quantikinetm elisa human il 6 immunoassay kit  (R&D Systems)

Journal: bioRxiv

Article Title: Gut Microbiota Production of Phenylacetate Programs Vascular Niche Senescence and Drives Atherosclerosis

doi: 10.64898/2026.02.27.708541

Figure Lengend Snippet: a, Young mice were pretreated with ABx for 2 weeks, and then colonized with Clos for 4 weeks ( n □=□6). b, qPCR shows transcriptional alterations of CDK inhibitors Cdkn2a , Cdkn2d , and Cdkn1a in tPVAT from Clos -mice ( n = 6) and vehicle-mice ( n = 6). c, Representative confocal immunofluorescence images of p16 INK4A in tPVAT from these mice ( n = 5). d, Relative protein expression analysis of SASP components in tPVAT from Clos -mice ( n = 5) and vehicle-mice ( n = 5). e, Young mice were administered to PAA (50 mg/kg, i.p. ) daily for 4 weeks ( n □=□6). f, Representative immunoblots and quantification of intensities for CDK inhibitors p16 INK4A , p19 INK4D , and p21 WAF1/Cip1 and DNA damage marker γ-H2A.X in tPVAT from these mice ( n = 6). g, Representative confocal images of p16 INK4A in tPVAT from these mice ( n = 6). h, Immunoblotting represents the expression of the SASP components IL-1β, IL-6, and CCL2 in tPVAT from PAA-or vehicle-treated mice ( n = 6). Data were determined in 8-10 micrographs and represent triplicated biologically independent experiments ( c,g ). Scale bars, 20 and 200 μm ( c,g ). Error bars represent SD ( b,d,f,h ). P values were calculated using a two-tailed unpaired Student’s t -test ( b,d,f,h ). Images created with https://BioRender.com ( a,e ). (* P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001).

Article Snippet: Cell culture supernatants were assessed for IL-6 using a QuantikineTM ELISA Human IL-6 Immunoassay kit (R&D Systems, D6050), according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Expressing, Western Blot, Marker, Two Tailed Test

Journal: bioRxiv

Article Title: Gut Microbiota Production of Phenylacetate Programs Vascular Niche Senescence and Drives Atherosclerosis

doi: 10.64898/2026.02.27.708541

Figure Lengend Snippet: a, IL-6 concentration in culture medium derived from replicative senescent or proliferating ECs treated with PAA (10 μM) or vehicle for 72 h ( n □=□10 biologically independent samples). b, Representative immunoblots demonstrate the expression of NOTCH1 and downstream targets N1ICD and HES1 in hADSC-adipocytes exposed to CM derived from vehicle- or PAA-treated ECs ( n = 6). c,d, Immunoblotting for NOTCH1 ( c ) and the insulin signaling pathway ( d ) in insulin-stimulated adipocytes treated with PAA-CM in the presence or absence of anti-IL6R neutralizing antibody, Tocilizumab (100 μg/mL) ( n = 6). e,f, Immunoblotting for the insulin signaling pathway ( e ) and thermogenic markers UCP1 and PGC1α ( f ) in adipocytes treated with PAA-CM in the presence or absence of Notch inhibitor, DAPT (10 µM) ( n = 6). g, qPCR represents transcriptional changes of thermogenic markers Ucp1 and Ppargc1a in PAA-CM-exposed adipocytes transfected with siHES1 or siNeg ( n = 6). h, Summary scheme outlining the mechanisms of microbial metabolite PAA for triggering adipocyte dysfunction. PAA indirectly activates NOTCH1 and its downstream HES1 in adipocytes by releasing senescence-messaging secretome containing IL6 from adjacent ECs. This both downregulates thermogenic function and the insulin signaling pathway in adipocytes. Error bars represent SD ( a-g ). P values were calculated using one-way ANOVA followed by Tukey’s post hoc test ( a ) and a two-tailed unpaired Student’s t -test ( b-g ). Image created with https://BioRender.com ( h ).

Article Snippet: Cell culture supernatants were assessed for IL-6 using a QuantikineTM ELISA Human IL-6 Immunoassay kit (R&D Systems, D6050), according to the manufacturer’s instructions.

Techniques: Concentration Assay, Derivative Assay, Western Blot, Expressing, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: Gut Microbiota Production of Phenylacetate Programs Vascular Niche Senescence and Drives Atherosclerosis

doi: 10.64898/2026.02.27.708541

Figure Lengend Snippet: a, Clos -colonized young mice received a senolytic cocktail containing Dasatinib + Quercetin (5 + 50 mg/kg/d) for 3 days, followed by a 7-day resting period ( n □=□6). b, Representative immunoblots for CDK inhibitors p16 INK4A , p19 INK4D , and p21 WAF1/Cip1 and DNA damage marker γ-H2A.X in tPVAT from Clos -colonized mice treated with D+Q or vehicle ( n = 6). c,d, Representative confocal immunofluorescence images of IL-6 ( c ) and bright-field SA-β-gal staining images ( d ) in tPVAT from these mice ( n = 6). e, Representative confocal images of NOTCH1 in tPVAT from these mice ( n = 6). f,g, Immunoblotting for the insulin signaling pathway ( f ) and thermogenic markers UCP1 and PGC1α ( g ) in tPVAT from Clos -colonized mice treated with D+Q or vehicle ( n = 6). Data were determined in 8-10 micrographs and represent triplicated biologically independent experiments. Scale bar, 50, 100, and 200 μm ( c,d,e ). Error bars represent SD ( b,d,f,g ). P values were calculated using a two-tailed unpaired Student’s t -test ( b,d,f,g ). Images created with https://BioRender.com ( a ).

