Journal: Inflammation
Article Title: Narciclasine Alleviates Endothelial Inflammation and Atherosclerosis Initiation by Inhibiting Histone Lactylation-Mediated NF-κB Activation
doi: 10.1007/s10753-025-02446-7
Figure Lengend Snippet: Narciclasine-mediated H3K18la is associated with the activation of the NF-κB pathway in ECs . ( A ) Schematic diagram of histone H3 lysine-18 lactylation (H3K18la) inhibition. ( B - D ) Western blotting analysis of VCAM1 (110 kDa), ICAM1 (90 kDa), and H3K18la (15 kDa) in HUVECs under the following conditions: ( B ) Stimulation with ox-LDL (100 µg/mL) in the presence or absence of 2-deoxy-D-glucose (2-DG, 0.5 mM), or sodium lactate (NaLac, 10 mM) for 24 h; ( C ) Transfection with negative control siRNA (siNC), siLDHA, or siP300 for 48 h, followed by stimulation with ox-LDL (100 µg/mL) for 24 h; ( D ) Stimulation with ox-LDL (100 µg/mL) in the presence or absence of dichloroacetate (DCA, 10 mM) for 24 h. Protein band intensities were quantified using ImageJ (version 1.48); H3K18la was normalized to H3 (15 kDa), and VCAM1 and ICAM1 to β-actin (42 kDa). Quantitative data are shown in Supplementary Fig. S14B-D ( n = 3 biological replicates). ( E ) Schematic diagram of H3K18la promotion. ( F - G ) Western blotting analysis of VCAM1 and H3K18la in HUVECs under the following conditions: ( F ) Pretreatment with or without narciclasine (Narc, 20 nM) for 6 h, followed by stimulation with ox-LDL (100 µg/mL) in combination with either NaLac (10 mM) or sodium acetate (NaAc, 10 mM) for 24 h; ( G ) Pretreatment with or without Narc (20 nM) for 6 h, followed by stimulation with ox-LDL (100 µg/mL) in the presence or absence of entinostat (MS275, 0.5 µM) for 24 h. Protein band intensities were quantified using ImageJ, with H3K18la was normalized to H3, VCAM1 to β-actin. Quantitative data are shown in Supplementary Fig. S14E and F ( n = 3 biological replicates). ( H - I ) HUVECs were pretreated with or without 20 nM Narc for 6 h, followed by stimulation with ox-LDL (100 µg/mL) in the presence or absence of 10 mM NaLac for 24 h. ( H ) Representative images of PKH26-labeled THP-1 monocyte adhesion to endothelial cells (scale bar = 250 μm, 10× magnification). The right panel shows the quantification of adherent monocytes from 10 random fields per replicate, expressed as cells per field ( n = 3 biological replicates). ( I ) Representative images of RAW 264.7 macrophage chemotaxis toward HUVEC-conditioned medium (Scale bar = 50 μm, 20× magnification). The right panel shows the quantification of migrated cells from 5 random fields per well, expressed as cells per field ( n = 3 biological replicates). ( J ) Western blotting analysis of p-p65 (65 kDa), and p65 (65 kDa) in HUVECs pretreated with or without Narc (20 nM) for 6 h, followed by stimulation with ox-LDL (100 µg/mL) in combination with either NaLac (10 mM) or MS275 (0.5 µM) for 1 h. Protein band intensities were quantified using ImageJ, with the p-p65/p65 ratio (right panel) calculated after normalizing both p-p65 and total p65 to β-actin ( n = 3 biological replicates). ( K ) Genome browser tracks of CUT&Tag signals for H3K18la at representative target gene loci in HUVECs. The tracks display normalized signal intensity (Reads Per Kilobase per Million mapped reads, RPKM) from a single biological replicate, aligned to the GRCh38/hg38 genome build. ( L ) KEGG pathway analysis of the 1147 genes overlapping between H3K18la-bound genes and genes downregulated by Narc. The Narc-downregulated gene set was defined from RNA-seq data (FDR < 0.05, |fold change| > 1.5; n = 3 biological replicates). The underlying RNA-seq data are available under GEO accession GSE202556 . ( M ) ChIP-qPCR analyses of H3K18la enrichment at the promoters of the indicated genes in HUVECs treated with ox-LDL (100 µg/mL, 24 h) in the presence or absence of Narc (20 nM). Data were normalized to input chromatin and are presented as fold enrichment relative to the IgG control ( n = 4 biological replicates). All data were represented as mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparisons test
Article Snippet: The human monocyte cell line THP-1 (ATCC ® TIB202TM) was cultured in RPMI 1640 medium (Gibco, CA) supplemented with 10% fetal bovine serum (Gibco), 0.05 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and 1% (v/v) penicillin/streptomycin (Solarbio Science & Technology Co., Ltd., Beijing, China) at 37 °C and 5% CO 2 .
Techniques: Activation Assay, Inhibition, Western Blot, Transfection, Negative Control, Labeling, Chemotaxis Assay, RNA Sequencing, ChIP-qPCR, Control
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