pkn1  (MedChemExpress)

Journal: Journal of Alzheimer's Disease Reports

Article Title: Identifying Alzheimer's disease genes in apolipoprotein E −/− mice brains with confirmed Porphyromonas gingivalis entry

doi: 10.1177/25424823251332874

Figure Lengend Snippet: Differential expression of genes induced by P. gingivalis FDC 381 (Pg) entry in the ApoE−/− mice versus the sham control groups in the cerebral region of the brain.

Article Snippet: Protein kinase N1 , Pkn1-Mm00723995_m1 , Kinase enzyme , No change.

Techniques: Quantitative Proteomics, Control, Binding Assay, Membrane, Activity Assay, Glycoproteomics, Protein Binding, Ubiquitin Proteomics

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , HUAECs were transfected with a control siRNA or siRNAs directed against different protein kinases and were exposed to disturbed flow (DF). Thereafter, flow-induced FOS and FOSB expression was determined by qRT-PCR. The plot shows the ranked average ratios of at least 3 independent experiments. b , HUAECs were transfected with control siRNA or different siRNAs against PKN1 and were exposed to disturbed flow (DF) for 3 hrs. FOS and FOSB levels were determined by qRT-PCR ( n = 3 independent experiments). c , HUAECs were transfected with control siRNA or siRNA against PKN1 and were kept under static conditions (-) or were exposed to disturbed flow (DF) for 3 hours. The heatmap shows normalized expression of upregulated and downregulated genes. d , HUAECs were transfected with control siRNA or an siRNA against JNK and were exposed to disturbed flow (DF) for 3 hours. Thereafter, inflammatory gene expression was determined by qRT-PCR ( n = 3 independent experiments). e-h , HUAECs were transfected with control siRNA or siRNA against PKN1 and were exposed to disturbed flow (DF) for 30 min or were kept under static conditions (e-g) or were incubated in the absence or presence of 10 ng/ml TNF for 30 min (h). Thereafter, phosphorylated p65 or PKN1 were determined by immunoblotting (e, h), or nuclear p65 localization was determined by staining (f) or immunoblotting (g) ( n = 4 independent experiments). Scale bar: 50 μm. Data are shown as mean ± s.e.m.; the p-values are given in the Fig. (2-way ANOVA with Bonferroni’s post hoc test (a), Kruskal-Wallis test (b, d-h)).

Article Snippet: The samples were then permeabilized and incubated with antibodies directed against CD31 (R&D Systems, cat. no. AF806), PKN1 (Santa Cruz Biotechnology, cat. no. sc-271594), VCAM1 (Abcam, cat. no. ab134047) and phospho-H3.3S31 (Thermo Fisher Scientific, cat. no. 44-244G) and co-stained with DAPI (Invitrogen, cat. no. D1306).

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Gene Expression, Incubation, Western Blot, Staining

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a – c , HUAECs were transfected with control siRNA or an siRNA against PKN1 and were exposed to disturbed flow (DF) for 3 h as described above. FOS and FOSB levels were determined by qRT–PCR ( a ) or immunoblotting ( b ), and AP-1 promoter activity ( c ) was determined by luciferase reporter assay after transfection of cells with AP-1 promoter luciferase reporter construct ( n = 3 independent experiments). d , HUAECs were transfected with control siRNA or siRNA against JNK or PKN1 and were exposed to DF for 30 min. Phosphorylated PKN1, JNK or c-JUN was determined by immunoblotting ( n = 5 independent experiments). e , HUAECs were transfected with control siRNA or an siRNA against JNK and were exposed to DF for 3 h. FOS and FOSB expression was determined by qRT–PCR ( n = 5 independent experiments). f , Volcano plot depicting results from TOBIAS. Red points depict motifs that are enriched and have a transcription factor (TF) footprint in control cells exposed to disturbed flow; blue points depict motifs that are enriched and have a TF footprint in cells exposed DF after knockdown of PKN1. g , h , HUAECs were transfected with control siRNA or siRNA against PKN1 and were exposed to DF for 3 h. Inflammatory gene expression was analyzed by qRT–PCR ( g , n = 4 independent experiments) or immunoblotting ( h , n = 3 independent experiments). i , The constitutively active mutant of PKN1 (PKN-CA) was expressed by lentiviral transduction in HUAECs, and expression of inflammatory genes was analyzed by qRT–PCR. Non-transducing lentivirus was used as a control ( n = 5 independent experiments). Data are shown as mean ± s.e.m.; the P values are given in the figure (Kruskal–Wallis test ( a – c , g , h ) and one-way ANOVA with Tukey’s post hoc test ( d , e , i )). NS, not significant.

Article Snippet: The samples were then permeabilized and incubated with antibodies directed against CD31 (R&D Systems, cat. no. AF806), PKN1 (Santa Cruz Biotechnology, cat. no. sc-271594), VCAM1 (Abcam, cat. no. ab134047) and phospho-H3.3S31 (Thermo Fisher Scientific, cat. no. 44-244G) and co-stained with DAPI (Invitrogen, cat. no. D1306).

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Activity Assay, Luciferase, Reporter Assay, Construct, Expressing, Knockdown, Gene Expression, Mutagenesis, Transduction

