pkh26 red fluorescent cell linker kit  (Merck & Co)

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 inhibits endothelial inflammation in vitro . ( A - F ) HUVECs were pretreated with narciclasine (Narc, 20 nM, 6 h) and subsequently treated with ox-LDL (100 µg/mL, 24 h); control groups received PBS or vehicle (0.1% DMSO). ( A ) Protein levels of VCAM1 (110 kDa) and ICAM1 (90 kDa) were analyzed by Western blotting. Quantification of VCAM1 and ICAM1 (normalized to β-actin, 42 kDa) as determined by densitometry (ImageJ 1.48v) is shown in the lower panel ( n = 3 biological replicates). ( B ) The mRNA levels of VCAM1 , ICAM1 , SELE , and CCL2 were measured by qPCR using the 2 –ΔΔCt method with β-actin as the reference gene ( n = 3 biological replicates). ( C ) 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 = 4 biological replicates). ( D ) 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). ( E ) H₂O₂ production was measured using the Amplex Red assay. Data are presented as background-subtracted relative fluorescence units (RFU) normalized to the total cell count ( n = 4 biological replicates). The p -values on the graph correspond to the 120-min time point. ( F ) Representative images of MitoSOX Red (mtROS, red) and Hoechst (nuclei, blue) staining. Scale bar = 25 μm. The right panel shows the quantification of MitoSOX Red fluorescence intensity from 10 random fields per group, analyzed using ImageJ. Fluorescence intensity was normalized to the number of nuclei per field ( n = 3 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: HUVECs were pretreated with or without 20 nM narciclasine for 6 h and then exposed to 100 μg/mL ox-LDL for 24 h. THP-1 monocytes were fluorescently labeled with the PKH26 Red Fluorescent Cell Linker Kit (Solarbio Science & Technology Co., Ltd.) and co-cultured with HUVECs in 24-well plates for 1 h. This experiment was independently repeated four times ( n = 4 biological replicates).

Techniques: In Vitro, Control, Western Blot, Labeling, Chemotaxis Assay, Amplex Red Assay, Fluorescence, Cell Characterization, Staining

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: HUVECs were pretreated with or without 20 nM narciclasine for 6 h and then exposed to 100 μg/mL ox-LDL for 24 h. THP-1 monocytes were fluorescently labeled with the PKH26 Red Fluorescent Cell Linker Kit (Solarbio Science & Technology Co., Ltd.) and co-cultured with HUVECs in 24-well plates for 1 h. This experiment was independently repeated four times ( n = 4 biological replicates).

Techniques: Activation Assay, Inhibition, Western Blot, Transfection, Negative Control, Labeling, Chemotaxis Assay, RNA Sequencing, ChIP-qPCR, Control