Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Prematurity reduces citrulline-arginine-nitric oxide production and precedes the onset of necrotizing enterocolitis in piglets

doi: 10.1152/ajpgi.00198.2018

Figure Lengend Snippet: Multiple end-point measurements of necrotizing enterocolitis (NEC) incidence and severity in piglets. A: Kaplan-Meir graph detailing piglet survival. B: gross NEC score in piglets of advancing gestational age exposed to the NEC protocol. A threshold of 3 in any one segment was considered positive for NEC (dashed line). C: histological NEC score was determined in hematoxylin-eosin-stained tissue sections, and a threshold of ≥2 in any tissue section was considered positive for NEC (dashed line). D: plasma intestinal fatty-acid binding protein (iFABP) concentration in the final plasma sample for piglet groups in response to the NEC protocol. B–D: *P < 0.05, significant differences between groups, and error bars show 95% confidence interval. Data analyzed by Kruskal-Wallis test and Dunn’s multiple comparison test. NS, not significant.

Article Snippet: Amino acid concentrations in plasma and feed were measured using phenyl isothiocyanate derivatives relative to a methionine sulfone internal standard by HPLC ( 6 ). iFABP was measured using a human iFABP Quantikine ELISA Kit (R&D Systems).

Techniques: Staining, Clinical Proteomics, Binding Assay, Concentration Assay, Comparison

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Generation of humanized Zt/g4 antibody and characterization of RON-targeted antibody-drug conjugates: ( a ) Modeling of CDRs from mouse Zt/g4 in the variable regions of human IgG heavy chain and light chain. The framework of human IgG1 molecule was used for Zt/g4 humanization. The models of Zt/g4 CDRs grafted in the variable regions of human IgG1 heavy chain and light chain were generated by using the software PIGS from Automatic Predictions of Immunoglobulin Structures (Tramontano at University of Rome, Italy). ( b ) Binding of subclone H-Zt/g4 molecules to human RON. Different amounts of individual H-Zt/g4 s were incubated with NIH-3 T3 cells expressing human RON followed by addition of goat anti-human IgG1 antibody coupled with FITC. ( c ) Kinetic characterization of H-Zt/g4 interaction with human RON proteins by Octet RED96 system. Pure RON proteins from lysates of NIH3T3 cells expressing RON were immobilized onto the amine reactive sensor and assayed against individual H-Zt/g4 molecules in duplicate. The data set is analyzed with global fitting to produce the antibody-receptor binding affinity ( K D ). Blue curves represent experimental data and red curves represent the statistical fitting of curves. ( d ) Interaction of H-Zt/g4 H1L3 with RONs from different species. NIH3T3 cells expressing human, monkey, or mouse RON were incubated with H-Zt/g4 H1L3 followed by goat anti-human IgG coupled with FITC. Immunofluorescent intensities from individual samples were determined by flow cytometric analysis. ( e ) Schematic representation of H-Zt/g4-MMAE structure. MMAE was conjugated to H-Zt/g4 by the valine-citruline dipeptide linker according to the manufacturer’s instruction ( www.concortis.com ). ( f ) HIC analysis of MMAE conjugated to H-Zt/g4: Individual Zt/g4-MMAEs with different numbers of MMAE (0 to 8) are marked as P0 to P8. A DAR combining P2, P4, and P6 at 3.77:1 was achieved. ( g ) Free MMAE dissociated from H-Zt/g4-MMAE in human plasma. H-Zt/g4-MMAE at 10 μg per ml was incubated with fresh human plasma at 37 °C for 20 days. The amount of free MMAE in plasma was determined using the LC-MS/MS method with slight modifications. ( h ) Samples from ( g ) were used also for measuring MMAE conjugated H-Zt/g4 as detailed in Materials and Methods. A ratio from free MMAE to the total MMAE in H-Zt/g4-MMAE was calculated to determine the percentages of MMAE dissociated from H-Zt/g4-MMAE

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Generated, Software, Binding Assay, Incubation, Expressing, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Effect of H-Zt/g4-MMAE on RON internalization, cell viability, and death: ( a ) H-Zt/g4-induced cell surface RON internalization. PDAC cell lines BxPC-3, FG and L3.6pl (1 × 10 6 cells per dish) were treated at 37 °C with 5 μg/ml of H-Zt/g4-MMAE, collected at different time points, washed with acidic buffer to eliminate cell surface bound IgG , and then incubated with 2 μg/mL of anti-RON mAb Zt/c1 . Immunofluorescence was analyzed by flow cytometer using FITC-coupled anti-mouse IgG. Immunofluorescence from cells treated with H-Zt/g4 at 4 °C was set as 100%. Internalization efficiency (IC 50 ) was calculated as the time required achieving 50% reduction of cell surface RON. ( b ) Intracellular localization of internalized RON. FG cells in a 6-well plate were treated with 5 μg/ml of H-Zt/g4 at 4 °C or 37 °C for 12 h followed by mouse anti-human IgG1-coupled with FITC. Nuclear DNAs were stained with DAPI. LAMP-1 was used as a marker for protein cytoplasmic localization. Similar results also observed in additional three PDAC cell lines (data not shown). ( c ) Effect of H-Zt/g4-MMAE on viability of PDAC cells. Three PDAC cell lines (8000 cells per well in a 96-well plate in triplicate) were treated with different amounts of H-Zt/g4-MMAE for 96 h. Panc-1 cells served as the negative control. Cell viability was determined by the MTS assay. ( d ) Death of PDAC cells after H-Zt/g4-MMAE treatment. PDAC cells were treated with different amounts of H-Zt/g4-MMAE for 96 h. The percentages of cell death were determined by the trypan blue exclusion method. Data shown in ( c ) and ( d ) are derived from one of three experiments with similar results