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , HUAECs were plated on fibronectin (FN), collagen IV (Col.IV) or poly- l -lysine (PLL) and were kept under static conditions (−) or were exposed to disturbed flow (DF) for 60 min. Total and phosphorylated PKN1 was determined by immunoblotting ( n = 3 independent experiments). b , c , HUAECs were exposed to DF for 30 min ( b ) or 3 h ( c ) or were kept under static conditions after pre-treatment for 30 min without or with the integrin α5β1 antagonist ATN-161 (10 μM). Total and phosphorylated PKN1 was determined by immunoblotting ( b ), and FOS / FOSB expression was analyzed by qRT–PCR ( c ) ( n = 3 independent experiments). d , HUAECs on FN or PLL were exposed to static conditions or DF for 1 h, after which total and phosphorylated PKN1 distribution in the nucleus and cytoplasm was analyzed by immunoblotting ( n = 3 independent experiments). e , HUAECs were exposed to DF for 1 h or kept under static conditions and then stained with antibodies against PKN1 (red) and p-PKN1 (green), along with DAPI (blue). The ratio of nuclear and cytoplasmic PKN1 or p-PKN1 was quantified ( n = 3 independent experiments). Scale bar, 20 μm. f , g , Cross-sections of the inner ( f , g ) and outer ( f ) curvatures of aortic arches from wild-type (WT) ( f , g ) or EC-Itga5-KO ( g ) mice were stained with antibodies against phosphorylated PKN1 (green) and CD31 (purple) as well as with DAPI (red). The bar diagrams show percentage of phosphorylated PKN1-positive area per endothelial DAPI-positive area. Scale bar, 20 μm ( n = 6 mice per group; at least three sections were analyzed per animal). h , Representative immunofluorescence confocal images of sections of the human aorta. Sections were stained with antibodies against phosphorylated PKN1 (purple), CD31 (green) and VCAM1 (red) as well as with DAPI (blue). Arrows indicate endothelial marker-positive cells. The Spearman correlation analysis showed a significant relationship ( r = 0.7747, P = 0.0028) ( n = 13 different patients; at least three sections per patient sample were analyzed). Scale bar, 20 µm. Data are shown as mean ± s.e.m.; the P values are given in the figure (Kruskal–Wallis test ( a – c ), Mann–Whitney two-sided test ( d , e ) or unpaired two-sided t -test ( f , g )). NS, not significant; Con., control; ATN., ATN-161.

Article Snippet: The samples were then permeabilized and incubated with antibodies directed against CD31 (R&D Systems, cat. no. AF806), PKN1 (Santa Cruz Biotechnology, cat. no. sc-271594), VCAM1 (Abcam, cat. no. ab134047) and phospho-H3.3S31 (Thermo Fisher Scientific, cat. no. 44-244G) and co-stained with DAPI (Invitrogen, cat. no. D1306).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Staining, Immunofluorescence, Marker, MANN-WHITNEY, Control

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , HUAECs were transfected with control siRNA or different siRNA against PKN1 and were exposed to disturbed flow (DF) for 30 min. Histone H3.3 phosphorylation, H3K27 acetylation or H3K36 trimethylation was determined by immunoblotting. b - d , HUAECs were transfected with control siRNA (Con.) or siRNA directed against histone H3.3, and cells were infected with control (non-transducing) lentivirus (Con.) or lentivirus transducing wild-type (WT) H3.3 or the indicated mutants of H3.3. Shown is the relative H3.3 mRNA expression (b), ( n = 5 independent experiments), the protein level determined by immunoblotting using an antibody directed against histone H3.3 (c) ( n = 3 independent experiments) and the analysis of phosphorylation of histone H3 at the indicated phosphorylation sites using phosphosite-specific antibodies after exposure of HUAECs to disturbed flow for 60 min (d) ( n = 3 independent experiments). e , Wild-type (WT) human H3.3 or the indicated mutants of H3.3 were expressed by lentiviral transduction after siRNA-mediated H3.3 knock-down in HUAECs, and cells were kept under static conditions or were exposed to disturbed flow (DF) for 3 h. Thereafter, ICAM1 and CCL2 expression was determined by qRT-PCR ( n = 5 independent experiments). f , HUAECs were exposed to disturbed flow (4 dynes/cm 2 ) for 24 hours, and phosphorylation of histone H3.3 at serine 31 was determined by immunoblotting ( n = 4 independent experiments). The control immunoblot for GAPDH is from the same experiment as the one shown in Extended Data Fig. . g , Recombinant histone H3.3 or H4 were incubated in kinase buffer without or with recombinant PKN1 and 50 µM [γ- 32 P]-ATP for 30 min at 30 °C. Thereafter, samples were separated by SDS-PAGE and analyzed by autoradiography and Ponceau staining. h , HUAECs were transfected with control siRNA or siRNA directed against RNAs encoding the kinases CHEK or IKKα ( CHEK1 or CHUK , respectively). Thereafter, cells were kept under static conditions (-) or were exposed to disturbed flow (DF) for 60 min. Phosphorylation level of H3.3S31 in lysates was determined by immunoblotting ( n = 3 independent experiments). The bar diagrams show the statistical evaluation as well as the knock-down efficiency. Data are shown as mean ± s.e.m.; the p-values are given in the Fig. (1-way ANOVA with Tukey’s post hoc test (b, e), Mann-Whitney two-sided test (f), Kruskal-Wallis test (h)).

Article Snippet: The samples were then permeabilized and incubated with antibodies directed against CD31 (R&D Systems, cat. no. AF806), PKN1 (Santa Cruz Biotechnology, cat. no. sc-271594), VCAM1 (Abcam, cat. no. ab134047) and phospho-H3.3S31 (Thermo Fisher Scientific, cat. no. 44-244G) and co-stained with DAPI (Invitrogen, cat. no. D1306).

Techniques: Transfection, Control, Phospho-proteomics, Western Blot, Infection, Expressing, Transduction, Knockdown, Quantitative RT-PCR, Recombinant, Incubation, SDS Page, Autoradiography, Staining, MANN-WHITNEY

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , HUAECs transfected with control siRNA or PKN1 siRNA were exposed to disturbed flow for specified time periods, and histone H3 phosphorylation was assessed by immunoblotting ( n = 3 independent experiments). The presented immunoblots are from the same experiment as blots shown in Fig. and include the same H3.3 loading control. b – f , Wild-type (WT) human H3.3 or mutants were expressed in HUAECs via lentiviral transduction after siRNA-mediated H3.3 knockdown; cells were subjected to static conditions or disturbed flow for 3 h; and mRNA levels of VCAM1 ( b ). SELE ( c ), FOS ( d ) and FOSB ( e ) were analyzed by qRT–PCR ( n = 5 independent experiments) or protein levels were determined by immunoblotting ( f , n = 3 independent experiments). g , Recombinant histone H3.3 or polynucleosomes were incubated with or without recombinant PKN1 and ATP and analyzed by immunoblotting for H3.3S31P, PKN1 and H3.3 ( n = 3 independent experiments). h , Representative immunofluorescence confocal images of human aorta stained for phosphorylated H3.3S31 (green), CD31 (red), VCAM1 (cyan) and DAPI (blue). Arrows indicate endothelial marker (CD31)-positive cells. Shown is the Spearman correlation analysis ( n = 13 different patients, at least three sections per patient). i , Representative en face immuno-confocal microscopy images of the aortic inner curvature from WT and EC-Pkn1-KO mice ( n = 4 per group) stained with anti-H3.3S31P or anti-CD31 antibodies and DAPI. Immunofluorescence was quantified as the percentage of H3.3S31P-positive cells among CD31-positive cells per view field ( n = 4 mice, at least three areas per mouse). j , k , Atheroprone Ldlr −/− mice were infected with AAV2-QuadYF virus to transduce WT H3.3 or the phosphosite mutant H3.3S31A. En face images of the aortic inner curvature were obtained after VCAM1, CD31 and DAPI staining ( j ). Immunofluorescence was quantified as the percentage of VCAM1-positive cells among CD31-positive cells per field ( j , n = 5 mice per group, at least three areas per mouse). Two weeks after carotid artery ligation, mice were euthanized, and light microscopy images of carotid arteries were analyzed for the percentage of neointima between the aorta and the ligation site ( k , n = 6 mice per group). Bar lengths, 20 µm ( h ), 50 µm ( i , j ) and 5 mm ( k ). Data are shown as mean ± s.e.m.; the P values are given in the figure (Kruskal–Wallis test ( a , d – f ), one-way ANOVA with Tukey’s post hoc test ( b , c ), Mann–Whitney two-sided test ( i , j ) or unpaired two-sided t- test ( k )). NS, not significant.

