout, wild-type Washc1 (OE-WT-Flag) or mutant Washc1 (OE-Mut-Flag) was then overexpressed. Co-IP experiments were conducted using anti-Flag antibodies, and subsequent WB analysis was used to detect the expression of WASH1-Flag and p62 (n = 3). (E) MMQ and AtT-20 cells with p300 overexpression and Washc1 knockout in the vector group, OE-WT-Flag group, or OE-Mut-Flag group were treated with BRC (10 μM) for 48 h, followed by WB analysis to detect the expression of LC3BI/II, p62, TOM20, TIM23, and COX IV (n = 3). (F) MMQ and AtT-20 cells with p300 overexpression and Washc1 knockout in the vector group, OE-WT-Flag group, or OE-Mut-Flag group were treated with BRC (10 μM) for 24 h, followed by mt-Keima ratiometric analysis to quantify mitophagy levels (n = 4). (G) MMQ and AtT-20 cells in the vector group, OE-WT-Flag group, or OE-Mut-Flag group were treated with BRC (10 μM) for 24 h, and confocal fluorescence microscopy was used to observe the co-localization of mitochondria (TOM20) and lysosomes (lysosome-associated membrane protein 1, LAMP1) (n = 3). (H–I) MMQ and AtT-20 cells with p300 overexpression and Washc1 knockout in the vector group, OE-WT-Flag group, or OE-Mut-Flag group were treated with BRC (10 μM) for 48 h, followed by flow cytometry to detect mitochondrial ROS and cell apoptosis levels (n = 3). One-way ANOVA was used for comparison among multiple groups. Data are presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Article Snippet: Cell apoptosis was investigated by using an Annexin V-FITC/PI apoptosis assay kit (Vazyme) according to the manufacturer's instructions.
Techniques: Binding Assay, Ubiquitin Proteomics, Plasmid Preparation, Knockdown, Expressing, Membrane, Knock-Out, Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Over Expression, Mutagenesis, Co-Immunoprecipitation Assay, Fluorescence, Microscopy, Flow Cytometry, Comparison
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![Upregulation of p300 synergizes with DA to promote cell <t>apoptosis</t> by increasing mitochondrial ROS in pituitary tumor cells. (A-B) Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability in MMQ and AtT-20 cells transfected with empty vector or p300 overexpression plasmid (OE-p300) treated with BRC (10 μM) for different durations (0, 3, 6, 9, 12, 15, 18, 21, 24, 30, 36, 42, 48 h) (n = 3). (C-D) CCK-8 assay was used to detect cell viability in MMQ and AtT-20 cells (transfected with empty vector or OE-p300) treated with BRC (10 μM) or CAB (25 μM) for 48 h (n = 5). (E) AtT-20 cells (transfected with empty vector or OE-p300) were treated with BRC (10 μM) for 24 h, followed by bulk RNA sequencing and KEGG pathway enrichment analysis of differentially upregulated genes (n = 3). (F–I) Flow cytometry was used to detect mitochondrial ROS levels in MMQ and AtT-20 cells (transfected with empty vector or OE-p300) treated with BRC (10 μM) or CAB (25 μM) for 48 h. Flow cytometry was used to detect mitochondrial ROS levels in PA cells treated with BRC (10 μM), CAB (25 μM), N-(4-chloro-3-triffuoromethyl-phenyl)-2- ethoxy-benzamide (CTB, 50 μM), BRC (10 μM) + CTB (50 μM), or CAB (25 μM) + CTB (50 μM) for 48 h (n = 3). (J-L) Flow cytometry was used to detect cell apoptosis levels in MMQ and AtT-20 cells (transfected with empty vector or OE-p300) treated with BRC (10 μM) alone or combined with N-acetylcysteine (NAC, 5 mM) or glutathione (GSH, 2 mM) for 48 h (n = 3). (M) Schematic diagram of molecular structures of wild-type p300 and histone acetyltransferase (HAT) domain-mutated p300. (N–S) Flow cytometry was used to detect mitochondrial ROS levels and apoptosis levels in MMQ and AtT-20 cells (transfected with empty vector, wild-type p300 overexpression plasmid [OE-WT-p300], or HAT domain-mutated p300 overexpression plasmid [OE-Mut-p300]) treated with BRC (10 μM) for 48 h (n = 3). One-way ANOVA was used for comparison among multiple groups. Data are presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0476/pmc12950476/pmc12950476__gr2.jpg)