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Incubation, Immunofluorescence, Flow Cytometry, Staining, Marker, Negative Control, MTS Assay, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Pharmacokinetic profiles of H-Zt/g4-MMAE in both mouse and cynomolgus monkey: ( a ) PK profiles of H-Zt/g4-MMAE in mouse. Tumor-bearing and -nonbearing mice (athymic nude, 5 mice per group) were injected with a single dose of H-Zt/g4-MMAE at 3, 10, and 20 mg/kg, respectively. Collected blood samples were analyzed using the MMAE ADC ELISA kit (Eagle Biosciences, Inc., Nashua, NH). Various PK parameters were calculated using the software provided by Eagle Biosciences. ( b ) Free MMAE dissociated from H-Zt/g4-MMAE in monkey plasma. A single dose of H-Zt/g4-MMAE at 10 or 30 mg/kg was injected into cynomolgus monkey (3 animals per group). Free MMAE from individual blood samples collected at different time intervals were subjected to the LC-MS/MS analysis. ( c ) PK profiles of H-Zt/g4-MMAE in cynomolgus monkey. Blood samples from ( b ) were analyzed for MMAE coupled H-Zt/g4 using the MMAE ADC ELISA kit as described in ( a ) to obtain various PK parameters

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Software, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Therapeutic efficacy of H-Zt/g4-MMAE in PDAC xenograft tumor models: ( a ) Dose-dependent effect of H-Zt/g4-MMAE: Athymic nude mice (5 mice per group) were subcutaneously inoculated with 5 × 10 6 FG cells. H-Zt/g4-MMAE at 1, 3, 7, 10, and 15 mg/kg was injected through tail vein in the Q6 × 5 regimen after tumors volumes reached to ~ 150 mm 3 . Mice injected with CmIgG-MMAE at 10 mg/kg were used as the control. Xenografts initiated by HT-29 cells served for comparison. ( b ) Effect of H-Zt/g4-MMAE in PDAC xenograft growth and eradication. Individual tumors from different groups described in (A) were collected from euthanized mice. Control mice bearing FG xenografts were sacrificed at day 24 due to rapid growth of tumors. Mice from other groups were killed at day 28 or day 44 dependent on the size of tumors. All tumors were weighted to reach the average tumor weight per group. The number of tumors from individual groups also was counted to determine the eradicating effect of H-Zt/g4-MMAE. NA, no tumors were found in the injected site. ( c ) Effect of H-Zt/g4-MMAE in three PDAC xenograft models: Xenograft tumors in mice (five animals per group) initiated by four PDAC cell lines were used for study. H-Zt/g4-MMAE was used at 20 mg/kg in the Q12 × 2 schedules. To establish the dose-effect relationship, the estimated reduction of H-Zt/g4-MMAE in vivo according to the t½ was marked as red circles. ( d ) Effect of H-Zt/g4-MMAE in tumor growth and eradication: Tumors were collected from mice described in ( b ). Tumor weight, count, and calculation were performed as described in ( b ). NA, no tumors were observed in the injected site

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Drug discovery, Injection, Control, Comparison, In Vivo

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Therapeutic Effect of H-Zt/g4-MMAE on xenograft tumors mediated by PDAC stem-like cells and primary PDX cells: ( a ) Effect of H-Zt/g4-MMAE on PDAC stem-like cell derived xenografts: Athymic nude mice (five mice per group) were subcutaneously inoculated with 5 × 10 5 PSC + 24/44/ESA prepared from BxPc-3, FG, and L3.6pl cells. H-Zt/g4-MMAE at 20 mg/kg was injected through tail vein in the Q12 × 2 regimen after tumors volumes reached to ~ 150 mm 3 . Mice injected with CmIgG-MMAE at 20 mg/kg were used as the control. ( b ) The eradicating effect of H-Zt/g4-MMAE on PDAC stem-like cell derived xenografts. Tumors were collected from mice as described in Fig. b. Tumor weight, count, and calculation were performed as described in Fig. b. ( c ) Mice were injected with individual primary PDX cell lines at 5 × 10 6 cells in 0.1 ml in PBS. H-Zt/g4-MMAE at 10 mg/kg was injected through tail vein in the Q12 × 2 regimen after tumors volumes reached to 150 to 200 mm 3 . Mice injected with CmIgG-MMAE at 10 mg/kg were used as the control. ( d ) Individual tumors were collected from each group of mice as described in Fig. b. Average tumor weight and number per group were measured to determine levels of inhibition and eradication