Article Snippet: The samples were then permeabilized and incubated with antibodies directed against CD31 (R&D Systems, cat. no. AF806), PKN1 (Santa Cruz Biotechnology, cat. no. sc-271594), VCAM1 (Abcam, cat. no. ab134047) and phospho-H3.3S31 (Thermo Fisher Scientific, cat. no. 44-244G) and co-stained with DAPI (Invitrogen, cat. no. D1306).

Techniques: Transfection, Control, Phospho-proteomics, Western Blot, Transduction, Knockdown, Quantitative RT-PCR, Recombinant, Incubation, Immunofluorescence, Staining, Marker, Confocal Microscopy, Infection, Virus, Mutagenesis, Ligation, Light Microscopy, MANN-WHITNEY

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , HUAECs were transfected with a control siRNA or siRNAs directed against PKN1 . Thereafter, cells were exposed to disturbed flow (DF) for the indicated time periods. H3K27 acetylation or H3K36 tri-methylation in lysates was determined by immunoblotting ( n = 3 independent experiments). The presented immunoblots are from the same experiment as the blots shown in Fig. and include the same H3.3 loading control. b , Wild-type (WT) or mutant (S31A) human H3.3 was expressed by lentiviral transduction after siRNA-mediated H3.3 knockdown in HUAECs. Thereafter, cells were exposed to DF for 3 h or were kept under static conditions. H3K27 acetylation or H3K36 tri-methylation in lysates was determined by immunoblotting ( n = 3 independent experiments). c – g , HUAECs were transfected with a control siRNA or siRNAs directed against PKN1 ( c , e ), SETD2 ( e ) or EP300 ( g ) and were then exposed to DF for 90 min. Alternatively, wild-type (WT) or mutant (S31A) human H3.3 was expressed by lentiviral transduction after siRNA-mediated knockdown of H3.3 ( d , f ) in HUAECs. ChIP assay was performed to detect the enrichment of H3K27 acetylation ( c , d , g ) or H3K36 tri-methylation ( e , f ) in the FOS / FOSB promoter ( c , d , g ) or gene body ( e , f ) ( n = 3 independent experiments). h , i , HUAECs were transfected with control siRNA or siRNAs directed against EP300 or SETD2 and were exposed to DF for 3 h. DF-induced expression of FOS and FOSB ( h ) or inflammatory genes ( i ) was analyzed by qRT–PCR ( n = 3 independent experiments). j , Schematic representation of the role of PKN1 in mediating DF-induced inflammatory gene expression in endothelial cells. Data are shown as mean ± s.e.m.; the P values are given in the figure (Kruskal–Wallis test ( a – i )). NS, not significant.

Article Snippet: The samples were then permeabilized and incubated with antibodies directed against CD31 (R&D Systems, cat. no. AF806), PKN1 (Santa Cruz Biotechnology, cat. no. sc-271594), VCAM1 (Abcam, cat. no. ab134047) and phospho-H3.3S31 (Thermo Fisher Scientific, cat. no. 44-244G) and co-stained with DAPI (Invitrogen, cat. no. D1306).

Techniques: Transfection, Control, Methylation, Western Blot, Mutagenesis, Transduction, Knockdown, Expressing, Quantitative RT-PCR, Gene Expression

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , Shown are representative en face immuno-confocal microscopy images of the inner and outer curvature of the aortic arch (left panels), or of the origin of an intercostal artery of the descending aorta (right panels) from wild-type mice stained with antibodies directed against H3.3S31P and CD31 as well as with DAPI. Immunofluorescence staining was quantified as the percentage of phosphorylated H3.3S31-positive cells among CD31-positive cells per view field ( n = 3 mice per group; at least 3 areas were analyzed per animal). b , Pkn1 expression in mouse lung endothelial cells (MLECs) from wild-type (WT) and EC-Pkn1-KO mice ( n = 3 mice per group). c , Map of vectors used to generate AAV2 particles transducing wild-type (WT) and mutant H3.3. Histone 3.3B(WT) or the H3.3(S31A) mutant are C-terminally fused with EGFP, and their expression is driven by the Tie1 promoter. The constructs were used to generate quadruplet mutant AAV2 viral particles referred to as AAV2-QuadYF. d , Relative histone H3.3 mRNA expression in mouse lung endothelial cells (MLECs) prepared from Ldlr -/- mice, which had been infected with empty, non-transducing AAV2-QuandYF virus (control) or AAV2-QuandYF-transducing wild-type H3.3 (WT) or the phospho-site mutant H3.3S31A ( n = 5 different mice). e , Images of carotid artery sections from AAV2-QuadYF-infected or non-infected mice stained for EGFP (green), CD31 (red) and with DAPI (blue) (shown is a representative of 3 independently performed experiments). f , g , Atheroprone Ldlr −/− mice were infected with AAV2-QuadYF virus transducing wild-type (WT) H3.3 or the phosphosite mutant H3.3(S31A). Cross sections of the left common carotid artery (ligated artery) were stained with antibodies against VCAM1 (f) or CD68 (g) and against CD31 (f) as well as with DAPI (blue). Immunofluorescence staining was quantified ( n = 5 mice per group; at least 3 areas per animal were analyzed). The boundary of the vessel area in (g) is indicated by a dashed line. Scale bars: 50 μm (a), 20 μm (e-g). Data are shown as mean ± s.e.m.; the p-values are given in the figure (Mann-Whitney two-sided test (a, b), unpaired two-sided t test (d, f, g)).

