Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: ( a ) Median expression of the KIM-1 gene in different cancer and normal tissues obtained from the TCGA dataset using Gene Expression Profiling Interactive Analysis (GEPIA). ( b ) Representative images for immunostaining of kidney tissue microarray by anti-KIM-1 (red) antibody and DAPI (blue). Tissue specimens were collected from randomized patients based on age, sex, and stage of cancer. The normal adjacent tissue (NAT) and tumor tissue were collected from the same patients.

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: Expressing, Gene Expression, Immunostaining, Microarray

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: ( a ) Median expression of the KIM-1 gene in different cancer and normal tissues obtained from the TCGA dataset using Gene Expression Profiling Interactive Analysis (GEPIA). Inset: The pictorial representation of the KIM-1 expression in cancer (red) and normal (green) organs. The figure was generated using the GEPIA platform. ( b ) Immunostaining of kidney tissue microarray by anti-KIM-1 (red) antibody, and DAPI (blue). Tissue specimens were collected from randomized patients based on age, sex, and stage of cancer. The normal adjacent tissue (NAT) and tumor tissue were collected from the same patients. The image shows the majority of the tumor expressing high KIM-1. 16 such images were combined to form a single circular tissue image. ( c ) Bar plot showing relative KIM-1 expression in cancer and normal adjacent tissue (NAT). Each data point represents one patient sample. The data were represented as mean ± SEM (n = 16, paired t-test, P < 0.01 **).

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: Expressing, Gene Expression, Generated, Immunostaining, Microarray

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: ( a ) Schematic representation of the synthesis of KIM-1 targeting ADC, LT-025, by using transglutaminase. First, the native monoclonal KIM-1 antibody was deglycosylated in the presence of the PNGase F enzyme, and then the payload-linker was attached to the antibody at Q295 in the presence of the microbial transglutaminase enzyme (MTGase). The payload used was DM1. ( b ) Data shows the difference in the retention time of KIM-1 antibody, deglycosylated KIM-1 antibody, and ADC (LT-025) using hydrophobic interaction chromatography (HIC). Data was recorded under physiological conditions in PBS (pH 7.4) at 280 nm. 10 μg of each sample was injected for analysis. The chromatograms were baseline corrected using Origin 8.0 and plotted using GraphPad Prism 10 software. ( c ) Mass spectral analysis of the antibody drug conjugate. The data were recorded using ESI-MS after DTT reduction of the LT-025. Deconvoluted mass spectra of light chain and heavy chain, of KIM-1 antibody, deglycosylated KIM-1 antibody, and LT-025. 5 μg of each sample was injected for ESI-MS analysis. ( d ) The schematic shows the importance of the deglycosylation of the antibody for ADC production. The upper histogram represents the HIC chromatogram of the native antibody. The lower histogram is the product after the conjugation reaction without performing the deglycosylation step. The chromatogram shows the presence of unreacted antibody with some additional product, which does not match the desired LT-025 chromatogram. ( e ) The data represent the stability of the LT-025 for 15 days. The area under the curve from the HIC chromatogram of the ADC was normalized to day 1 and plotted with increasing days. 10 μL of 1 mg/mL of each sample was injected for analysis. The data shows no significant change in the peak area, indicating the stability of the ADC.

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: Hydrophobic Interaction Chromatography, Injection, Software, Conjugation Assay

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: Calculation of DAR using UV-visible spectroscopy. (a) The absorption of spectra of LT-025, DM1 linker, and KIM-1 antibody are given. (b) The equation for the calculation of DAR. (c) Table showing the calculation of DAR using the equation by measuring the absorbance of the antibody and linker from the ADC. The extinction coefficients of the antibody and the linker were calculated by measuring the absorbance at known concentrations of antibody and linker. The absorption maxima of the linker and antibody were obtained as 254 nm, and 280 nm, respectively. The DAR was finally calculated by the ratio of C drug to C mAb. Notation ‘l’ in the equation corresponds to the path length, which was considered as 1 cm.

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: Spectroscopy

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: ( a ) The antigen-binding capacity of LT-025, and the KIM-1 antibody was studied using ELISA. The immobilization of the LT-025 was checked in a KIM-1 protein-coated ELISA plate. The presence of the LT-025 was measured using an HRP-conjugated secondary antibody. The data shows a dose-dependent increase in the absorption, representing the successful antigen binding by the LT-025. The antigen recognition of the LT-025 shows a similar dose response curve to that of the monoclonal KIM-1 antibody, signifying no loss of the antigen-binding capability of the ADC. ( b ) The comparison of the binding of LT-025 alone with ADC in the presence of KIM-1 free antibody was studied using ELISA. The data shows a 50% reduction in the immobilization of both ADC in the presence of free antibody. ( c ) Quantitative estimation of the antigen binding of the LT-025 using Bio-layer interferometry (BLI). The data shows dose-dependent increase in the association and dissociation events. ( d ) Comparison of the antigen binding of LT-025 and KIM-1 antibody using BLI. 50 nM of each sample was used for the study. ( e ) Equilibrium constants and rate constants of ADC and the antibody (K D : Dissociation equilibrium constant, k a : association rate constant, and k dis : dissociation rate constant.

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: (a) Schematic structure of KIM-1 Cy5 AFC and the structure of Cy5 linker (b) Characterization data of AFC using HIC. Method used for the HPLC is a gradient method from 100 % of 25 mM phosphate buffer pH 7 with 1.5 M ammonium sulphate to 100 % of 25 mM phosphate buffer pH 7 with 20 % isopropanol. The absorption was recorded at 280 nm and 649 nm (absorption maxima of Cy5 linker). The distinguished peak in the 649 nm chromatogram represents the presence of the Cy5 fluorophore in the AFC.

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques:

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: ( a ) Schematic representation of the synthesis of an antibody fluorophore conjugate (AFC) by using the MTGase reaction as described for the ADC. First, the native monoclonal antibody was deglycosylated in the presence of the PNGase F enzyme, and then the Cy5-fluorophore-linker was attached to the antibody at Q295 in the presence of the MTGase. ( b ) SDS-PAGE (fluorescence imaging and Coomassie staining) of KIM-1 antibody, deglycosylated antibody, LT-025, and Cy5-AFC. The data shows attachment of the Cy5 fluorophore in the heavy chain of the antibody. No conjugation of Cy5 was observed in the light chain. ( c ) Schematic representation of the cellular internalization of the ADC and AFC. The AFC first binds with the KIM-1 receptor on the cell surface, and it gets endocytosed into the cell and is transported to the lysosomal compartment. ( d ) Internalization study of Cy5 AFC at different time points. 50 μg/ml of AFC. An increase in MFI (Cy5) over time means increased binding/internalization of AFC onto the Renca RCC cell line. ( e ) Confocal microscopy images showing the internalization of the AFC in Renca cells. The images show successful cellular internalization of AFC (50 μg/ml) within 0.5 h and 4 h. An increased cellular internalization and colocalization at the lysosomal compartment were observed in 4 h. Scale bar = 5 μm.