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Derivative Assay, Injection, Control, Inhibition

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Therapeutic effect of H-Zt/g4-MMAE in comparison with H-Zt/g4-DM1 in inhibition of xenograft tumors derived from human pancreatic and colorectal cancer cells a

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Comparison, Inhibition, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Toxicological activities of H-Zt/g4-MMAE in mouse and cynomolgus monkey. ( a ) and ( b ) Adverse activities of H-Zt/g4-MMAE in blood leukocytes in cynomolgus monkey. H-Zt/g4-MMAE at 10 or 30 mg/kg in a single dose was injected once into cynomolgus monkey. Monkeys without ADC injection served as the control. Peripheral blood samples were collected at different time intervals. Total numbers of leukocytes ( a ) including neutrophil, lymphocytes, and monocytes from each group were countered accordingly. The percentages of blood leukocytes ( b ) were also determined. ( c ) Adverse effect of H-Zt/g4-MMAE on blood erythrocytes and reticulocytes in cynomolgus monkey. Total numbers of erythrocytes and reticulocytes from blood samples collected from each group as described in ( b ) were counted accordingly. ( d ) Adverse effects of H-Zt/g4-MMAE on various enzymes in plasma of cynomolgus monkey. Six enzymatic activities from each group were quantitatively measured using the blood samples collected from individual monkeys as described in ( b )

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Injection, Control, Clinical Proteomics

Journal: Kidney international

Article Title: Targeted reduction of advanced glycation improves renal function in obesity.

doi: 10.1038/ki.2011.57

Figure Lengend Snippet: Figure 2 | Murine renal and biochemical parameters at study completion. Groups of mice were followed for 16 weeks. (a) Urinary albumin excretion rate (AER) over 24 h measured by enzyme-linked immunosorbent assay (ELISA). (b) Creatinine clearance (CrCl) as determined by high-performance liquid chromatography (HPLC) following correction for body surface area. (c–e) N-Carboxymethyllysine (CML) analyzed by ELISA in plasma (c), dietary CML consumption over 24 h (d), and kidney cortex protein (e). (f) CML immunohistochemistry staining on paraffin-fixed kidney sections from (A) lean low advanced glycation end-product (AGE), (B) obese, (C) obese alagebrium (ALA), and (D) obese RAGE–/–. Obese (high AGE/high-fat diet), ALA (AGE-lowering therapy, alagebrium chloride 1 mg/kg/day), and RAGE–/– (RAGE deletion). Data for AER were logarithmically transformed as these were not normally distributed. Other data are presented as mean±s.d. *Po0.05 vs lean low AGE, **Po0.01 vs lean low AGE, ***Po0.001 vs lean low AGE, wPo0.05 vs obese.

Article Snippet: Albumin excretion rate was assessed using a mouse albumin ELISA kit according to the manufacturer’s instructions (Bethyl Laboratories, Montgomery, TX).

Techniques: Enzyme-linked Immunosorbent Assay, High Performance Liquid Chromatography, Clinical Proteomics, Immunohistochemistry, Staining, Transformation Assay

Journal: Cell host & microbe

Article Title: Antiretroviral therapy reveals triphasic decay of intact SIV genomes and persistence of ancestral variants.

doi: 10.1016/j.chom.2023.01.016

Figure Lengend Snippet: Figure 4. Comparison of gag ddPCR and IPDA quantification of SIV DNA (A) Correlation between gag+ SIV DNA copies and intact proviruses quantified using the IPDA. Individual datapoints represent the number of copies detected using either an amplicon in gag (y-axis)65 or the IPDA (x-axis).20 Each point is the geometric mean of 3 replicates. Intact proviruses are corrected for shearing (DSI) and env+2LTR circles. The correlation between the two variables was calculated using Pearson’s coefficient. Data from one animal, T624, were excluded from this analysis due to failure of the gag amplicon resulting from mutations or deletion. (B) Correlation between gag+ SIV DNA copies and intact proviruses, with gag values corrected using the same env+2LTR correction factor applied to the IPDA data, calculated using Pearson’s coefficient. (C) Comparison of the decay of SIV gag copies and intact proviruses for the animals in cohort 18–02. IPDA data are corrected for env+2LTR circles and DNA shearing. Vertical lines represent the standard deviations. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and virus strains SIVmac251 swarm 56 Dr. Dan Barouch, BIDMC N/A Biological samples N/A SIVmac251-infected Rhesus macaque PBMCs Dr. Dan Barouch, BIDMC N/A Chemicals, peptides, and recombinant proteins Platinum TaqDNA Polymerase High Fidelity ThermoFisher Scientific Cat # 11304011 SuperScript III Reverse Transcriptase ThermoFisher Scientific Cat # 18-080-044 dNTP Mix (10 mM each) ThermoFisher Scientific Cat # 18-427-088 UltraPure 1M Tris-HCl Buffer (pH 8.0) ThermoFisher Scientific Cat # 15-568-025 RNaseOUT Recombinant Ribonuclease Inhibitor ThermoFisher Scientific Cat # 10777019 Critical commercial assays QIAamp DNA Mini Kit Qiagen Cat # 51304 CD4+ T Cell Isolation Kit, Non-Human Primate Miltenyi Biotec Cat # 130-092-144 2X ddPCR Supermix for Probes (no dUTPs) Bio-Rad Cat # 186-3024 Deposited data SIV – plasma RNA sequences This paper Genbank: OQ168641-OQ168979 SIV – non-defective proviral DNA sequences This paper Genbank: OQ168980-OQ170751 SIV – hypermutated (defective) proviral sequences This paper Genbank: OQ170752-OQ170785 Experimental models: Organisms/strains Rhesus macaque (Macaca mulatta) infected with SIVmac251 Indian origin Animals T523, T530, T537, T544, T545, T623, T624, T625, T627 & T628 Oligonucleotides Oligonucleotides, standard desalting (Sanger sequencing & ddPCR) – see STAR Methods and Table S8 IDT N/A FAM/VIC labeled probes with MGB quencher, HPLC purified (see Table S8) Applied Biosystems N/A Unlabeled competition probes w/MGB quencher, HPLC purified (see Table S8) Applied Biosystems N/A FAM/HEX labeled probes w/MGB-NFQ quencher, HPLC purified (see Table S8) IDT N/A Recombinant DNA Synthetic double-stranded DNA controls (gBlocks) – see STAR Methods IDT N/A Software and algorithms QuantaSoft Analysis-Pro Bio-Rad http://www.bio-rad.com/en-us/ product/qx200-droplet-digitalpcr-system?ID=MPOQQE4VY Geneious Prime Dotmatics https://www.geneious.com/prime/ MEGA v7.0 & v11 MEGA https://www.megasoftware.net/ (Continued on next page) Cell Host & Microbe 31, 356–372.e1–e5, March 8, 2023 e1