Article Snippet: The samples were then permeabilized and incubated with antibodies directed against CD31 (R&D Systems, cat. no. AF806), PKN1 (Santa Cruz Biotechnology, cat. no. sc-271594), VCAM1 (Abcam, cat. no. ab134047) and phospho-H3.3S31 (Thermo Fisher Scientific, cat. no. 44-244G) and co-stained with DAPI (Invitrogen, cat. no. D1306).

Techniques: Confocal Microscopy, Staining, Immunofluorescence, Expressing, Mutagenesis, Construct, Infection, Virus, Control, Phospho-proteomics, MANN-WHITNEY

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , b , Shown are representative en face immuno-confocal microscopy images of the inner curvature from 12-week-old atherosclerosis-prone Ldlr −/− mice without (Ldlr-KO) or with endothelium-specific Pkn1 deficiency (Ldlr-KO;EC-Pkn1-KO). En face aortic arch preparations were stained with anti-CD31, anti-VCAM1 ( a ) or anti-SELE antibodies ( b ) as well as with DAPI. Immunofluorescence staining was quantified as the percentage of VCAM1-positive or SELE-positive endothelial cells (ECs) among CD31-positive cells per view field ( n = 5 mice per condition; at least three areas were analyzed per animal). c – i , Atherosclerosis-prone Ldlr −/− mice without (Ldlr-KO) or with endothelium-specific PKN1 deficiency (Ldlr-KO;EC-Pkn1-KO) underwent partial carotid artery ligation. Seven days after ligation, cross-sections of the left common carotid artery (ligated artery) were stained with antibodies against H3.3S31P ( c ), H3K27Ac ( d ) or H3K36me3 ( e ) and against CD31 as well as with DAPI. f , The staining for H3.3S31P, H3K27Ac or H3K36me3 was quantified by determining the percentage of positively stained cells per field of view ( n = 5 mice per condition; at least three sections were analyzed per animal). g , Relative expression of mRNAs encoding FOS and FOSB in ECs of carotid arteries 7 days after ligation analyzed by qRT–PCR ( n = 5 mice per group). h , i , Twenty-eight days after ligation, light microscopical images of carotid arteries ( h ) were taken, or en face immunofluorescence staining for the expression of VCAM1 in ECs was performed ( i ). Bar diagrams show the statistical evaluation ( n = 8 ( h ) and n = 5 ( i ) mice per group). j – l , Ldlr −/− mice without (Ldlr-KO) or with induced endothelium-specific Pkn1 deficiency (Ldlr-KO;EC-Pkn1-KO) were fed an HFD for 16 weeks. Thereafter, aortas and adjacent vessels were analyzed. Shown are representative images of atherosclerotic plaques observed in brachiocephalic arteries (innominate arteries) ( j ) of Oil Red O-stained atherosclerotic lesions in the aortic valve region ( k ) and of whole aortas prepared en face and stained with Oil Red O ( l ) ( n = 9 mice per group). Bar lengths, 50 µm ( a , b ), 20 µm ( c – e ), 5 mm ( h , l ), 100 µm ( i , j ) and 250 µm ( k ). Data are shown as mean ± s.e.m.; the P values are given in the figure (unpaired two-sided t -test ( a , b , f , h – l ) and Kruskal–Wallis test ( g )). NS, not significant.

Article Snippet: The samples were then permeabilized and incubated with antibodies directed against CD31 (R&D Systems, cat. no. AF806), PKN1 (Santa Cruz Biotechnology, cat. no. sc-271594), VCAM1 (Abcam, cat. no. ab134047) and phospho-H3.3S31 (Thermo Fisher Scientific, cat. no. 44-244G) and co-stained with DAPI (Invitrogen, cat. no. D1306).

Techniques: Confocal Microscopy, Staining, Immunofluorescence, Ligation, Expressing, Quantitative RT-PCR

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , b , Shown are representative en face immuno-confocal microscopy images of the outer curvature from 12-week-old atherosclerosis-prone Ldlr -/- mice without (Ldlr-KO) or with endothelium-specific Pkn1 deficiency (Ldlr-KO;EC-Pkn1-KO). En face aortic arch preparations were stained with antibodies against CD31, VCAM1 (a) or SELE (b) as well as with DAPI (a, b). Immunofluorescence staining was quantified as the percentage of VCAM1- or SELE-positive cells among CD31-positive cells per view field ( n = 5 mice per group; at least 3 areas were analyzed per animal). c , d , 1 day after partial carotid ligation, immunofluorescence staining for the phosphorylation of histone H3.3 at S31 or for expression of VCAM1 in endothelial cells was performed using specific antibodies. Shown are representative sections of the ligated and unligated (control) carotid artery (c) and the statistical analysis ( n = 5; d). Bar length: 50 µm. Data are shown as mean ± s.e.m.; the p-values are given in the figure (unpaired two-sided t test (a,b,d)).

Article Snippet: The samples were then permeabilized and incubated with antibodies directed against CD31 (R&D Systems, cat. no. AF806), PKN1 (Santa Cruz Biotechnology, cat. no. sc-271594), VCAM1 (Abcam, cat. no. ab134047) and phospho-H3.3S31 (Thermo Fisher Scientific, cat. no. 44-244G) and co-stained with DAPI (Invitrogen, cat. no. D1306).

Techniques: Confocal Microscopy, Staining, Immunofluorescence, Ligation, Phospho-proteomics, Expressing, Control

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , b , THP-1 cells were transfected with a control siRNA or siRNAs directed against PKN1 . Phosphorylated H3.3S31, PKN1 and GAPDH in lysates were determined by immunoblotting ( a ). Inflammatory gene expression in response to LPS (1 µg ml −1 , 1 h) was analyzed by qRT–PCR ( b ) ( n = 3 independent experiments). c , d , BMDMs were isolated from wild-type (WT) or LysM-Cre; Pkn1 flox/flox (KO) mice and were stimulated for 1 h with 1 µg ml −1 LPS. Phosphorylated H3.3S31, PKN1 and GAPDH in lysates were determined by immunoblotting ( c ). Inflammatory gene expression was analyzed by qRT–PCR ( d ) ( n = 3 mice per group). Data are shown as mean ± s.e.m.; the P values are given in the figure (Kruskal–Wallis test ( a – d )). NS, not significant.