![Upregulation of p300 synergizes with DA to elevate mitochondrial ROS levels via H3K18la-mediated transcription of Ndufs7 and Washc1 in pituitary tumor cells. (A-D) AtT-20 cells (transfected with empty vector or OE-p300) were treated with sodium lactate (5 mM) or BRC (10 μM) for 24 h, followed by bulk RNA sequencing. (A) Volcano plot showing the distribution of differentially expressed genes in the vector + sodium lactate group (Vec + L-Na) compared with the vector group (Vec). (B) Volcano plot showing the distribution of differentially expressed genes in the OE-p300+BRC group compared with the Vec + BRC group. (C) Venn diagram of the intersection of upregulated differentially expressed genes from groups A and B. (D) KEGG pathway enrichment of the 2273 genes obtained from the intersection in C (n = 3). (E) Peak plot showing H3K18la enrichment in genome-wide promoter regions of cells from the OE-p300+BRC group and Vec + BRC group in AtT-20 cells (transfected with empty vector or OE-p300) treated with BRC (10 μM) for 24 h. (F) Venn diagram of the intersection between the 2273 genes obtained from the intersection in C and the upregulated differentially expressed genes enriched by CUT&Tag using anti-H3K18la antibody in AtT-20 cells (transfected with OE-p300) treated with BRC (10 μM) for 24 h. (G) Venn diagram of the intersection between the 36 genes obtained from the intersection in F and the ROS-related gene set (KEGG: mmu05208). (H) Peak plot showing H3K18la enrichment in the NADH dehydrogenase [ubiquinone] Fe–S protein 7 (Ndufs7) promoter region in the Vec + BRC group and OE-p300+BRC group in AtT-20 cells. (I) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) of H3K18la enrichment in the Ndufs7 promoter region in the Vec + BRC group, OE-p300+BRC group, and OE-p300+BRC + Gal group in AtT-20 cells (n = 3). AtT-20 cells in the vector group were treated with BRC (10 μM) or BRC (10 μM) + CTB (50 μM) for 48 h. AtT-20 cells in the OE-p300 group, OE-p300 with Ndufs7 knockdown group 1 (sh1+OE), and OE-p300 with Ndufs7 knockdown group 2 (sh2+OE) were treated with BRC (10 μM) for 48 h, followed by flow cytometry to detect mitochondrial ROS levels (J), hydrogen peroxide detection kit to measure intracellular hydrogen peroxide levels (K), and flow cytometry to detect cell apoptosis levels (L) (n = 3). (M) After AtT-20 cells in the vector group, OE-p300 group, OE-p300 with Ndufs7 knockdown group 1 (sh1+OE), and OE-p300 with Ndufs7 knockdown group 2 (sh2+OE) were treated with BRC (10 μM) for 36 h, electron leak was detected via high-resolution respirometry combined with fluorometry (n = 3). (N) AtT-20 cells (transfected with empty vector or OE-p300) were treated with BRC (10 μM) for 24 h, followed by bulk RNA sequencing and KEGG pathway enrichment analysis of differentially downregulated genes (n = 3). (O) TEM was used to observe mitochondrial morphological changes in AtT-20 cells (transfected with empty vector or OE-p300) treated with BRC (10 μM) for 24 h, the process indicated by the red arrows is mitophagy. (n = 3). (P) Venn diagram of the intersection between the 36 genes obtained from the intersection in F and the autophagy-related gene set (GO:0010506). (Q) Peak plot showing H3K18la enrichment in the WASH complex subunit 1 (Washc1) promoter region in the vector group and OE-p300+BRC group in AtT-20 cells. (R) ChIP-qPCR of H3K18la enrichment in the Washc1 promoter region in the Vec + BRC group, OE-p300+BRC group, and OE-p300+BRC + Gal group in AtT-20 cells (n = 3). AtT-20 cells in the vector group were treated with BRC (10 μM) or BRC (10 μM) + CTB (50 μM) for 48 h. AtT-20 cells in the OE-p300 group, OE-p300 with Washc1 knockdown group 1 (sh1+OE), and OE-p300 with Washc1 knockdown group 2 (sh2+OE) were treated with BRC (10 μM) for 48 h, followed by flow cytometry to detect mitochondrial ROS levels (S), hydrogen peroxide detection kit to measure intracellular hydrogen peroxide levels (T), and flow cytometry to detect cell apoptosis levels (U) (n = 3). (V) Schematic diagram showing that NDUFS7 increases mitochondrial ROS and WASH1 inhibits mitophagy, thereby inducing cell apoptosis. One-way ANOVA was used for comparison among multiple groups. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0476/pmc12950476/pmc12950476__gr4.jpg)