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: SDS Page, Fluorescence, Imaging, Staining, Conjugation Assay, Binding Assay, Confocal Microscopy

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: Characterization of transduction of KIM-1 on RENCA cells using ( a ) Western blotting and ( b ) flow cytometry. The mean fluorescence intensity (MFI) was plotted in the bar graph. FITC conjugated anti-mouse KIM-1 antibody was used to probe the KIM-1 expression ( c ) The KIM-1 AFC internalization in Normal Renca and Renca KIM-1++ by flow cytometry. The histogram shows a notable increase in the cellular internalization both at 1 and 4 h. ( d ) The bar graph represents the MFI of AFC internalization in Renca and Renca KIM-1++ by flow cytometry. The data show a significant increase in cellular internalization. The data were represented as mean ± SEM (n = 3, two-way ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: Transduction, Western Blot, Flow Cytometry, Fluorescence, Expressing, Comparison

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: Cell viability and IC 50 of ADC on ( a ) normal Renca and ( b ) Renca KIM-1++, obtained by MTT assay. The LT-025 exhibits significant cell death with the IC 50 values in the nanomolar range. A lower IC 50 value was observed in Renca KIM-1++ than in normal Renca because of increased receptor-mediated cellular internalization of the ADC. The data were represented as mean ± SEM (n = 3). ( c ) Comparison of the cell viability of LT-025, free antibody, and the KIM-1 MMAE ADC by MTT assay. The data showed significantly higher cell death by LT-025 in comparison to free KIM-1 antibody and KIM-1 MMAE ADC. The data were represented as mean ± SEM (n = 3, two-way ANOVA followed by Tukey’s multiple comparison test). ( d ) Confocal images of Renca with and without treatment of LT-025 (100 μg/ml) displaying tubulin disruption. The images showed disruption of the tubulin (green) crosslinking by DM1, a known microtubule inhibitor. Sacale bar = 10 μm. ( e ) Quantification of tubulin in untreated and LT-025 treated cells from the confocal images. The mean pixel intensity of the tubulin (green) was plotted after normalizing with the mean pixel intensity of actin (red). The data were represented as mean ± SEM (n = 23, Unpaired t -test with Welch’s correction). ( f ) Quantification of the area of the control and treated Renca cells. The data showed a significant reduction in the area of the cells. The data were represented as mean ± SEM (n = 23, Unpaired t -test with Welch’s correction). ( g ) The bar plot showing the % of apoptotic cells measured with flow cytometry using Annexin-V AF645 apoptosis kit. The data represent significantly higher apoptosis in normal Renca by LT-025 compared to the control and sunitinib-treated group. (n = 3, one-way ANOVA followed by Tukey’s multiple comparison test). ( h ) The bar plot showing the % of apoptotic sunitinib-resistant Renca measured with flow cytometry using Annexin V-AF645 apoptosis kit. The data represent significantly higher apoptosis in sunitinib-resistant Renca by treatment with dual therapy (LT-025 + sunitinib) compared to the control and monotherapy. 12.5 μM of sunitinib and 100 nM of ADC were used for the study (n = 3, one-way ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: MTT Assay, Comparison, Disruption, Control, Flow Cytometry

Journal: Cancer Research

Article Title: The Myc–miR-17∼92 Axis Blunts TGFβ Signaling and Production of Multiple TGFβ-Dependent Antiangiogenic Factors

doi: 10.1158/0008-5472.can-10-2412

Figure Lengend Snippet: Figure 1. Clusterin inhibits tumor growth and angiogenesis. A, growth of subcutaneous tumors derived from RasMyc cells transduced with a retrovirus expressing murine clusterin and/or the puromycin resistance gene. Inset, immunoblot of clusterin levels in Ras and RasMyc colonocytes. B, growth of subcutaneous tumors derived from HCT116 cells transduced with an empty vector or the retrovirus expressing human clusterin. Regression lines represent average rates of growth. The P value refers to the difference in regression coefficients. Inset, immunoblot of clusterin levels in transduced cells. C, in vivo bioluminescent imaging of representative animals from the experiment in B. Mice were photographed at days 3 and 21 after injection. No less than five animals per group were used in all these experiments. D, immunohistochemical staining of Ki-Ras and Ki-Ras/Myc tumors with an anti-clusterin antibody. Cytoplasmic staining for clusterin is depicted in brown whereas nuclei are counterstained in blue. E, alignment of the second TSR of murine thrombospondin-1 with the C9 protein and clusterin. F, hemoglobin content of Matrigel pellets 7 d after injection. Matrigels were admixed with p53-null mouse colonocytes transduced with either empty vector (blue bar) or clusterin retrovirus (red bar) and injected s.c. into syngeneic host animals. G, H&E staining of RasMyc/vector (top) and RasMyc/clusterin (bottom) tumor sections. Perfused blood vessels contain numerous RBC, which could be clearly seen under higher magnification (inset). The scatter plot represents densities of perfused blood vessels in four individual tumors. H, immunohistochemical staining of control (top) and clusterin-overexpressing (bottom) HCT116 tumor sections with antibody recognizing the endothelial cell surface antigen CD31. The scatter plot represents the densities of CD31-positive blood vessels in four individual tumors.

Article Snippet: For thrombospondin-1 expression is, either cell lysates or conditioned media were used. ranes were probed with antibodies to clusterin, , TGFBR2 (Santa Cruz Biotechnology and Abcam), 4 (Santa Cruz Biotechnology), Smad2 and Smad3 ogen), phosphorylated Smad3 (Cell Signaling), and (Ab-11, Lab Vision) according to the recommendaof the manufacturer.

Techniques: Derivative Assay, Transduction, Expressing, Western Blot, Plasmid Preparation, In Vivo, Imaging, Injection, Immunohistochemical staining, Staining, Control