Article Snippet: The samples were then permeabilized and incubated with antibodies directed against CD31 (R&D Systems, cat. no. AF806), PKN1 (Santa Cruz Biotechnology, cat. no. sc-271594), VCAM1 (Abcam, cat. no. ab134047) and phospho-H3.3S31 (Thermo Fisher Scientific, cat. no. 44-244G) and co-stained with DAPI (Invitrogen, cat. no. D1306).

Techniques: Transfection, Control, Western Blot, Gene Expression, Quantitative RT-PCR, Isolation

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , HUAECs were transfected with a control siRNA or siRNAs directed against different protein kinases and were exposed to disturbed flow (DF). Thereafter, flow-induced FOS and FOSB expression was determined by qRT-PCR. The plot shows the ranked average ratios of at least 3 independent experiments. b , HUAECs were transfected with control siRNA or different siRNAs against PKN1 and were exposed to disturbed flow (DF) for 3 hrs. FOS and FOSB levels were determined by qRT-PCR ( n = 3 independent experiments). c , HUAECs were transfected with control siRNA or siRNA against PKN1 and were kept under static conditions (-) or were exposed to disturbed flow (DF) for 3 hours. The heatmap shows normalized expression of upregulated and downregulated genes. d , HUAECs were transfected with control siRNA or an siRNA against JNK and were exposed to disturbed flow (DF) for 3 hours. Thereafter, inflammatory gene expression was determined by qRT-PCR ( n = 3 independent experiments). e-h , HUAECs were transfected with control siRNA or siRNA against PKN1 and were exposed to disturbed flow (DF) for 30 min or were kept under static conditions (e-g) or were incubated in the absence or presence of 10 ng/ml TNF for 30 min (h). Thereafter, phosphorylated p65 or PKN1 were determined by immunoblotting (e, h), or nuclear p65 localization was determined by staining (f) or immunoblotting (g) ( n = 4 independent experiments). Scale bar: 50 μm. Data are shown as mean ± s.e.m.; the p-values are given in the Fig. (2-way ANOVA with Bonferroni’s post hoc test (a), Kruskal-Wallis test (b, d-h)).

Article Snippet: The anti-PKN1 antibody (cat. no. 610687, 1:1,000) was obtained from BD Biosciences, and an antibody against phosphorylated PKN1 (cat. no. AB-PK781, 1:1,000) was obtained from Kinexus Bioinformatics Corporation.

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Gene Expression, Incubation, Western Blot, Staining

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a – c , HUAECs were transfected with control siRNA or an siRNA against PKN1 and were exposed to disturbed flow (DF) for 3 h as described above. FOS and FOSB levels were determined by qRT–PCR ( a ) or immunoblotting ( b ), and AP-1 promoter activity ( c ) was determined by luciferase reporter assay after transfection of cells with AP-1 promoter luciferase reporter construct ( n = 3 independent experiments). d , HUAECs were transfected with control siRNA or siRNA against JNK or PKN1 and were exposed to DF for 30 min. Phosphorylated PKN1, JNK or c-JUN was determined by immunoblotting ( n = 5 independent experiments). e , HUAECs were transfected with control siRNA or an siRNA against JNK and were exposed to DF for 3 h. FOS and FOSB expression was determined by qRT–PCR ( n = 5 independent experiments). f , Volcano plot depicting results from TOBIAS. Red points depict motifs that are enriched and have a transcription factor (TF) footprint in control cells exposed to disturbed flow; blue points depict motifs that are enriched and have a TF footprint in cells exposed DF after knockdown of PKN1. g , h , HUAECs were transfected with control siRNA or siRNA against PKN1 and were exposed to DF for 3 h. Inflammatory gene expression was analyzed by qRT–PCR ( g , n = 4 independent experiments) or immunoblotting ( h , n = 3 independent experiments). i , The constitutively active mutant of PKN1 (PKN-CA) was expressed by lentiviral transduction in HUAECs, and expression of inflammatory genes was analyzed by qRT–PCR. Non-transducing lentivirus was used as a control ( n = 5 independent experiments). Data are shown as mean ± s.e.m.; the P values are given in the figure (Kruskal–Wallis test ( a – c , g , h ) and one-way ANOVA with Tukey’s post hoc test ( d , e , i )). NS, not significant.

Article Snippet: The anti-PKN1 antibody (cat. no. 610687, 1:1,000) was obtained from BD Biosciences, and an antibody against phosphorylated PKN1 (cat. no. AB-PK781, 1:1,000) was obtained from Kinexus Bioinformatics Corporation.

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Activity Assay, Luciferase, Reporter Assay, Construct, Expressing, Knockdown, Gene Expression, Mutagenesis, Transduction

