mouse acth  (Bio-Rad)

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: Upregulation of p300 synergizes with DA to promote cell apoptosis 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.

Article Snippet: Rat prolactinoma cell line MMQ, growth hormone adenoma cell line GH3, and mouse corticotropin adenoma cell line AtT-20 were obtained from the American Type Culture Collection (ATCC).

Techniques: Cell Counting, CCK-8 Assay, Transfection, Plasmid Preparation, Over Expression, RNA Sequencing, Flow Cytometry, Comparison

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: Upregulation of p300 synergizes with DA to promote histone H3K18 lactylation in pituitary tumor cells. (A) Transmission electron microscopy (TEM) was used to observe mitochondrial morphological changes in MMQ cells (transfected with empty vector or OE-p300) treated with BRC (10 μM) for 48 h, the structures indicated by the red arrows are the mitochondria. (n = 3). (B) MMQ cells (transfected with empty vector or OE-p300) were treated with BRC (10 μM) for 48 h, followed by detection of extracellular acidification rate (ECAR) using a Seahorse cell energy metabolism assay. (C) Glycolytic baseline and glycolytic capacity were calculated based on ECAR (n = 3). (D-G) MMQ and AtT-20 cells (transfected with empty vector or OE-p300) were treated with BRC (10 μM) for different durations (0, 3, 6, 12, 18, 24, 48, 72 h). (D, F) Intracellular lactic acid and acetyl-CoA in MMQ and AtT-20 cells were detected using a lactic acid detection kit and acetyl-CoA detection kit, and the OE-p300 group at each time point was normalized using the Vec + BRC group ("nc" in the figure) from the corresponding time point. (E, G) The ratios of lactic acid to acetyl-CoA contents in MMQ and AtT-20 cells were calculated separately (n = 3). (H–I) WB analysis was used to detect pan lactyl-lysine and pan acetyl-lysine protein expression in MMQ (H) and AtT-20 (I) cells (transfected with empty vector or OE-p300) treated with BRC (10 μM) for 24 h or 48 h (n = 3). (J) WB analysis was used to detect pan lactyl-lysine and pan acetyl-lysine protein expression in PA cells treated with BRC (10 μM) or BRC (10 μM) + CTB (50 μM) for 24 h or 48 h (n = 3). (K-L) WB analysis was used to detect H3K18 lactylation (H3K18la) expression 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 = 3). (M) WB analysis was used to detect H3K18la expression in PA cells treated with BRC (10 μM), CAB (25 μM), CTB (50 μM), BRC (10 μM) + CTB (50 μM), or CAB (25 μM) + CTB (50 μM) for 48 h (n = 3). (N) Schematic diagram of molecular structures of wild-type p300 and HAT domain-mutated p300. (O) WB analysis was used to detect H3K18la expression in MMQ and AtT-20 cells (transfected with empty vector, OE-WT-p300, or OE-Mut-p300) treated with BRC (10 μM) for 48 h (n = 3). (P) Co-IP experiments were performed using anti-p300 antibodies in MMQ and AtT-20 cells (transfected with OE-WT-p300 or OE-Mut-p300) treated with BRC (10 μM) for 48 h, followed by WB analysis to detect H3K18la expression (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: Rat prolactinoma cell line MMQ, growth hormone adenoma cell line GH3, and mouse corticotropin adenoma cell line AtT-20 were obtained from the American Type Culture Collection (ATCC).

Techniques: Transmission Assay, Electron Microscopy, Transfection, Plasmid Preparation, Expressing, Co-Immunoprecipitation Assay, Comparison

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: 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.

Article Snippet: Rat prolactinoma cell line MMQ, growth hormone adenoma cell line GH3, and mouse corticotropin adenoma cell line AtT-20 were obtained from the American Type Culture Collection (ATCC).

Techniques: Transfection, Plasmid Preparation, RNA Sequencing, Genome Wide, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Knockdown, Flow Cytometry, Comparison

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: Rat prolactinoma cell line MMQ, growth hormone adenoma cell line GH3, and mouse corticotropin adenoma cell line AtT-20 were obtained from the American Type Culture Collection (ATCC).