Journal: Cancer Research

Article Title: The Myc–miR-17∼92 Axis Blunts TGFβ Signaling and Production of Multiple TGFβ-Dependent Antiangiogenic Factors

doi: 10.1158/0008-5472.can-10-2412

Figure Lengend Snippet: Figure 2. Clusterin is regulated by miR-17~92 via the TGFβ pathway. A, immunoblotting analysis of clusterin expression levels in the following cell lines. Left, Ras-only mouse colonocytes transduced with either empty vector (Ras/Puro) or the miR-17~92–encoding retrovirus (Ras/miR-17~92). Right, RasMyc cells transfected with scrambled or anti–miR-17~92 2′-O-methyl oligoribonucleotides. B, changes in expression levels of thrombospondin-1 (THBS1) and clusterin mRNAs in HCT116 Dicerhypo (left) and A172 (right) cells after transfection with the indicated microRNA mimics. mRNA levels in HCT116 and A172 cells were profiled using Affymetrix microarrays and qPCR as described in Materials and Methods. C, activation of TGFβ signaling in Ras colonocytes. Ras cells treated with vehicle or 10 ng/mL of TGFβ1 for 30 min were analyzed by immunoblotting for phosphorylated Smad3 (pSmad3) and total Smad3. D, measurement of TGFβ effects on TSR proteins. Ras cells were treated with increasing doses of TGFβ1 for 48 h and lysates were immunoblotted for clusterin and CTGF proteins. Tsp-1 was detected in conditioned medium. E, immunoblotting analysis of TSR proteins in Ras/vector, Ras/miR-17~92 or c-Myc cells cultured in the absence or presence of TGFβ1 (5 ng/mL) for 48 h. F, CLU mRNA levels in Ras/vector and Ras/17~92 cells before and after (24 h) stimulation with TGFβ, as measured by qPCR. Expression levels of CLU are adjusted to those of glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: For thrombospondin-1 expression is, either cell lysates or conditioned media were used. ranes were probed with antibodies to clusterin, , TGFBR2 (Santa Cruz Biotechnology and Abcam), 4 (Santa Cruz Biotechnology), Smad2 and Smad3 ogen), phosphorylated Smad3 (Cell Signaling), and (Ab-11, Lab Vision) according to the recommendaof the manufacturer.

Techniques: Western Blot, Expressing, Transduction, Plasmid Preparation, Transfection, Activation Assay, Cell Culture

Journal: Cancer Research

Article Title: The Myc–miR-17∼92 Axis Blunts TGFβ Signaling and Production of Multiple TGFβ-Dependent Antiangiogenic Factors

doi: 10.1158/0008-5472.can-10-2412

Figure Lengend Snippet: Figure 3. miR-17~92 targets endogenous TGFβ receptor II. A, luciferase sensor assay. Constructs tested were psiCHECK-2 derivatives containing a single miR-17/20a binding site from TGFBR2 3′-UTR in either wild-type (wt) or seed-mutated (mut) conformation. Cells were additionally cotransfected with miR-17 or control mimic. Results are expressed as ratios of renilla to firefly luciferase, the latter being constitutively expressed from the same vector and serving as a control for transfection efficiency. Sequence alignment corresponds to positions 268 to 274 of TGFBR2 3′-UTR and mature hsa-miR-17. Arrows indicate mutated nucleotides. B, expression levels of TGFBR2 mRNA in DLD1 Dicerhypo cells 10 h after transfection with microRNA mimics (25 nmol/L). mRNA levels were quantified by microarray. C, immunoblotting analysis of TGFBR2 and clusterin expression levels in Ras cells transfected with 10 nmol/L of nontargeting siRNA pool or siRNA pool targeting mouse TGFBR2. D and E, immunoblotting analysis of TGFBR2 and Smad3 in Ras cells 20 h after transfection with microRNA mimics (25 nmol/L). Bottom, quantitations of Western blots above.

Article Snippet: For thrombospondin-1 expression is, either cell lysates or conditioned media were used. ranes were probed with antibodies to clusterin, , TGFBR2 (Santa Cruz Biotechnology and Abcam), 4 (Santa Cruz Biotechnology), Smad2 and Smad3 ogen), phosphorylated Smad3 (Cell Signaling), and (Ab-11, Lab Vision) according to the recommendaof the manufacturer.

Techniques: Luciferase, Construct, Binding Assay, Control, Plasmid Preparation, Transfection, Sequencing, Expressing, Microarray, Western Blot

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: miR-199 Family Contributes to Regulation of Sonic Hedgehog Expression During Craniofacial Development

doi: 10.1002/dvdy.191

Figure Lengend Snippet: A) RNA-sequencing of chicken ectoderm (n=4/stage, each sample was pooled from 2 individuals) before (HH18) and after (HH22) SHH expression in the FEZ, showing many differentially expressed miRNAs in the chick between these two stages. B) Microarray clustering shows differentially expressed miRNAs from chick ectoderm (n=4/treatment, each sample was pooled from 8 individuals; mean data shown) during three experiments: HH18 and HH22, and after activating (SHH-N) or blocking (5E1) SHH in the neural tube. Only the miR-199 family correctly correlates to expected changes in SHH. hsa: Homo sapiens, dre: Danio rerio, gga: Gallus gallus.

Article Snippet: 5 , 60 SHH-N treatment was performed using affi-Gel Blue beads (50 – 100 mesh, 200 – 250 diameter; Biorad) soaked in recombinant SHH-N protein (400 ng/ml in PBS with 0.1% bovine serum albumin (BSA); Ontogeny) or 0.1% BSA (control) at 37°C for one hour.

Techniques: RNA Sequencing Assay, Expressing, Microarray, Blocking Assay

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: miR-199 Family Contributes to Regulation of Sonic Hedgehog Expression During Craniofacial Development

doi: 10.1002/dvdy.191

Figure Lengend Snippet: DF1 cells respond to exogenous SHH (+ SHH-N) by increasing GLI1 and PTC expression; however, infection with RCAS-miR-199b (+ SHH-N + RCAS-miR-199b), reduces SHH signal transduction as seen by a significant decrease in GLI1 and PTC expression. Error bars represent standard error (4 biological replicates). Significance is based on a two-way ANOVA with Tukey’s multiple comparisons test. * : p<0.05; ** : p<0.01.

Article Snippet: 5 , 60 SHH-N treatment was performed using affi-Gel Blue beads (50 – 100 mesh, 200 – 250 diameter; Biorad) soaked in recombinant SHH-N protein (400 ng/ml in PBS with 0.1% bovine serum albumin (BSA); Ontogeny) or 0.1% BSA (control) at 37°C for one hour.

Techniques: Expressing, Infection, Transduction

Journal: iScience

Article Title: Sirtuin 2 regulates neutrophil functions through NAD + synthesis pathway in virus infection

doi: 10.1016/j.isci.2024.110184

Figure Lengend Snippet: SIRT2 expression is associated with NAD + synthesis and neutrophil function during virus infection (A) C57BL/6 mice were infected with or without the PR8 virus for 48 h, after which the lungs were collected. RNA was analyzed via microarray analysis to compare the expression profiles of control and virus-infected cells from the lung with those of certain genes involved in surface and intracellular signaling pathways. (B) The expression of the indicated molecules was examined via qPCR analyses of neutrophils in BALF from PBS (ctrl)-treated or virus-infected mice (the expression in the control groups was set to 1). (C) Expressions of SIRT2 and NAMPT in neutrophils in BALF from PBS (ctrl)- or virus (virus)-infected mice by flow cytometry. (D) Sirt2 mRNA expression was examined by qPCR analyses in the indicated cell populations isolated from BALF from virus-infected mice. (E) Sirt2 and Nampt mRNA expression in neutrophils isolated from BALF was examined via qPCR at different time points following virus infection in mice. (F and G) Changes in the NAD + (F) and Qprt mRNA levels (G) in neutrophils isolated from BALF at different time points following virus infection. (H and I) The correlation between the percent of TNF-α + cells among neutrophils (H) or the dry/wet weight ratio of the lung (I) and the expression of Sirt2 in neutrophils during virus infection. The data are representative of three or four independent experiments ( n = 4–10 mice per group). ∗∗ p < 0.01 and ∗∗∗ p < 0.001, compared with the indicated groups.