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , HUAECs were plated on fibronectin (FN), collagen IV (Col.IV) or poly- l -lysine (PLL) and were kept under static conditions (−) or were exposed to disturbed flow (DF) for 60 min. Total and phosphorylated PKN1 was determined by immunoblotting ( n = 3 independent experiments). b , c , HUAECs were exposed to DF for 30 min ( b ) or 3 h ( c ) or were kept under static conditions after pre-treatment for 30 min without or with the integrin α5β1 antagonist ATN-161 (10 μM). Total and phosphorylated PKN1 was determined by immunoblotting ( b ), and FOS / FOSB expression was analyzed by qRT–PCR ( c ) ( n = 3 independent experiments). d , HUAECs on FN or PLL were exposed to static conditions or DF for 1 h, after which total and phosphorylated PKN1 distribution in the nucleus and cytoplasm was analyzed by immunoblotting ( n = 3 independent experiments). e , HUAECs were exposed to DF for 1 h or kept under static conditions and then stained with antibodies against PKN1 (red) and p-PKN1 (green), along with DAPI (blue). The ratio of nuclear and cytoplasmic PKN1 or p-PKN1 was quantified ( n = 3 independent experiments). Scale bar, 20 μm. f , g , Cross-sections of the inner ( f , g ) and outer ( f ) curvatures of aortic arches from wild-type (WT) ( f , g ) or EC-Itga5-KO ( g ) mice were stained with antibodies against phosphorylated PKN1 (green) and CD31 (purple) as well as with DAPI (red). The bar diagrams show percentage of phosphorylated PKN1-positive area per endothelial DAPI-positive area. Scale bar, 20 μm ( n = 6 mice per group; at least three sections were analyzed per animal). h , Representative immunofluorescence confocal images of sections of the human aorta. Sections were stained with antibodies against phosphorylated PKN1 (purple), CD31 (green) and VCAM1 (red) as well as with DAPI (blue). Arrows indicate endothelial marker-positive cells. The Spearman correlation analysis showed a significant relationship ( r = 0.7747, P = 0.0028) ( n = 13 different patients; at least three sections per patient sample were analyzed). Scale bar, 20 µm. Data are shown as mean ± s.e.m.; the P values are given in the figure (Kruskal–Wallis test ( a – c ), Mann–Whitney two-sided test ( d , e ) or unpaired two-sided t -test ( f , g )). NS, not significant; Con., control; ATN., ATN-161.

Article Snippet: The anti-PKN1 antibody (cat. no. 610687, 1:1,000) was obtained from BD Biosciences, and an antibody against phosphorylated PKN1 (cat. no. AB-PK781, 1:1,000) was obtained from Kinexus Bioinformatics Corporation.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Staining, Immunofluorescence, Marker, MANN-WHITNEY, Control

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , HUAECs were transfected with control siRNA or different siRNA against PKN1 and were exposed to disturbed flow (DF) for 30 min. Histone H3.3 phosphorylation, H3K27 acetylation or H3K36 trimethylation was determined by immunoblotting. b - d , HUAECs were transfected with control siRNA (Con.) or siRNA directed against histone H3.3, and cells were infected with control (non-transducing) lentivirus (Con.) or lentivirus transducing wild-type (WT) H3.3 or the indicated mutants of H3.3. Shown is the relative H3.3 mRNA expression (b), ( n = 5 independent experiments), the protein level determined by immunoblotting using an antibody directed against histone H3.3 (c) ( n = 3 independent experiments) and the analysis of phosphorylation of histone H3 at the indicated phosphorylation sites using phosphosite-specific antibodies after exposure of HUAECs to disturbed flow for 60 min (d) ( n = 3 independent experiments). e , Wild-type (WT) human H3.3 or the indicated mutants of H3.3 were expressed by lentiviral transduction after siRNA-mediated H3.3 knock-down in HUAECs, and cells were kept under static conditions or were exposed to disturbed flow (DF) for 3 h. Thereafter, ICAM1 and CCL2 expression was determined by qRT-PCR ( n = 5 independent experiments). f , HUAECs were exposed to disturbed flow (4 dynes/cm 2 ) for 24 hours, and phosphorylation of histone H3.3 at serine 31 was determined by immunoblotting ( n = 4 independent experiments). The control immunoblot for GAPDH is from the same experiment as the one shown in Extended Data Fig. . g , Recombinant histone H3.3 or H4 were incubated in kinase buffer without or with recombinant PKN1 and 50 µM [γ- 32 P]-ATP for 30 min at 30 °C. Thereafter, samples were separated by SDS-PAGE and analyzed by autoradiography and Ponceau staining. h , HUAECs were transfected with control siRNA or siRNA directed against RNAs encoding the kinases CHEK or IKKα ( CHEK1 or CHUK , respectively). Thereafter, cells were kept under static conditions (-) or were exposed to disturbed flow (DF) for 60 min. Phosphorylation level of H3.3S31 in lysates was determined by immunoblotting ( n = 3 independent experiments). The bar diagrams show the statistical evaluation as well as the knock-down efficiency. Data are shown as mean ± s.e.m.; the p-values are given in the Fig. (1-way ANOVA with Tukey’s post hoc test (b, e), Mann-Whitney two-sided test (f), Kruskal-Wallis test (h)).

Article Snippet: The anti-PKN1 antibody (cat. no. 610687, 1:1,000) was obtained from BD Biosciences, and an antibody against phosphorylated PKN1 (cat. no. AB-PK781, 1:1,000) was obtained from Kinexus Bioinformatics Corporation.

Techniques: Transfection, Control, Phospho-proteomics, Western Blot, Infection, Expressing, Transduction, Knockdown, Quantitative RT-PCR, Recombinant, Incubation, SDS Page, Autoradiography, Staining, MANN-WHITNEY

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , HUAECs transfected with control siRNA or PKN1 siRNA were exposed to disturbed flow for specified time periods, and histone H3 phosphorylation was assessed by immunoblotting ( n = 3 independent experiments). The presented immunoblots are from the same experiment as blots shown in Fig. and include the same H3.3 loading control. b – f , Wild-type (WT) human H3.3 or mutants were expressed in HUAECs via lentiviral transduction after siRNA-mediated H3.3 knockdown; cells were subjected to static conditions or disturbed flow for 3 h; and mRNA levels of VCAM1 ( b ). SELE ( c ), FOS ( d ) and FOSB ( e ) were analyzed by qRT–PCR ( n = 5 independent experiments) or protein levels were determined by immunoblotting ( f , n = 3 independent experiments). g , Recombinant histone H3.3 or polynucleosomes were incubated with or without recombinant PKN1 and ATP and analyzed by immunoblotting for H3.3S31P, PKN1 and H3.3 ( n = 3 independent experiments). h , Representative immunofluorescence confocal images of human aorta stained for phosphorylated H3.3S31 (green), CD31 (red), VCAM1 (cyan) and DAPI (blue). Arrows indicate endothelial marker (CD31)-positive cells. Shown is the Spearman correlation analysis ( n = 13 different patients, at least three sections per patient). i , Representative en face immuno-confocal microscopy images of the aortic inner curvature from WT and EC-Pkn1-KO mice ( n = 4 per group) stained with anti-H3.3S31P or anti-CD31 antibodies and DAPI. Immunofluorescence was quantified as the percentage of H3.3S31P-positive cells among CD31-positive cells per view field ( n = 4 mice, at least three areas per mouse). j , k , Atheroprone Ldlr −/− mice were infected with AAV2-QuadYF virus to transduce WT H3.3 or the phosphosite mutant H3.3S31A. En face images of the aortic inner curvature were obtained after VCAM1, CD31 and DAPI staining ( j ). Immunofluorescence was quantified as the percentage of VCAM1-positive cells among CD31-positive cells per field ( j , n = 5 mice per group, at least three areas per mouse). Two weeks after carotid artery ligation, mice were euthanized, and light microscopy images of carotid arteries were analyzed for the percentage of neointima between the aorta and the ligation site ( k , n = 6 mice per group). Bar lengths, 20 µm ( h ), 50 µm ( i , j ) and 5 mm ( k ). Data are shown as mean ± s.e.m.; the P values are given in the figure (Kruskal–Wallis test ( a , d – f ), one-way ANOVA with Tukey’s post hoc test ( b , c ), Mann–Whitney two-sided test ( i , j ) or unpaired two-sided t- test ( k )). NS, not significant.