Article Snippet: Cell culture supernatants were assessed for IL-6 using a QuantikineTM ELISA Human IL-6 Immunoassay kit (R&D Systems, D6050), according to the manufacturer’s instructions.

Techniques: Western Blot, Marker, Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Gut Microbiota Production of Phenylacetate Programs Vascular Niche Senescence and Drives Atherosclerosis

doi: 10.64898/2026.02.27.708541

Figure Lengend Snippet: a,b, Plasma samples from aged ASCVD patients (>80 years old) enrolled in the ASCVD cohort ( n = 110; male and female) and healthy controls ( n = 77; male and female) were subjected to targeted metabolomics for PAA quantification. c, Adjusted regression models for the association of PAA with atherosclerosis in the ASCVD cohort ( n □=□187). Effect estimates were controlled for age, sex, smoking, alcohol, family history of CVD, LDL-C, triglycerides, HDL-C, Hb1Ac, CRP, troponin T, and NT-proBNP. Error bars show 95% confidence intervals. OR, odds ratio. d, Ldlr −/− and WT mice were fed chow (for 8 weeks) or Western diet (for 12 weeks). WC: WT + chow diet; WW: WT + Western diet; LC: Ldlr −/− + chow diet; LW: Ldlr −/− + Western diet. e, Plasma PAA levels in these mice quantified by LC-MS/MS targeted metabolomics ( n □=□5). f, Representative images (left) and quantification (right) of H&E staining of aortic root lesions ( n □=□5). Arrowheads indicate plaque areas. g, Correlation of plasma PAA levels with aortic root lesion area and luminal occlusion (%). h, Representative immunoblots and quantification of intensities for the CDK inhibitor p16 INK4A , the SASP component IL-6, DNA damage marker γ-H2A.X, and the endothelial function marker phosphorylated eNOS S1177 in aortas from Ldlr −/− and WT mice fed a chow or Western diet for 12 weeks ( n = 5). i, Expression of CDKN1A , IL1B , and IL6 genes (upper) and co-expression of the senescence-associated genes in atherosclerotic aortic wall (AOR) of patients with CAD ( n □=□600) and healthy individuals ( n □=□250) in the STARNET database. j,k,l, Representative immunoblots and quantification of intensities for senescence hallmarks ( j ), insulin signaling pathway ( k ), and thermogenic markers ( l ) in tPVAT from Ldlr −/− and WT mice fed a chow or Western diet for 12 weeks ( n = 5). m, Plasma and aortic PVAT samples were collected from the validation study, including aged CAD patients undergoing CABG surgery ( n □=□5) and non-CAD controls ( n □=□5), for LC-MS/MS PAA quantification and senescence studies. n, Plasma PAA concentrations in aged CAD patients ( n □=□5) and non-CAD controls ( n □=□5). o,p,q, Representative immunoblots and quantification of intensities for senescence hallmarks ( o ), NOTCH1 and thermogenic markers ( p ), and insulin signaling pathway ( q ) in aortic PVAT from the individuals in the validation study ( n = 5). r, PAA was administered (PAA) or not (Ctrl) to chow-fed Ldlr −/− mice for 8 weeks. s, Quantification of H&E-stained aortic root lesion area (left) and aortic occlusion (right). Total cholesterol concentrations in plasma ( n = 5). t, Representative immunoblots and quantification of intensities for senescence hallmarks p16 INK4A , IL-6, and γ-H2A.X in aortas (left) and tPVAT (right). Data were determined in 8-10 micrographs and represent triplicated biologically independent experiments. Scale bar, 200 μm ( f ). Error bars represent SD ( h,j-l,o-t ). P values were calculated using two-tailed Mann–Whitney U -test ( b ), one-way ANOVA followed by Tukey’s post hoc test ( e,f,h,j-l ), Welch’s t-test ( i ), and a two-tailed unpaired Student’s t -test ( n-t ). Correlation coefficient and P values were calculated by Spearman’s rank-order correlation test ( g ). Data are shown as median with min–max; each violin represents interquartile range (IQR); center lines indicate the median; upper and lower lines are bounded by 25th and 75th percentiles ( b , n ). Images created with https://BioRender.com ( a,d,m,r ).

Article Snippet: Cell culture supernatants were assessed for IL-6 using a QuantikineTM ELISA Human IL-6 Immunoassay kit (R&D Systems, D6050), according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Western Blot, Liquid Chromatography with Mass Spectroscopy, Staining, Marker, Expressing, Biomarker Discovery, Two Tailed Test, MANN-WHITNEY