Article Snippet: SIRT2 deficiency inhibits neutrophil functions through the QA-QPRT-NAD + synthesis pathway (A–F) WT mice were challenged with the PR8 virus at 48 h and treated with PBS (vehicle), AGK2 (5 mg/kg, Selleck), AGK2+FK866 (10 mg/kg, Selleck) or AGK2+FK866+QA (10 mg/kg, Selleck) (A). (B) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with the PR8 virus. (C) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with the PR8 virus. (D) Flow cytometry analysis of CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (E) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (F–I) Human CD34 + hematopoietic stem cells were induced with G-CSF (10 ng/mL, Sigma) for 7 days.

Techniques: Expressing, Virus, Infection, Microarray, Control, Protein-Protein interactions, Flow Cytometry, Isolation

Journal: iScience

Article Title: Sirtuin 2 regulates neutrophil functions through NAD + synthesis pathway in virus infection

doi: 10.1016/j.isci.2024.110184

Figure Lengend Snippet: SIRT2 deficiency limits neutrophil infiltration and function in ameliorating viral infection-related inflammation WT and Sirt2 −/− mice were challenged with PBS (ctrl) or subjected to PR8 virus infection for 48 h. Clinical score of infected mice (A) and the ratio of dry and wet weights of lungs (B) from mice. (C) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with PBS or the PR8 virus. (D) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with PBS or the PR8 virus. (E) Flow cytometry analysis of CD11b + Ly6G + cells in the bronchoalveolar lavage fluid (BALF) or lungs of WT and Sirt2 −/− mice challenged with PBS (ctrl) or PR8 virus infection for 48 h. Dot plots present representative flow cytometry data. (F) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT and Sirt2 −/− mice challenged with PBS (ctrl) or PR8 virus infection for 48 h. Dot plots present representative flow cytometry data. (G) NETs from CD11b + Ly6G + neutrophils were isolated from BALF from WT and Sirt2 −/− mice at 48 h after PR8 infection. Typical NET images are displayed. Scale bars, 10 μm; original magnification, 630×. (H) Intracellular staining of CitH3 in Ly6G + cells in BALF from WT and Sirt2 −/− mice at 48 h after PR8 infection. Dot plots present representative flow cytometry data (upper panel), and the statistical results are shown (lower panel). (I) Western blot analysis of the CitH3 in Ly6G + cells sorted from BALF from WT and Sirt2 −/− mice 48 h after challenge with PBS (ctrl) or PR8 infection. The data are representative of three or four independent experiments ( n = 3–6 mice per group). ∗∗∗ p < 0.001, compared with the indicated groups.

Article Snippet: SIRT2 deficiency inhibits neutrophil functions through the QA-QPRT-NAD + synthesis pathway (A–F) WT mice were challenged with the PR8 virus at 48 h and treated with PBS (vehicle), AGK2 (5 mg/kg, Selleck), AGK2+FK866 (10 mg/kg, Selleck) or AGK2+FK866+QA (10 mg/kg, Selleck) (A). (B) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with the PR8 virus. (C) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with the PR8 virus. (D) Flow cytometry analysis of CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (E) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (F–I) Human CD34 + hematopoietic stem cells were induced with G-CSF (10 ng/mL, Sigma) for 7 days.

Techniques: Infection, Virus, Staining, Immunohistochemical staining, Flow Cytometry, Isolation, Western Blot

Journal: iScience

Article Title: Sirtuin 2 regulates neutrophil functions through NAD + synthesis pathway in virus infection

doi: 10.1016/j.isci.2024.110184

Figure Lengend Snippet: CXCR2 is critically involved in regulating neutrophil function induced by Sirt2 −/− during virus infection Percent (A) and mean fluorescent intensity (MFI; B) of CXCR2 surface expressions among CD11b + Ly6G + neutrophils were isolated from bronchoalveolar lavage fluid (BALF) of WT and Sirt2 −/− mice at 48 h after challenge with PBS (ctrl) or PR8 infection. Representative flow cytometry data (left) and statistical results are shown (right). (C–J) WT and Sirt2 −/− mice were challenged with PBS (ctrl) or PR8 virus infection for 48 h with a CXCR2 inhibitor (CXCR2i; SB265610, 5 mg/kg, intraperitoneal injected daily; Tocris Biosciences) or vehicle (PBS). Clinical score of infected mice (C) and the ratio of dry and wet weights of lungs (D) from mice. Percent of CD11b + Ly6G + cells in the BALF (E) and lungs (F) of mice. Intracellular staining of TNF-α in Ly6G + cells in the BALF (G) and lungs (H) of mice. (I) NETs from CD11b + Ly6G + neutrophils were isolated from the BALF of mice. The percent of NET + cells were quantified. (J) Intracellular staining of CitH3 in Ly6G + cells in BALF from mice. The data are representative of three or four independent experiments ( n = 3–4 mice per group). ∗∗ p < 0.01 and ∗∗∗ p < 0.001, compared with the indicated groups.

Article Snippet: SIRT2 deficiency inhibits neutrophil functions through the QA-QPRT-NAD + synthesis pathway (A–F) WT mice were challenged with the PR8 virus at 48 h and treated with PBS (vehicle), AGK2 (5 mg/kg, Selleck), AGK2+FK866 (10 mg/kg, Selleck) or AGK2+FK866+QA (10 mg/kg, Selleck) (A). (B) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with the PR8 virus. (C) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with the PR8 virus. (D) Flow cytometry analysis of CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (E) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (F–I) Human CD34 + hematopoietic stem cells were induced with G-CSF (10 ng/mL, Sigma) for 7 days.