Article Snippet: The anti-PKN1 antibody (cat. no. 610687, 1:1,000) was obtained from BD Biosciences, and an antibody against phosphorylated PKN1 (cat. no. AB-PK781, 1:1,000) was obtained from Kinexus Bioinformatics Corporation.

Techniques: Transfection, Control, Phospho-proteomics, Western Blot, Transduction, Knockdown, Quantitative RT-PCR, Recombinant, Incubation, Immunofluorescence, Staining, Marker, Confocal Microscopy, Infection, Virus, Mutagenesis, Ligation, Light Microscopy, MANN-WHITNEY

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , HUAECs were transfected with a control siRNA or siRNAs directed against PKN1 . Thereafter, cells were exposed to disturbed flow (DF) for the indicated time periods. H3K27 acetylation or H3K36 tri-methylation in lysates was determined by immunoblotting ( n = 3 independent experiments). The presented immunoblots are from the same experiment as the blots shown in Fig. and include the same H3.3 loading control. b , Wild-type (WT) or mutant (S31A) human H3.3 was expressed by lentiviral transduction after siRNA-mediated H3.3 knockdown in HUAECs. Thereafter, cells were exposed to DF for 3 h or were kept under static conditions. H3K27 acetylation or H3K36 tri-methylation in lysates was determined by immunoblotting ( n = 3 independent experiments). c – g , HUAECs were transfected with a control siRNA or siRNAs directed against PKN1 ( c , e ), SETD2 ( e ) or EP300 ( g ) and were then exposed to DF for 90 min. Alternatively, wild-type (WT) or mutant (S31A) human H3.3 was expressed by lentiviral transduction after siRNA-mediated knockdown of H3.3 ( d , f ) in HUAECs. ChIP assay was performed to detect the enrichment of H3K27 acetylation ( c , d , g ) or H3K36 tri-methylation ( e , f ) in the FOS / FOSB promoter ( c , d , g ) or gene body ( e , f ) ( n = 3 independent experiments). h , i , HUAECs were transfected with control siRNA or siRNAs directed against EP300 or SETD2 and were exposed to DF for 3 h. DF-induced expression of FOS and FOSB ( h ) or inflammatory genes ( i ) was analyzed by qRT–PCR ( n = 3 independent experiments). j , Schematic representation of the role of PKN1 in mediating DF-induced inflammatory gene expression in endothelial cells. Data are shown as mean ± s.e.m.; the P values are given in the figure (Kruskal–Wallis test ( a – i )). NS, not significant.

Article Snippet: The anti-PKN1 antibody (cat. no. 610687, 1:1,000) was obtained from BD Biosciences, and an antibody against phosphorylated PKN1 (cat. no. AB-PK781, 1:1,000) was obtained from Kinexus Bioinformatics Corporation.

Techniques: Transfection, Control, Methylation, Western Blot, Mutagenesis, Transduction, Knockdown, Expressing, Quantitative RT-PCR, Gene Expression

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , Shown are representative en face immuno-confocal microscopy images of the inner and outer curvature of the aortic arch (left panels), or of the origin of an intercostal artery of the descending aorta (right panels) from wild-type mice stained with antibodies directed against H3.3S31P and CD31 as well as with DAPI. Immunofluorescence staining was quantified as the percentage of phosphorylated H3.3S31-positive cells among CD31-positive cells per view field ( n = 3 mice per group; at least 3 areas were analyzed per animal). b , Pkn1 expression in mouse lung endothelial cells (MLECs) from wild-type (WT) and EC-Pkn1-KO mice ( n = 3 mice per group). c , Map of vectors used to generate AAV2 particles transducing wild-type (WT) and mutant H3.3. Histone 3.3B(WT) or the H3.3(S31A) mutant are C-terminally fused with EGFP, and their expression is driven by the Tie1 promoter. The constructs were used to generate quadruplet mutant AAV2 viral particles referred to as AAV2-QuadYF. d , Relative histone H3.3 mRNA expression in mouse lung endothelial cells (MLECs) prepared from Ldlr -/- mice, which had been infected with empty, non-transducing AAV2-QuandYF virus (control) or AAV2-QuandYF-transducing wild-type H3.3 (WT) or the phospho-site mutant H3.3S31A ( n = 5 different mice). e , Images of carotid artery sections from AAV2-QuadYF-infected or non-infected mice stained for EGFP (green), CD31 (red) and with DAPI (blue) (shown is a representative of 3 independently performed experiments). f , g , Atheroprone Ldlr −/− mice were infected with AAV2-QuadYF virus transducing wild-type (WT) H3.3 or the phosphosite mutant H3.3(S31A). Cross sections of the left common carotid artery (ligated artery) were stained with antibodies against VCAM1 (f) or CD68 (g) and against CD31 (f) as well as with DAPI (blue). Immunofluorescence staining was quantified ( n = 5 mice per group; at least 3 areas per animal were analyzed). The boundary of the vessel area in (g) is indicated by a dashed line. Scale bars: 50 μm (a), 20 μm (e-g). Data are shown as mean ± s.e.m.; the p-values are given in the figure (Mann-Whitney two-sided test (a, b), unpaired two-sided t test (d, f, g)).

Article Snippet: The anti-PKN1 antibody (cat. no. 610687, 1:1,000) was obtained from BD Biosciences, and an antibody against phosphorylated PKN1 (cat. no. AB-PK781, 1:1,000) was obtained from Kinexus Bioinformatics Corporation.