Techniques: Comparison, Amplification

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

Journal: Communications Biology

Article Title: Paradoxical regulation of IGF2 in promoting lipid metabolism in adipose tissues

doi: 10.1038/s42003-025-08458-1

Figure Lengend Snippet: A , B “U” and inverted “U” shaped relationship between IGF2 levels and the lipid species detected by LC-MS/MS assay utilizing a polynomial fourth order equation to fit the non-linear regression curve, where R² (R-squared) represents the coefficient of determination, and Sy.x represents the standard deviation of the residuals, n = 200. C , D “U” and inverted “U” shaped relationship between IGF2 levels and the lipid species detected by ELISA kit assay utilizing a polynomial fourth order equation to fit the non-linear regression curve, n = 200. E , F Pearson correlation analyses of L-IGF2 levels and H-IGF2 levels with triglyceride conducted by LC-MS/MS assay, n = 200, all p < 0.01. G , H Pearson correlation analyses of L-IGF2 levels and H-IGF2 levels with HDL-c conducted by LC-MS/MS assay, n = 200, all p < 0.05. I , J Pearson correlation analyses of L-IGF2 levels and H-IGF2 levels with triglyceride conducted by ELISA kit assay, n = 200, all p < 0.001. K , L Pearson correlation analyses of L-IGF2 levels and H-IGF2 levels with HDL-c conducted by ELISA kit assay, n = 200, all p < 0.05. M , N Multiple stepwise logistic regression analysis of MetS, HOMA-IR, and other metabolic subgroups (central obesity, hypertension, hyperglycemia, hypertriglyceridemia, and low HDL-c) connected with L-IGF2 and H-IGF2 levels conducted by LC-MS/MS assay and ELISA kit assay, respectively.

Article Snippet: The measurement of serum IGF2 level was conducted with an ELISA kit (E-EL-H6037 for human samples and E-EL-M3078 for mice samples, Elabscience, China).

Techniques: Liquid Chromatography with Mass Spectroscopy, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: Paradoxical regulation of IGF2 in promoting lipid metabolism in adipose tissues

doi: 10.1038/s42003-025-08458-1

Figure Lengend Snippet: A The diagram of Ad-IGF2 and its control adenovirus were injected into the eWAT of DIO mice at multi-point (2 × 10 10 pfu/mouse, injected two times for a week, a total of 4 weeks of injection). B In vivo imaging was employed to track the expression sites of the overexpressing adenovirus constructs tagged with GFP, including Ad-IGF2 and Ad-GFP. C , D Representative western blot results of IGF2 protein levels in iWAT, eWAT, liver, skeletal muscle and pancreas tissues. E For a duration of 8 weeks, eWAT and iWAT of the mice were carefully dissected and visually documented using photography. F ELISA results of IGF2 protein levels in the serum. n = 6, ** p < 0.01 by two-tailed, unpaired Student’s t test. G – J The weight of eWAT and iWAT, liver and the whole-body weight of mice were compared, n = 6, * p < 0.05, n.s not significant (unpaired Student’s t test). K Hematoxylin/Eosin representative staining of adipocytes in iWAT, eWAT, prWAT, liver, and muscle tissue. Magnification: 20×. L – N Adipocyte size distribution curves of eWAT, iWAT and prWAT between Ad-GFP group mice (gray) and Ad-IGF2 group mice (blue), respectively. n = 6, Data represent the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001 analyzed by a one-way ANOVA with a Tukey’s multiple comparisons test. O , P The measurement analysis of blood glucose levels was conducted at specific times in obese mice after IGF2 overexpression by GTT and ITT assays, n = 6, * p < 0.05, analyzed by a two-way ANOVA with Bonferroni’s multiple comparisons test. Q , R The area under curve (AUC) analysis of GTT and ITT assays were conducted in obese mice after IGF2 overexpression, n = 6, *** p < 0.001 by two-tailed, unpaired Student’s t test. S , T Plasma TC, TG, HDL-c, LDL-c, ALT, AST, and ALP contents have been identified following injection of Ad-GFP and Ad-IGF2, n = 6, Data represent the mean ± SD; * p < 0.05 (unpaired Student’s t test).