Techniques: Virus, Infection, Isolation, Flow Cytometry, Injection, Staining

Journal: iScience

Article Title: Sirtuin 2 regulates neutrophil functions through NAD + synthesis pathway in virus infection

doi: 10.1016/j.isci.2024.110184

Figure Lengend Snippet: Epithelial cells help neutrophils use QA for NAD + synthesis induced by Sirt2 −/− following virus infection (A) TDO and 3-HA in the NAD + synthesis pathway. (B) Single-cell suspension was prepared from lung tissue. Mass spectrometry analysis of the metabolite abundance of 3-HA in neutrophils from BALF and in lung tissue from WT and Sirt2 −/− mice challenged with the PR8 virus at 48 h. (C) Mass spectrometry analysis of the metabolite abundance of 3-HA in Epcam1 + and Epicam1 - cells from lung tissue from WT and Sirt2 −/− mice challenged by PR8 virus infection at 48 h. (D) Flow cytometry analysis of TDO expression in neutrophils and epithelial cells in the lungs of WT and Sirt2 −/− mice challenged with the PR8 virus at 48 h. (E) Ido1/2 and Tdo mRNA expression of neutrophils or cells from lung tissue from WT and Sirt2 −/− mice challenged by PR8 virus infection at 48 h. (F–H) Neutrophils were isolated from the spleens of WT and Sirt2 −/− mice cocultured with vehicle (in PBS), epithelial cells or epithelial cells treated with a TDO inhibitor (LM10, 10 nM, MCE) and stimulated with virus for different durations. Ratio of the NAD + level in Sirt2 −/− and WT mice (G) and the ratio of TNF-α + cells in neutrophils from Sirt2 −/− and WT mice (H). The data are representative of three or four independent experiments ( n = 3–4 mice per group). ∗∗ p < 0.01 and ∗∗∗ p < 0.001, compared with the indicated groups. n.s., not significant.

Article Snippet: SIRT2 deficiency inhibits neutrophil functions through the QA-QPRT-NAD + synthesis pathway (A–F) WT mice were challenged with the PR8 virus at 48 h and treated with PBS (vehicle), AGK2 (5 mg/kg, Selleck), AGK2+FK866 (10 mg/kg, Selleck) or AGK2+FK866+QA (10 mg/kg, Selleck) (A). (B) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with the PR8 virus. (C) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with the PR8 virus. (D) Flow cytometry analysis of CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (E) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (F–I) Human CD34 + hematopoietic stem cells were induced with G-CSF (10 ng/mL, Sigma) for 7 days.

Techniques: Virus, Infection, Suspension, Mass Spectrometry, Flow Cytometry, Expressing, Isolation

Journal: iScience

Article Title: Sirtuin 2 regulates neutrophil functions through NAD + synthesis pathway in virus infection

doi: 10.1016/j.isci.2024.110184

Figure Lengend Snippet: SIRT2 deficiency inhibits neutrophil functions through the QA-QPRT-NAD + synthesis pathway (A–F) WT mice were challenged with the PR8 virus at 48 h and treated with PBS (vehicle), AGK2 (5 mg/kg, Selleck), AGK2+FK866 (10 mg/kg, Selleck) or AGK2+FK866+QA (10 mg/kg, Selleck) (A). (B) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with the PR8 virus. (C) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with the PR8 virus. (D) Flow cytometry analysis of CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (E) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (F–I) Human CD34 + hematopoietic stem cells were induced with G-CSF (10 ng/mL, Sigma) for 7 days. Neutrophils were treated with AGK2 (0–10 μM, Selleck) at the indicated dose and stimulated with virus for 24 h (F). (G) Sirt2 mRNA expression in neutrophils. (H) TNF-α production in the supernatant, as determined by ELISA. (I) Qprt and Nampt mRNA expression in neutrophils. The data are representative of three or four independent experiments ( n = 3 per group). ∗ p < 0.05 and ∗∗∗ p < 0.001, compared with the indicated groups.

Article Snippet: SIRT2 deficiency inhibits neutrophil functions through the QA-QPRT-NAD + synthesis pathway (A–F) WT mice were challenged with the PR8 virus at 48 h and treated with PBS (vehicle), AGK2 (5 mg/kg, Selleck), AGK2+FK866 (10 mg/kg, Selleck) or AGK2+FK866+QA (10 mg/kg, Selleck) (A). (B) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with the PR8 virus. (C) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with the PR8 virus. (D) Flow cytometry analysis of CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (E) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (F–I) Human CD34 + hematopoietic stem cells were induced with G-CSF (10 ng/mL, Sigma) for 7 days.

Techniques: Virus, Staining, Immunohistochemical staining, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: Lz expansion is associated with increasing levels of circulating and endothelial IGF2 (A) Weights of micro-dissected Lz. (B) Linear correlation analyses between fetal and Lz weights: p = 0.002 (E14), p < 0.0001 (E16), and p < 0.0001 (E19) (n = 46–189 placentae from n > 10 L per group in [A] and [B]). (C) Levels of IGF2 (ng/mL) in plasma of wild-type fetuses. (D) Linear correlation analyses between fetal weights and circulating IGF2: p < 0.0001 (E16 and E19) (n = 70–79 per group in [C] and [D]). (E) Igf2 mRNA in situ hybridization (blue) in E14 wild-type Lz (red arrows—FPEC, feto-placental endothelial cells; AS, antisense probe; inset with S, sense probe; scale bars, 20 μm). (F) Relative Igf2 mRNA expression levels measured by qRT-PCR in FPEC from wild-type Lz (n = 6–7 per group). (G) Imprinted genes that rank within top 100 expressed genes in E16 wild-type FPEC (FPKM, fragments per kilobase million; n = 4). (H) Double immunostaining for IGF2 and CD31 in E19 wild-type placenta. Endothelial cells are very thin and hard to detect except where the cytoplasm is more voluminous around the nucleus, with intense IGF2 stain (white arrows). Transmembrane glycoprotein CD31 immunostaining is in the membrane and largely marks endothelial intercellular junctions (scale bars, 20 μm). (I) Semi-quantitative measurement of IGF2 protein in FPEC versus trophoblast cells (E19 wild-type Lz, n = 60 cells per group from two placentae). White arrows—endothelial cells; scale bars, 50 μm. For (E), (H), and (I): FC, fetal capillaries; MBS, maternal blood spaces; LT, labyrinthine trophoblast cells; S-TGC, sinusoidal trophoblast giant cells. Data in (A), (C), (F), (G), and (I) are presented as averages ± standard deviation (SD); ∗∗∗ p < 0.001 calculated by one-way ANOVA plus Tukey’s multiple comparisons test in (A) and (F) or by unpaired t test with Welch’s correction in (C) and (I). See also .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Clinical Proteomics, In Situ Hybridization, Expressing, Quantitative RT-PCR, Double Immunostaining, Staining, Immunostaining, Membrane, Standard Deviation