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

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: p300 activator YF-2 combined with DA exerts a synergistic anti-pituitary adenoma effect. (A) CCK-8 assay was used to detect cell viability in MMQ and AtT-20 cells treated with YF-2 at different concentrations (0, 1.25, 2.5, 5, 10, 20 μM) for 48 h, after which dose-response curves were fitted based on cell viability and half-maximal inhibitory concentration (IC50) was calculated (n = 4). (B–C) Synergy indices for MMQ cells treated with combinations of YF-2 (0, 2.5, 5, 10 μM) and either BRC (0, 5, 10, 20 μM) or CAB (0, 12.5, 25, 50 μM) for 48 h were calculated using the ZIP synergy scoring model via SynergyFinder Version 3.0 ( https://synergyfinder.fimm.fi ), with a synergy index >10 indicating synergy and a white dashed box denoting the concentration range with the highest potential for maximum synergy (n = 4). (D-G) MMQ and AtT-20 cells were treated with BRC (10 μM), CAB (25 μM), YF-2 (5 μM), BRC (10 μM) + YF-2 (5 μM), or CAB (25 μM) + YF-2 (5 μM) for 48 h. (D, F) CCK-8 assay was used to detect cell viability in MMQ and AtT-20 cells (n = 5). (E) PRL concentration in the supernatant of MMQ cells was detected using a PRL ELISA kit (n = 4). (G) ACTH concentration in the supernatant of AtT-20 cells was detected using an ACTH ELISA kit (n = 4). (H–I) Nude mice were subcutaneously implanted with MMQ and AtT-20 cells, followed by i.p. injection of PBS, BRC (10 mg/kg/d), YF-2 (20 mg/kg/d), or BRC (10 mg/kg/d) + YF-2 (20 mg/kg/d) for 2 weeks. Representative images of subcutaneous xenograft tumors (left), average volume of excised tumors (middle), and average weight of excised tumors (right) (n = 6). (J-K) Quantification of IF staining for Ki-67 expression in tumor tissue sections (n = 6). (L-O) MMQ and AtT-20 cells in the vector group were treated with BRC (10 μM), YF-2 (5 μM) or BRC (10 μM) + YF-2 (5 μM) for 48 h. MMQ and AtT-20 cells transfected with wild-type p300 overexpression plasmid (OE-WT), or HAT domain-mutated p300 overexpression plasmid (OE-Mut) treated with BRC (10 μM) + YF-2 (5 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: Rat prolactinoma cell line MMQ, growth hormone adenoma cell line GH3, and mouse corticotropin adenoma cell line AtT-20 were obtained from the American Type Culture Collection (ATCC).

Techniques: CCK-8 Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Injection, Staining, Expressing, Plasmid Preparation, Transfection, Over Expression, Flow Cytometry, Comparison

Journal: Molecular Psychiatry

Article Title: Regulation of stress susceptibility by chromatin-binding protein PHF6 in the pituitary intermediate lobe

doi: 10.1038/s41380-025-03300-w

Figure Lengend Snippet: a Representative images of c-Fos (green) and DAPI (blue) staining in the pituitary under RS-, FS-, and 2MT-stress conditions, as well as basal conditions, in control and Phf6 cKO mice. White dotted lines indicate the IL. Scale bars, 100 μm. b-d Quantification of c-Fos-positive cells in the IL under RS ( b ), FS ( c ), and 2MT ( d ) stress conditions. Following stress exposure, Phf6 cKO mice showed a significant reduction in the number of c-Fos-positive cells in the IL compared to control mice (RS: n = 4 per group, FS: n = 3 per group under basal and n = 4 per group under stress; 2MT: n = 3 per group under basal and n = 4 per group under stress; * p < 0.05, ** p < 0.01, two-way ANOVA with Bonferroni’s multiple comparisons test). e Representative images of FosB (green) and DAPI (blue) staining in the pituitary under basal and RS-stress conditions in control and Phf6 cKO mice. White dotted lines indicate the IL. Scale bars, 100 μm. f Quantification of FosB-positive cells in the IL under RS-stress conditions. Following RS-stress exposure, Phf6 cKO mice showed a significant reduction in the number of FosB-positive cells in the IL compared to control mice ( n = 4 per group, *** p < 0.001, two-way ANOVA with Bonferroni’s multiple comparisons test). g Experimental design for assessing stress-induced hormone release in control and Phf6 cKO mice. h Plasma concentrations of α-MSH, β-endorphin, and corticosterone were significantly reduced in Phf6 cKO mice compared to control mice following RS stress (Ctrl-base, n = 11; Ctrl-stress, n = 11; Phf6 cKO-base, n = 12; Phf6 cKO-stress, n = 12, * p < 0.05, two-way ANOVA with Bonferroni’s multiple comparisons test). The stress-induced increase in plasma ACTH levels was comparable in Phf6 cKO mice and control mice (Ctrl-base, n = 8; Ctrl-stress, n = 8; Phf6 cKO-base, n = 7; Phf6 cKO-stress, n = 8; p = 0.1238). Data are presented as mean ± SEM.

Article Snippet: ELISA kits were used to quantify α-MSH (EK-043-01, Phoenix Pharmaceuticals, USA), β-endorphin (EK-022-06, Phoenix Pharmaceuticals, USA), corticosterone (AR-E-8100, LDN, Germany), and ACTH (E-EL-M0079, Elabscience, China).

Techniques: Staining, Control, Clinical Proteomics