Article Snippet: The measurement of serum IGF2 level was conducted with an ELISA kit (E-EL-H6037 for human samples and E-EL-M3078 for mice samples, Elabscience, China).

Techniques: Control, Injection, In Vivo Imaging, Expressing, Construct, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Staining, Over Expression, Clinical Proteomics

Journal: Communications Biology

Article Title: Paradoxical regulation of IGF2 in promoting lipid metabolism in adipose tissues

doi: 10.1038/s42003-025-08458-1

Figure Lengend Snippet: A The diagram of IGF2-RNAi and its control lentivirus NC-RNAi were injected into the tail vein of 3-4-week-old mice (1 × 10 10 pfu/mouse, injected two times for a week, collectively a 4 weeks of injection). B Representative western blot results of IGF2 protein levels in liver tissues when 16 weeks old. C Representative western blot results of IGF2 protein levels in eWAT, iWAT, liver, skeletal muscle, pancreas and kidney tissues. D ELISA results of IGF2 protein level in serum samples, n = 12, *** p < 0.001 (unpaired Student’s t test). E At 16 weeks, liver tissues, iWAT, and eWAT of mice underwent dissection and photography. The representative images were depicted. F – J The whole-body weight, the liver, iWAT, eWAT ( n = 12) and quadriceps ( n = 6) weight of mice were compared, the p -values have been annotated, n.s not significant (unpaired Student’s t test). K Representative H&E staining of iWAT, eWAT, liver and muscle tissues, and the Oil Red staining of liver and muscle tissues. Magnification: 20×. L , M Adipocyte size distribution curves of eWAT and iWAT between NC-RNAi group mice (gray) and IGF2-RNAi group mice (yellow), respectively. n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001 analyzed by a one-way ANOVA with a Tukey’s multiple comparisons test. N , O Plasma TC, TG, HDL-c, LDL-c, ALT, AST, and ALP contents have been identified following injection of IGF2-RNAi and NC-RNAi, n = 6, * p < 0.05, ** p < 0.01 (unpaired Student’s t test). P , R The levels of blood glucose underwent measurement at specific times by GTT and ITT assays, n = 6, * p < 0.05, ** p < 0.01 analyzed by a two-way ANOVA with Bonferroni’s multiple comparisons test. Q , S The area under curve (AUC) analysis of GTT and ITT assays were conducted in obese mice after IGF2 knockdown, n = 6, Data represent the mean ± SD; *** p < 0.001 by two-tailed, unpaired Student’s t test.

Article Snippet: The measurement of serum IGF2 level was conducted with an ELISA kit (E-EL-H6037 for human samples and E-EL-M3078 for mice samples, Elabscience, China).

Techniques: Control, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Dissection, Staining, Clinical Proteomics, Knockdown, Two Tailed Test

Journal: Communications Biology

Article Title: Paradoxical regulation of IGF2 in promoting lipid metabolism in adipose tissues

doi: 10.1038/s42003-025-08458-1

Figure Lengend Snippet: A The utilization of Nile red staining and Oil red O staining techniques revealed the presence of lipid droplets in 3T3-L1 adipocytes after IGF2 overexpression and knockdown treatment. B , C Quantification of cellular triglyceride (TG) content and the levels of free fatty acid (FFA) in the medium in 3T3-L1 adipocytes after IGF2 overexpression and knockdown treatment. * p < 0.05, ** p < 0.01. D , E The mRNA levels of genes associated with adipogenesis, lipogenesis, and lipolysis in 3T3-L1 cells transfected with Ad-IGF2 or IGF2-RNAi as well as the corresponding controls by RT-qPCR assays, using Ppia as internal controls, n = 6, Data represent the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001 by two-tailed, unpaired Student’s t test. F Protein expression levels of genes related to adipogenesis, lipogenesis and lipolysis in the 3T3-L1 adipocytes by western blot assays. G Mice adipose tissue has been dissected and isolated for primary adipocyte culture, and the results of 0, 4, 8 and 12 days were induced by the classic “Cocktail” induction differentiation regimen. H Primal cell supernatant was extracted during the above induction differentiation process, and IGF2 concentration was detected by ELISA kit. n = 3. I 3T3-L1 preadipocytes underwent treatment via different concentration gradients of recombinant IGF2 protein powder and induced differentiation. Oil red O staining employment demonstrated that the deposition of lipid droplets within adipocytes.