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: Deletion of Igf2 in the epiblast or endothelium impairs Lz expansion (A) Left: schematic of Igf2 expression in conceptuses with conditional deletion driven by Meox2 Cre . Right: immunostaining for YFP (green) in a representative fetus and placenta paraffin section at E12 of gestation, double transgenic for Meox2 Cre and Rosa26 fl STOP fl YFP 10 reporter. YFP expression in the placenta is localized to the Lz and Cp (high magnification, inset). Blue—DAPI stain for nuclei; scale bars: 1 mm (low magnification) and 100 μm (high magnification). (B) Fetal and placental growth kinetics, measured as average wet-weights for each genotype per litter (E12: n = 10 L [n = 41 controls {C} and n = 32 Igf2 EpiKO ]; E14: n = 25 L [n = 114 C and n = 88 Igf2 EpiKO ]; E16: n = 37 L [n = 154 C and n = 127 Igf2 EpiKO ]; E19: n = 37 L [n = 164 C and n = 121 Igf2 EpiKO ]). (C) Absolute volumes of the placental layers (Db, decidua basalis; Jz, junctional zone; Lz, labyrinthine zone; Cp, chorionic plate), measured by stereology (n = 6 per group). (D) Absolute volumes of Lz components, measured by stereology (LT, labyrinthine trophoblast; MBSs, maternal blood spaces; FCs, fetal capillaries) (n = 6 per group). (E) Left: schematic representation of Igf2 expression in conceptuses with conditional deletion driven by Tek Cre . Right: representative confocal microscopy of frozen sections from a fetus and its corresponding placenta, double transgenic for TeK Cre and Ai9(RCL-tdT) reporter at E16 of gestation. Scale bars: 2 mm (fetus) and 1 mm (placenta). (F) Fetal and placental growth kinetics (E12: n = 5 L [n = 17 C and n = 16 Igf2 ECKO ]; E14: n = 8 L [n = 26 C and n = 34 Igf2 ECKO ]; E16: n = 13 L [n = 60 C and n = 46 Igf2 ECKO ]; E19: n = 7 L [n = 31 C and n = 27 Igf2 ECKO ]). (G) Absolute volumes of the placental layers measured by stereology (n = 5–7 per group). (H) Absolute volumes of Lz components, measured by stereology (n = 5–7 per group). (I) Double immunostaining for EPCAM (epithelial cell adhesion molecule) (red) and MCT1 (monocarboxylate transporter 1) (green) in a representative frozen placental section at E12 of gestation. EPCAM expression is observed as clusters of positive cells within the Lz placenta. Blue—DAPI (4′,6-diamidino-2-phenylindole) stain for nuclei; scale bars: 500 μm (left panel) and 20 μm (right panel). (J) Analysis of EPCAM high -positive cells by flow cytometry. Left panel: example of gating used to identify EPCAM high -positive cells (the viability dye 7-aminoactinomycin D [7-AAD] was used to exclude dead cells). Right: quantification of placental EPCAM high -positive cells at E12 in conceptuses with conditional Igf2 deletion driven by Meox2 Cre (n = 10 C and n = 9 Igf2 EpiKO from 2 L) or Tek Cre (n = 8 C and n = 8 Igf2 ECKO from 2 L). For all graphs data are shown as averages; error bars represent SD in (C), (D), (G), (H), and (J) or 95% confidence intervals (95% CI) in (B) and (F); N.S.—statistically not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 calculated by a mixed effects model in (B) and (F) (see STAR Methods), two-way ANOVA plus Sidak’s multiple comparisons tests in (D) and (H) or unpaired t tests in (C), (G), and (J). See also .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Expressing, Immunostaining, Paraffin Section, Transgenic Assay, Staining, Confocal Microscopy, Double Immunostaining, Flow Cytometry

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: Lack of fetus-derived IGF2 reduces the expansion of feto-placental microvasculature in late gestation (A) Functions enriched in DEGs at E19. (B) qRT-PCR analysis of angiopoietin-Tie2/TEK signaling components in Lz (n = 6–8 per group). (C) TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining in E16 Lz (arrows point to apoptotic cells) and data quantification (n = 6 samples per group); scale bars, 50 μm. (D) Left: representative double immunostaining for TUNEL (red) and laminin (green, marker of feto-placental capillaries) in the Lz of an E16 Igf2 EpiKO mutant placenta (DAPI, blue marks the nuclei; white and red arrows indicate TUNEL + FPECs and LT, respectively; scale bars, 25 μm). Right: quantification of TUNEL + cells that are positive or negative for laminin (n = 6 Igf2 EpiKO mutant placentae). (E) Feto-placental endothelial cell (FPEC) proliferation measured by flow cytometry (left—representative histograms at E16; right—data quantification; n = 4–11 per group). (F) qRT-PCR analysis of Adgre1 in Lz. (G) Representative F4/80 immunostainings in E16 Lz (arrows indicate macrophages). Scale bars, 100 μm. Right: percentage of macrophages/Lz at E16 (n = 6–8 samples per group). (H) Representative CD31 immunostaining in Lz (scale bars, 100 μm). (I) qRT-PCR analysis for SynT-II (syncytiotrophoblast layer II) marker genes. For all graphs, data are presented as averages or individual values; error bars are SD; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by two-way ANOVA plus Sidak’s multiple comparisons tests in (B), (C), (E), (F), and (I) or Mann-Whitney tests in (G). See also Figure S4 and .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Derivative Assay, Quantitative RT-PCR, TUNEL Assay, Staining, Double Immunostaining, Marker, Mutagenesis, Flow Cytometry, Immunostaining, MANN-WHITNEY

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: Genetic models of mismatched placental and fetal growth reveal circulating IGF2 as a major endocrine regulator of FPEC and Lz expansion (A–E) Column 1: schematic diagrams of the genetic models: Igf2 EpiKO (A), Igf2 ECKO (B), Igf2 TrKO (C), Igf2 UbKO (D), and H19 -DMD EpiKO (E). Columns 2 and 3: total numbers (column 2) and proportion of FPEC/Lz (column 3), measured by flow cytometry (n conceptuses per group: Igf2 EpiKO : n = 9–18; Igf2 ECKO : n = 5–11; Igf2 TrKO : n = 6–17; Igf2 UbKO : n = 3–26; H19 -DMD EpiKO : n = 9–15). Column 4: Lz growth kinetics ( Igf2 EpiKO : n = 9–20 L; Igf2 ECKO : n = 3–9 L; Igf2 TrKO : n = 4–9 L; Igf2 UbKO : n = 3–8 L; H19 -DMD EpiKO : n = 3–4 L). Column 5: IGF2 levels (ng/mL) in plasma (n per group: Igf2 EpiKO : n = 12; Igf2 ECKO : n = 9; Igf2 TrKO : n = 6–7; Igf2 UbKO : n = 7–11; H19 -DMD EpiKO : n = 9). Data are shown as averages or individual values and error bars are SD (columns 2, 3, and 5) and 95% CI (column 4). N.S.—not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 calculated by two-way ANOVA plus Sidak’s multiple comparisons tests (second and third columns), mixed effects model (fourth column) or Mann-Whitney tests (fifth column). See also A, S5B, and .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Flow Cytometry, Clinical Proteomics, MANN-WHITNEY