Techniques: Confocal Microscopy, Staining, Immunofluorescence, Expressing, Mutagenesis, Construct, Infection, Virus, Control, Phospho-proteomics, MANN-WHITNEY

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , b , Shown are representative en face immuno-confocal microscopy images of the inner curvature from 12-week-old atherosclerosis-prone Ldlr −/− mice without (Ldlr-KO) or with endothelium-specific Pkn1 deficiency (Ldlr-KO;EC-Pkn1-KO). En face aortic arch preparations were stained with anti-CD31, anti-VCAM1 ( a ) or anti-SELE antibodies ( b ) as well as with DAPI. Immunofluorescence staining was quantified as the percentage of VCAM1-positive or SELE-positive endothelial cells (ECs) among CD31-positive cells per view field ( n = 5 mice per condition; at least three areas were analyzed per animal). c – i , Atherosclerosis-prone Ldlr −/− mice without (Ldlr-KO) or with endothelium-specific PKN1 deficiency (Ldlr-KO;EC-Pkn1-KO) underwent partial carotid artery ligation. Seven days after ligation, cross-sections of the left common carotid artery (ligated artery) were stained with antibodies against H3.3S31P ( c ), H3K27Ac ( d ) or H3K36me3 ( e ) and against CD31 as well as with DAPI. f , The staining for H3.3S31P, H3K27Ac or H3K36me3 was quantified by determining the percentage of positively stained cells per field of view ( n = 5 mice per condition; at least three sections were analyzed per animal). g , Relative expression of mRNAs encoding FOS and FOSB in ECs of carotid arteries 7 days after ligation analyzed by qRT–PCR ( n = 5 mice per group). h , i , Twenty-eight days after ligation, light microscopical images of carotid arteries ( h ) were taken, or en face immunofluorescence staining for the expression of VCAM1 in ECs was performed ( i ). Bar diagrams show the statistical evaluation ( n = 8 ( h ) and n = 5 ( i ) mice per group). j – l , Ldlr −/− mice without (Ldlr-KO) or with induced endothelium-specific Pkn1 deficiency (Ldlr-KO;EC-Pkn1-KO) were fed an HFD for 16 weeks. Thereafter, aortas and adjacent vessels were analyzed. Shown are representative images of atherosclerotic plaques observed in brachiocephalic arteries (innominate arteries) ( j ) of Oil Red O-stained atherosclerotic lesions in the aortic valve region ( k ) and of whole aortas prepared en face and stained with Oil Red O ( l ) ( n = 9 mice per group). Bar lengths, 50 µm ( a , b ), 20 µm ( c – e ), 5 mm ( h , l ), 100 µm ( i , j ) and 250 µm ( k ). Data are shown as mean ± s.e.m.; the P values are given in the figure (unpaired two-sided t -test ( a , b , f , h – l ) and Kruskal–Wallis test ( g )). NS, not significant.

Article Snippet: The anti-PKN1 antibody (cat. no. 610687, 1:1,000) was obtained from BD Biosciences, and an antibody against phosphorylated PKN1 (cat. no. AB-PK781, 1:1,000) was obtained from Kinexus Bioinformatics Corporation.

Techniques: Confocal Microscopy, Staining, Immunofluorescence, Ligation, Expressing, Quantitative RT-PCR

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , b , Shown are representative en face immuno-confocal microscopy images of the outer curvature from 12-week-old atherosclerosis-prone Ldlr -/- mice without (Ldlr-KO) or with endothelium-specific Pkn1 deficiency (Ldlr-KO;EC-Pkn1-KO). En face aortic arch preparations were stained with antibodies against CD31, VCAM1 (a) or SELE (b) as well as with DAPI (a, b). Immunofluorescence staining was quantified as the percentage of VCAM1- or SELE-positive cells among CD31-positive cells per view field ( n = 5 mice per group; at least 3 areas were analyzed per animal). c , d , 1 day after partial carotid ligation, immunofluorescence staining for the phosphorylation of histone H3.3 at S31 or for expression of VCAM1 in endothelial cells was performed using specific antibodies. Shown are representative sections of the ligated and unligated (control) carotid artery (c) and the statistical analysis ( n = 5; d). Bar length: 50 µm. Data are shown as mean ± s.e.m.; the p-values are given in the figure (unpaired two-sided t test (a,b,d)).

Article Snippet: The anti-PKN1 antibody (cat. no. 610687, 1:1,000) was obtained from BD Biosciences, and an antibody against phosphorylated PKN1 (cat. no. AB-PK781, 1:1,000) was obtained from Kinexus Bioinformatics Corporation.

Techniques: Confocal Microscopy, Staining, Immunofluorescence, Ligation, Phospho-proteomics, Expressing, Control

Journal: Nature Cardiovascular Research

Article Title: Phosphorylation of endothelial histone H3.3 serine 31 by PKN1 links flow-induced signaling to proatherogenic gene expression

doi: 10.1038/s44161-024-00593-y

Figure Lengend Snippet: a , b , THP-1 cells were transfected with a control siRNA or siRNAs directed against PKN1 . Phosphorylated H3.3S31, PKN1 and GAPDH in lysates were determined by immunoblotting ( a ). Inflammatory gene expression in response to LPS (1 µg ml −1 , 1 h) was analyzed by qRT–PCR ( b ) ( n = 3 independent experiments). c , d , BMDMs were isolated from wild-type (WT) or LysM-Cre; Pkn1 flox/flox (KO) mice and were stimulated for 1 h with 1 µg ml −1 LPS. Phosphorylated H3.3S31, PKN1 and GAPDH in lysates were determined by immunoblotting ( c ). Inflammatory gene expression was analyzed by qRT–PCR ( d ) ( n = 3 mice per group). Data are shown as mean ± s.e.m.; the P values are given in the figure (Kruskal–Wallis test ( a – d )). NS, not significant.

Article Snippet: The anti-PKN1 antibody (cat. no. 610687, 1:1,000) was obtained from BD Biosciences, and an antibody against phosphorylated PKN1 (cat. no. AB-PK781, 1:1,000) was obtained from Kinexus Bioinformatics Corporation.

Techniques: Transfection, Control, Western Blot, Gene Expression, Quantitative RT-PCR, Isolation