Article Snippet: The measurement of serum IGF2 level was conducted with an ELISA kit (E-EL-H6037 for human samples and E-EL-M3078 for mice samples, Elabscience, China).

Techniques: Staining, Over Expression, Knockdown, Transfection, Quantitative RT-PCR, Two Tailed Test, Expressing, Western Blot, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Nutrients

Article Title: Lactoferrin Supplementation during Gestation and Lactation Is Efficient for Boosting Rat Pup Development

doi: 10.3390/nu14142814

Figure Lengend Snippet: Composition of the diets given to the control group of mothers (P20 Soy) or to the bLF-supplemented mothers (P20 Soy + bLF).

Article Snippet: Bovine lactoferrin concentration was measured in the plasma, milk, and urine of the mothers and in the plasma of the pups using a Bovine Lactoferrin ELISA Kit (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Control, Starch

Journal: Nutrients

Article Title: Lactoferrin Supplementation during Gestation and Lactation Is Efficient for Boosting Rat Pup Development

doi: 10.3390/nu14142814

Figure Lengend Snippet: Composition of body weight at postnatal day 17 (DEXA values) of pups with maternal bLF supplementation (bLF) or without supplementation (Cont). ( A ) Effect of maternal bLF supplementation on the lean mass. ( B ) Effect of maternal bLF supplementation on fat mass. ( C ) Effect of maternal bLF supplementation on adiposity. Data represented are means ± SEM (*** p < 0.001; n = 16 per group). The control and lactoferrin groups were compared by an unpaired two-tailed Student’s t -test.

Article Snippet: Bovine lactoferrin concentration was measured in the plasma, milk, and urine of the mothers and in the plasma of the pups using a Bovine Lactoferrin ELISA Kit (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Control, Two Tailed Test

Journal: Nutrients

Article Title: Lactoferrin Supplementation during Gestation and Lactation Is Efficient for Boosting Rat Pup Development

doi: 10.3390/nu14142814

Figure Lengend Snippet: Effects of bLF supplementation on pup bone mineral density (BMD) and bone markers. Pups were recovered from mothers receiving the control diet (Cont) or the bLF-supplemented diet (bLF). ( A ) Whole body BMD. ( B ) Femoral BMD. ( C ) Vertebral BMD. ( D ) Plasma marker of bone resorption CTX. ( E ) Plasma marker of bone formation PINP. ( F ) PINP/CTX ratio. Data presented are means ± SEM (*** p < 0.001, **** p < 0.0001; n = 16 per group). The control and lactoferrin groups were compared by an unpaired two-tailed Student’s t -test.

Article Snippet: Bovine lactoferrin concentration was measured in the plasma, milk, and urine of the mothers and in the plasma of the pups using a Bovine Lactoferrin ELISA Kit (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Control, Clinical Proteomics, Marker, Two Tailed Test

Journal: Nutrients

Article Title: Lactoferrin Supplementation during Gestation and Lactation Is Efficient for Boosting Rat Pup Development

doi: 10.3390/nu14142814

Figure Lengend Snippet: GC-MS and LC-MS multivariate analysis of metabolite profiles in plasma of pups recovered from mothers receiving bLF (LF) or mothers receiving no supplement (Cont). ( A ) GC-MS orthogonal projections of latent structures discriminant analysis (OPLS-DA) score plots of plasma metabolites in the control (green) and lactoferrin (blue) pups. LF vs. Cont ( p < 0.01). ( B ) Loadings plots highlighting discriminant metabolites. ( C ) LC-MS orthogonal projections of latent structures discriminant analysis (OPLS-DA) score plots of plasma metabolites in the control (green) and lactoferrin (blue) pups. LF vs. Cont ( p < 0.01). ( D ) Loadings plots highlighting discriminant metabolites.

Article Snippet: Bovine lactoferrin concentration was measured in the plasma, milk, and urine of the mothers and in the plasma of the pups using a Bovine Lactoferrin ELISA Kit (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Gas Chromatography-Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Clinical Proteomics, Control

Journal: Journal of Medicinal Chemistry

Article Title: Novel Targeted System To Deliver Chemotherapeutic Drugs to EphA2-Expressing Cancer Cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: Figure 1. EphA2 and EphA4 expression in 68 human cancer cell lines and 17 normal tissue RNA samples. (A) EphA2 and EphA4 receptor expression in normal and cancer cell lines. Bar graph shows the fold expression of both EphA2 and EphA4 receptors in MDA-MD-231, PC-3, and LNCaP cancer cell lines compared to cultured human dermal fibroblast (HDF) and to a total human prostate RNA sample (prostate). (B) Correlation between EphA2 mRNA expression and EphA2 protein levels in 30 selected human cancer cell lines. (C) Box and Whisker plots of the 10th to 90th percentile of EphA2 expression for the RNA samples grouped according to their tissue of origin. Dotted lines depict cutoffs for 7-fold and 14-fold expression levels observed in the reference HDF cell line, respectively.