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: IGF2 signaling regulates angiogenic properties of endothelial cells (A) Volcano plot representation of DEGs identified by RNA-seq in E16 FPEC ( Igf2 EpiKO versus controls). Significant upregulated and downregulated DEGs (false discovery rate [FDR] < 0.05) are shown with red and blue, respectively. (B) Top scoring biological processes enriched in DEGs. Biologically validated DEGS are listed in parentheses. The dotted line corresponds to FDR-corrected p value of 0.05. (C) Biological validation. Data are shown as averages (n = 11–12 samples per group); error bars are SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 calculated by Mann-Whitney tests. (D) Volcano plot representation of DEGs identified by RNA-seq in E16 FPEC ( Igf2 ECKO versus controls). Significant upregulated and downregulated DEGs (FDR < 0.05) are shown with red and blue, respectively. (E) Transcription factors (TFs) identified by analysis of motif enrichment (AME). (F) IPA regulatory network built with the four TFs identified using AME analysis. Proteins labeled with a star are known regulators of angiogenesis (angiostatic or pro-angiogenic factors) and key references are listed in . See also C and S5D and and .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: RNA Sequencing, Biomarker Discovery, MANN-WHITNEY, Labeling

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: IGF2 Acts on FPECs via IGF2R-ERK signaling ex vivo (A) Primary FPEC isolated from E16 Lz: D0—freshly isolated cells; D10—FPEC at passage one (P1, 10 days of culture). (B) Confocal imaging of passage one FPEC, stained for CD31 (scale bars, 20 μm). (C) Flow cytometry analysis of P1 FPEC stained for CD31, demonstrating that these are almost exclusively CD31 + . (D) qRT-PCR analysis for Igf1r , Igf2r , and Insr in FPECs isolated by FACS (n = 6–7 per group). (E) Relative expression of the three IGF receptors in P1 FPEC. (F) qRT-PCR analysis of Igf2 mRNA levels in P1 FPEC cultured in 5% O 2 versus primary FPEC isolated from E16 Lz by FACS. (G) Schematic representation of IGF2 and IGF receptors. IGF2 Leu27 analog acts specifically on IGF2R and picropodophyllin (PPP) inhibits phosphorylation of IGF1R. (H) Representative images of capillary-like tube formation assay in primary FPEC seeded on matrigel and exposed to exogenous IGF2, IGF2 Leu27 , PPP, or PPP+IGF2 (equal seeding of cell numbers at 30 min and tube formation at 8 h), and quantification of number of nodes, branches, and total length (n = 5–6 independent experiments). (I) qRT-PCR analysis of Igf2r mRNA levels in primary FPECs upon knockdown by siRNA (n = 8 samples/group). (J) Proliferation assay of primary FPEC with or without IGF2R siRNA knockdown, in presence or absence of IGF2, on 4 consecutive days after plating. Cells with IGF2R siRNA knockdown exhibit significant proliferation defects that are further accentuated upon IGF2 treatment (n = 5 biological replicates per group). (K) qRT-PCR analysis of Angpt2 mRNA levels in primary FPECs transfected with scrambled siRNA or IGF2R siRNA, upon 4 days of treatment with 50 ng/mL mouse recombinant IGF2 (n = 8 samples/group). (L) Left side: identification of delayed ERK1/2 phosphorylation in FPECs with IGF2R siRNA knockdown upon acute treatment with 50 ng/mL mouse recombinant IGF2. HSP90 was used as internal control for protein loading. Right side: quantification of ratios pERK1/2 to total ERK1/2 for n = 3 independent biological replicates. For all graphs, data are presented as averages or individual values and error bars represent SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 calculated by a Mann-Whitney test in (F), two-way ANOVA tests with Sidak’s multiple comparisons test in (H), (J), and (L), Wilcoxon matched-pairs signed rank test in (I) and paired Student’s t test in (K).

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Ex Vivo, Isolation, Imaging, Staining, Flow Cytometry, Quantitative RT-PCR, Expressing, Cell Culture, Phospho-proteomics, Capillary Tube Formation Assay, Knockdown, Proliferation Assay, Transfection, Recombinant, Control, MANN-WHITNEY

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: IGF2 acts on FPECs via IGF2R in vivo (A) Representative double immunostaining for IGF2R (red) and CD31 (green) in Igf2r ECKO mutant and control Lz at E16 (DAPI, blue; scale bars, 25 μm). (B) Flow cytometry analysis showing that the majority (>80%) of Igf2r ECKO mutant feto-placental endothelial cells (FPECs) express YFP (n = 6–14 per genotype). (C) Fetal and placental growth kinetics in Igf2r ECKO ( Igf2r fl/+ ; Tek +/Cre ) mutants compared with Igf2r fl/+ controls (n = 8–28 conceptuses from n = 3–8 L for each developmental stage). (D) Proportion and total numbers of FPEC/Lz measured by flow cytometry (n = 6–14 per group). (E) Representative CD31 staining in E16 Lz (scale bars, 100 μm). (F) Lz growth kinetics: Igf2r ECKO (n = 8–16 conceptuses per group). (G) IGF2 levels (ng/mL) in plasma at E16 (n = 9 per group). (H) Model summarizing the proposed actions of fetus-, endothelial-, and trophoblast-derived IGF2. For all graphs, data are presented as averages or individual values and error bars represent SD in (B), (D), and (G), or 95% CI in (C) and (F). N.S.— not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 calculated by two-way ANOVA tests in (B) and (D), mixed effects model in (C) and (F) and Mann-Whitney tests in (G). See also Figure S7 and .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: In Vivo, Double Immunostaining, Mutagenesis, Control, Flow Cytometry, Staining, Clinical Proteomics, Derivative Assay, MANN-WHITNEY

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet:

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Plasmid Preparation, Recombinant, Blocking Assay, BIA-KA, Western Blot, Stripping Membranes, Reverse Transcription, SYBR Green Assay, Red Blood Cell Lysis, Staining, Enzyme-linked Immunosorbent Assay, In Situ, TUNEL Assay, Imaging, Flow Cytometry, Transfection, Gene Expression, Expressing, Microarray, Isolation, Software

Journal: Analytical Biochemistry

Article Title: An expression system for screening of proteins for glycan and protein interactions

doi: 10.1016/j.ab.2010.12.036

Figure Lengend Snippet: Gateway-based cloning and expression system. (A) An entry clone containing the leader peptide and extracellular domain (ECD) of interest is recombined via the LR reaction with a destination vector to generate an expression clone. This incorporates the ECD fused to EGFP, an Avitag biotin acceptor peptide, a PP cleavage site, and a GPI signal sequence. (B) Following expression in mammalian cells, surface-presented protein can be cleaved with PP and biotinylated with BirA either at the cell surface or following cleavage. This versatile cloning vector enables multiple assays with either cell-expressed or soluble versions of the protein of interest.

Article Snippet: The slides were rinsed with blocking buffer and overlaid for 1.5 h with the biotinylated siglec–GFP constructs that had been precomplexed for 1 h at 4 °C with AlexaFluor-647–streptavidin (Molecular Probes) (1:1, w/w) and rabbit anti-GFP (Invitrogen) (1:6, w/w).