Article Snippet: The amount of EphA2 protein in each cell line was assayed using the human total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Cell Culture, Whisker Assay

Journal: Journal of Medicinal Chemistry

Article Title: Novel Targeted System To Deliver Chemotherapeutic Drugs to EphA2-Expressing Cancer Cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: Figure 2. YSA peptide targets cells expressing EphA2. (A) The YSA peptide targets quantum dots to EphA2 on the cell surface. COS cells transfected with either the extracellular and transmembrane portions of EphA2 fused to enhanced green fluorescent protein (EphA2-EGFP) or membrane targeted EGFP-F were incubated with YSA bound to red fluorescent quantum dots (YSA−Qdots). YSA−Qdots only bind to cells transfected with EphA2-EGFP. EGFP fluorescence is green. Nuclei are stained in blue with DAPI. (B) YSA targets quantum dots to endogenous EphA2 on the cell surface. HUVE cells were incubated at 4 °C with YSA conjugated Qdots (YSA−Qdots), an unrelated 12- mer control peptide that does not bind to EphA2 also conjugated to Qdots (Ctrl-Qdots), or unconjugated Qdots (Qdots). Only quantum dots conjugated to the YSA peptide bind to the HUVE cells. (C) YSA targets Qdots to lysosomes. MDA-MB-231 cells, which express high levels of endogenous EphA2, were incubated at 4 °C with YSA−Qdots followed by incubation at 37 °C for 0, 15, and 60 min. YSA−Qdots are seen on the cell surface at 0 min but become concentrated in structures near the nucleus at 15 and 60 min (arrow). Double labeling with the lysosomal marker Lamp1 (green) shows colocalization of the quantum dots in lysosomes at 60 min (arrows).

Article Snippet: The amount of EphA2 protein in each cell line was assayed using the human total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Transfection, Membrane, Incubation, Fluorescence, Staining, Control, Labeling, Marker

Journal: Journal of Medicinal Chemistry

Article Title: Novel Targeted System To Deliver Chemotherapeutic Drugs to EphA2-Expressing Cancer Cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: Figure 3. YSA−PTX retains high potency for inhibition of EphA2-ephrin-A5 binding and high EphA2 binding affinity. (A) General chemical structures of YSA and DYP peptide−drug conjugates. (B) YSA, YSA−PTX, and control DYP−PTX were incubated at the indicated concentrations together with a constant concentration of ephrin-A5 AP in ELISA wells precoated with EphA2 Fc. The ratio of ephrin-A5 AP bound in the presence and in the absence of peptide is shown. (C) Biotinylated YSA−PTX and control DYP−PTX were incubated at the indicated concentrations in EphA2 Fc-coated ELISA wells and were then detected with streptavidin−HRP. The graphs show averages ± SE from quadruplicate measurements in representative experiments, while the KD and IC50 values are calculated from three to five experiments. (D) ATP-Lite analysis of viability of PC-3 cells 72 h after the addition of DYP, DYP−PTX, YSA, YSA−PTX, or PTX at 10 nM (n = 3).

Article Snippet: The amount of EphA2 protein in each cell line was assayed using the human total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Inhibition, Binding Assay, Control, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Medicinal Chemistry

Article Title: Novel Targeted System To Deliver Chemotherapeutic Drugs to EphA2-Expressing Cancer Cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: Figure 4. YSA−PTX is internalized in cancer and endothelial cells expressing EphA2. (A) YSA−PTX coupled to fluorescent quantum dots, but not DYP−PTX, is internalized with EphA2 into lysosomes of prostate cancer cells expressing EphA2. PC3 prostate cancer cells, which express high levels of EphA2, or LNCaP prostate cancer cells, which do not detectably express EphA2 protein, were treated for 20 min with 100 μM YSA−PTX or control DYP−PTX, followed by a 20 min of incubation with streptavidin-conjugated quantum dots (Qdots). After removal of the solution containing the peptides and the quantum dots, the cells were incubated for 2 h at 37 °C to allow EphA2 internalization induced by YSA−PTX binding. The cells were stained for EphA2 (left) or the lysosomal marker Lamp1 (right). Qdots were also imaged, and nuclei were labeled with DAPI in blue. Representative fluorescence micrographs are shown. Scale bar = 25 μM. (B) Ephrin-A1 Fc and YSA−PTX, but not DYP−PTX, cause EphA2 internalization into lysosomes. PC3 cells were treated for 2 h with 0.2 μg/mL ephrin-A1 Fc, 100 μM YSA−PTX, or 100 μM control DYP−PTX. The cells were stained for Lamp1 (red) and EphA2 (green). Nuclei were labeled with DAPI (blue). Representative confocal micrographs are shown. Scale bar = 25 μm. (C) Ephrin-A1 Fc and YSA−PTX, but not DYP−PTX, cause EphA2 internalization into cells. PC3 prostate cancer and HUVE endothelial cells were treated for 1 h with 0.2 μg/mL ephrin-A1 Fc, 100 μM YSA−PTX, or 100 μM control DYP−PTX. Proteins present on the cell surface were then labeled with biotin. EphA2 immunoprecipitates were probed with antiphosphotyrosine antibody (PTyr), streptavidin−HRP (biotin), and EphA2 antibody. The amount of GAPDH in the cell lysates used for the immune precipitations is also shown as a control for equal amounts of protein in the lysates used for immunoprecipitation.

Article Snippet: The amount of EphA2 protein in each cell line was assayed using the human total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Control, Incubation, Binding Assay, Staining, Marker, Labeling, Fluorescence, Immunoprecipitation

Journal: Journal of Medicinal Chemistry

Article Title: Novel Targeted System To Deliver Chemotherapeutic Drugs to EphA2-Expressing Cancer Cells

doi: 10.1021/jm201743s

Figure Lengend Snippet: Figure 5. Expression of EphA2 and cellular and in vivo activity of drug conjugates. (A) Groups of five to seven SCID beige mice bearing pre- established subcutaneous PC3 tumors were treated with twice weekly with intravenous doses of vehicle (CT), paclitaxel (5 mg/kg), YSA− paclitaxel (equimolar doses of the paclitaxel dose), starting at day 0. Tumor sizes were measured, and averages ± SEM are shown. p < 0.05 for the comparison of YSA−paclitaxel with control, by repeated- measures two-way ANOVA using all the measurements as well as by one-way ANOVA and Dunnett’s post hoc test using the measurements at day 18. (B) Biodistribution analysis. PC3 tumors (n = 3, average volume of 200 mm3) were excised 30 min after equimolar amounts of YSA−PTX or PTX (25 mg/kg) were injected intravenously. LC−MS analysis was performed to quantify PTX levels from tumor extracts. The histogram shows averages ± SEM. p < 0.05 for the comparison of PTX levels, by t test analysis. Plasma levels of PTX were not significantly different (YSA−PTX = 998 ± 742 ng/mL; PTX = 771 ± 585 ng/mL).

Article Snippet: The amount of EphA2 protein in each cell line was assayed using the human total EphA2 DuoSet Elisa kit (R&D Systems, Minneapolis, MN).

Techniques: Expressing, In Vivo, Activity Assay, Comparison, Control, Injection, Liquid Chromatography with Mass Spectroscopy, Clinical Proteomics

Journal: Journal of Extracellular Biology

Article Title: Enrichment of Immune Cell‐Derived Extracellular Vesicles From Plasma Using 35 and 70 nm Size‐Exclusion Chromatography Columns of Different Sizes

doi: 10.1002/jex2.70098

Figure Lengend Snippet: Schematic overview of the experimental setup. Extracellular vesicle (EV) isolation from whole blood with different size exclusion chromatography (SEC) columns. Blood was collected in citrate tubes from healthy subjects and centrifuged at 1000 × g to generate platelet‐rich plasma (PRP). PRP was centrifuged at 2500 × g to generate platelet‐poor plasma (PPP), which was then filtered through a 1.2 µm filter to fully remove the residual platelets. EVs were then isolated with four different SEC columns, using the 35 or 70 nm IZON columns of two sizes: either qEV original (0.5 mL, small) or qEV10 (10 mL, large). After ultrafiltration (UF), the samples were stored in aliquots at –80°C and subsequently analysed by nanoparticle tracking analysis (NTA), electron microscopy, flow cytometry, MACSPlex, Western blot and LC‐MS/MS.

Article Snippet: The MACSPlex EV Kit IO, human (130‐108‐813, Miltenyi) was used for surface marker detection of EVs isolated from both large and small columns according to the manufacturer's instructions.

Techniques: Isolation, Size-exclusion Chromatography, Clinical Proteomics, Electron Microscopy, Flow Cytometry, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of Extracellular Biology

Article Title: Enrichment of Immune Cell‐Derived Extracellular Vesicles From Plasma Using 35 and 70 nm Size‐Exclusion Chromatography Columns of Different Sizes

doi: 10.1002/jex2.70098

Figure Lengend Snippet: Surface marker analysis of isolated EVs using Flow cytometry. (A) EVs isolated by the 35 or 70 nm column were stained for CD81 and analysed on the Sony ID 7000 Spectral Cell Analyzer, and gating was performed on the buffer and antibody control. All positive events are susceptible to 0.1% SDS treatment. (B) EVs isolated with large columns 35 and 70 nm, fractions 1–5 of triplicate donors were analysed using the MACSPlex EV Kit. Median Allophycocyanin (APC) Fluorescence Intensity (MFI) is plotted. For large columns, the median of triplicate donors was plotted. (C) EV markers (CD81, CD63, and CD9) for fractions 1–5 of the 35 and 70 nm large columns are shown as APC MFI. Individual donors are shown as dots and bars represent the mean with standard deviation shown as error bars. (D) Some platelet markers (CD41b, CD42a, CD29) and other cell surface markers (CD24, HLA‐I, HLA‐II, CD40) are shown as median APC MFI. Individual donors are shown as dots and the standard deviation is shown as error bars. Data were analysed using Two‐Way ANOVA (Šidák's test), with statistical significance indicated as p < 0.05 (*) and p < 0.001 (***).

Article Snippet: The MACSPlex EV Kit IO, human (130‐108‐813, Miltenyi) was used for surface marker detection of EVs isolated from both large and small columns according to the manufacturer's instructions.

Techniques: Marker, Isolation, Flow Cytometry, Staining, Control, Fluorescence, Standard Deviation