Techniques: Cloning, Expressing, Plasmid Preparation, Sequencing

Journal: Analytical Biochemistry

Article Title: An expression system for screening of proteins for glycan and protein interactions

doi: 10.1016/j.ab.2010.12.036

Figure Lengend Snippet: Expression of EGFP fusion proteins in CHO cells measured by flow cytometry. Cell surface expression of EGFP fusion proteins was measured on living cells using biotinylated anti-GFP followed by streptavidin–APC and detected on the FL-4 channel (emission maximum 660 nm). Total endogenous GFP was detected on the FL-1 channel (emission maximum 509 nm). CHO cells were either nontransfected (CHO) or CHO cells expressing siglec-7 (S7), siglec-8 (S8), siglec-9 (S9), BACE, JAM-B, or JAM-C.

Article Snippet: The slides were rinsed with blocking buffer and overlaid for 1.5 h with the biotinylated siglec–GFP constructs that had been precomplexed for 1 h at 4 °C with AlexaFluor-647–streptavidin (Molecular Probes) (1:1, w/w) and rabbit anti-GFP (Invitrogen) (1:6, w/w).

Techniques: Expressing, Flow Cytometry

Journal: Analytical Biochemistry

Article Title: An expression system for screening of proteins for glycan and protein interactions

doi: 10.1016/j.ab.2010.12.036

Figure Lengend Snippet: Binding of polymeric glycan probes to siglec-expressing CHO cells. Either nontransfected control cells (CHO) or CHO cells expressing siglec-7 (S7), siglec-8 (S8), or siglec-9 (S9) were incubated with biotinylated PAA probes carrying either lactose, Sia2,8Sia, SLe x , or 6′SU-SLe x and binding detected with streptavidin–APC. See for carbohydrate sequences.

Article Snippet: The slides were rinsed with blocking buffer and overlaid for 1.5 h with the biotinylated siglec–GFP constructs that had been precomplexed for 1 h at 4 °C with AlexaFluor-647–streptavidin (Molecular Probes) (1:1, w/w) and rabbit anti-GFP (Invitrogen) (1:6, w/w).

Techniques: Binding Assay, Glycoproteomics, Expressing, Control, Incubation

Journal: Analytical Biochemistry

Article Title: An expression system for screening of proteins for glycan and protein interactions

doi: 10.1016/j.ab.2010.12.036

Figure Lengend Snippet: Preparation of soluble siglecs and protein–glycan interactions measured on glycan arrays. (A) Western blot of soluble material released from siglec-expressing CHO cells either treated (+) or not treated (−) with PP. Supernatants containing cleaved siglec-7–EGFP (S7), siglec-8–EGFP (S8), and siglec-9–EGFP (S9) were probed with biotinylated goat anti-GFP followed by streptavidin–alkaline phosphatase. (B) Western blot of cleaved material either pretreated (+) or not pretreated (−) with BirA enzyme and probed with streptavidin–alkaline phosphatase. (C) Dose–response microarray analysis of the binding of siglecs to lipid-linked oligosaccharide probes. Microarrays of six oligosaccharide probes (structures are shown in ) were generated on nitrocellulose-coated glass slides. Each probe was printed in duplicate at 1 ( ), 1.33 ( ), 1.66 ( ), 2.33 ( ), 3.33 ( ), and 5 ( ) fmol/spot. Binding of biotinylated siglec-7–EGFP (top panel), siglec-8–EGFP (middle panel), and siglec-9–EGFP (bottom panel) chimeras was detected with AlexaFluor-647-labeled streptavidin as described in Materials and methods.

Article Snippet: The slides were rinsed with blocking buffer and overlaid for 1.5 h with the biotinylated siglec–GFP constructs that had been precomplexed for 1 h at 4 °C with AlexaFluor-647–streptavidin (Molecular Probes) (1:1, w/w) and rabbit anti-GFP (Invitrogen) (1:6, w/w).

Techniques: Glycoproteomics, Western Blot, Expressing, Microarray, Binding Assay, Generated, Labeling

Journal: Analytical Biochemistry

Article Title: An expression system for screening of proteins for glycan and protein interactions

doi: 10.1016/j.ab.2010.12.036

Figure Lengend Snippet: Measurement of protein–glycosaminoglycan interactions. Here 8mer saccharides derived from a partial heparinase I digestion of porcine mucosal heparin were spotted at different concentrations onto GAPS II microarray slides and incubated sequentially with 20 nM biotinylated protein–EGFP followed by AlexaFluor-546-labeled streptavidin. (A) Microarray image showing duplicate spots representative of 10 replicate spots. Contrast and brightness has been adjusted to show spot signal with respect to equivalent background signal. S9, siglec-9; −, streptavidin detection reagent applied to subarrays where no protein was incubated. (B) Graphs showing mean spot intensities against saccharide spotting concentration for different proteins. Spot intensity was normalized relative to background intensity by subtraction of the local background for each spot and the mean standard deviation of these values calculated for 10 replicate spots.

Article Snippet: The slides were rinsed with blocking buffer and overlaid for 1.5 h with the biotinylated siglec–GFP constructs that had been precomplexed for 1 h at 4 °C with AlexaFluor-647–streptavidin (Molecular Probes) (1:1, w/w) and rabbit anti-GFP (Invitrogen) (1:6, w/w).

Techniques: Derivative Assay, Microarray, Incubation, Labeling, Concentration Assay, Standard Deviation

Journal: Analytical Biochemistry

Article Title: An expression system for screening of proteins for glycan and protein interactions

doi: 10.1016/j.ab.2010.12.036

Figure Lengend Snippet: Binding of biotinylated JAM-B–EGFP and JAM-C–EGFP chimeras to CHO cells expressing JAM-B and JAM-C. (A) Western blot of biotinylated JAM-C or JAM-B probed with streptavidin–alkaline phosphatase shows the presence of a single species for each protein at the expected molecular weight. (B) Wild-type CHO cells (black line) or CHO cells expressing JAM-B (red and green lines) were incubated with either biotinylated JAM-C–EGFP (black and green lines) or biotinylated JAM-B–EGFP (red line) at 1 μg/ml. Binding was detected with streptavidin–APC, and cells were analyzed by flow cytometry. (C) Wild-type CHO cells (black line) or CHO cells expressing JAM-C (red and green lines) were incubated with either biotinylated JAM-B–EGFP (black and green lines) or biotinylated JAM-C–EGFP (red line). Binding was detected with streptavidin–APC, and cells were analyzed by flow cytometry. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The slides were rinsed with blocking buffer and overlaid for 1.5 h with the biotinylated siglec–GFP constructs that had been precomplexed for 1 h at 4 °C with AlexaFluor-647–streptavidin (Molecular Probes) (1:1, w/w) and rabbit anti-GFP (Invitrogen) (1:6, w/w).

Techniques: Binding Assay, Expressing, Western Blot, Molecular Weight, Incubation, Flow Cytometry