Journal: Cardiovascular Research

Article Title: Vascular endothelial tissue factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis

doi: 10.1093/cvr/cvab263

Figure Lengend Snippet: Effects of TMAO exposure on TF and VCAM1 expression in human endothelial cells. HMEC-1 was left untreated or exposed to 200 µM TMAO for 2, 4, and 6 h and mRNA expression for (A) flTF, (B) asTF, and (C) VCAM1 analysed. In addition, HMEC-1 was treated with vehicle or TMAO at different concentrations as indicated for 2 h and mRNA expression of (D) flTF, (E) asTF, and (F) VCAM1 assessed. (G) Protein amounts of flTF, asTF, and VCAM1 in HMEC treated with 200 µM TMAO for 6 h quantified via western blot. Human monocytic THP-1 cells were treated with TMAO or vehicle for 2 h and mRNA expression of (H) flTF and (I) asTF analysed. Results are presented as mean±SEM. Global P-values shown were obtained by non-parametric Kruskal–Wallis test with Dunn’s multiple comparisons post hoc test to compare different treatments. Differences between two groups were assessed using a Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Gene expression was determined using TaqMan Fast Universal PCR Master Mix (Catalogue #4366072, Thermofisher, Waltham, MA, USA) or TaqManTM RNA-to-CTTM 1-Step Kit (Catalogue #4392653, Thermofisher, Waltham, MA, USA) using the following FAM- or VIC-tagged TaqMan ® gene expression assays (all from Life Technologies, Carlsbad, CA, USA): Human GAPDH (Assay ID Hs99999905_m1), human asTF and flTF (Custom probes, for details see Reference 57 ) human VCAM1 (Assay ID Hs01003372_m1), mouse GAPDH (Assay ID Mm99999915_g1), mouse flTF (Assay ID Mm00438856_m1), mouse asTF (custom probe, Clone ID AP7DRF4), mouse VCAM1 (Assay ID Mm00449197_m1).

Techniques: Expressing, Western Blot, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Vascular endothelial tissue factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis

doi: 10.1093/cvr/cvab263

Figure Lengend Snippet: TMAO acutely raises TF and VCAM1 expression in aortic tissue in vivo. C57/BL6 mice were injected with TMAO or vehicle intraperitoneally for 1.5 h. Next, (A) plasma TMAO levels were quantified by LC/MS/MS and arotic mRNA expression of (B) flTF, (C) asTF, and (D) VCAM1 quantified via TaqMan rtPCR. To assess protein expression, the animals were injected with vehicle or TMAO for 6 h. Subsequently, (E) TMAO plasma levels and (F) protein amounts of TF and VCAM1 were quantified via western blot and (G) density of the protein bands quantified. (H) To analyse localization within the vessel wall, aortic tissue of TMAO-injected mice was probed for TF and VCAM1 expression 6 h post injection using immunofluorescence staining (TF red, upper panel, VCAM1 red, lower panel). The tissue was counterstained with DAPI (blue) and an f-actin probe (green). Results are presented as mean±SEM. Pairwise comparison was performed using a Mann–Whitney test.

Article Snippet: Gene expression was determined using TaqMan Fast Universal PCR Master Mix (Catalogue #4366072, Thermofisher, Waltham, MA, USA) or TaqManTM RNA-to-CTTM 1-Step Kit (Catalogue #4392653, Thermofisher, Waltham, MA, USA) using the following FAM- or VIC-tagged TaqMan ® gene expression assays (all from Life Technologies, Carlsbad, CA, USA): Human GAPDH (Assay ID Hs99999905_m1), human asTF and flTF (Custom probes, for details see Reference 57 ) human VCAM1 (Assay ID Hs01003372_m1), mouse GAPDH (Assay ID Mm99999915_g1), mouse flTF (Assay ID Mm00438856_m1), mouse asTF (custom probe, Clone ID AP7DRF4), mouse VCAM1 (Assay ID Mm00449197_m1).

Techniques: Expressing, In Vivo, Injection, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Comparison, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Vascular endothelial tissue factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis

doi: 10.1093/cvr/cvab263

Figure Lengend Snippet: TMAO chronically induces endothelial TF and VCAM1 expression in mouse aortas in vivo. C57/BL6 mice were either fed a control chow diet or a choline diet. After 10 days of diet, (A) plasma levels of TMAO were quantified and aortic mRNA expression for (B) flTF, (C)asTF, and (D) VCAM1 analysed. (E) Protein amounts of TF and VCAM1 in aortic tissue were measured via western blot and related to the corresponding plasma levels of TMAO. Aortas of LPS-injected mice (15 mg/kg for 6 h) as well as recombinant mouse TF were used as positive controls. (F) Density of the detected bands was quantified using an imaging software. (G) Plasma of the same animals was analysed with respect to TAT complexes via ELISA. LPS-injected animals served as a known positive control for TAT induction (H) Immunohistochemistry experiments using specific antibodies were used to assess protein expression of TF, VCAM1, and the endothelial marker CD31 in aortic tissue. (I) Mean OD of endothelial protein expression from three different anatomical sides was quantified using an imaging software and three data points for each animal were plotted. Results are presented as mean±SEM. Pairwise comparison was performed using a Mann–Whitney test.

Article Snippet: Gene expression was determined using TaqMan Fast Universal PCR Master Mix (Catalogue #4366072, Thermofisher, Waltham, MA, USA) or TaqManTM RNA-to-CTTM 1-Step Kit (Catalogue #4392653, Thermofisher, Waltham, MA, USA) using the following FAM- or VIC-tagged TaqMan ® gene expression assays (all from Life Technologies, Carlsbad, CA, USA): Human GAPDH (Assay ID Hs99999905_m1), human asTF and flTF (Custom probes, for details see Reference 57 ) human VCAM1 (Assay ID Hs01003372_m1), mouse GAPDH (Assay ID Mm99999915_g1), mouse flTF (Assay ID Mm00438856_m1), mouse asTF (custom probe, Clone ID AP7DRF4), mouse VCAM1 (Assay ID Mm00449197_m1).

Techniques: Expressing, In Vivo, Control, Clinical Proteomics, Western Blot, Injection, Recombinant, Imaging, Software, Enzyme-linked Immunosorbent Assay, Positive Control, Immunohistochemistry, Marker, Comparison, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Vascular endothelial tissue factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis

doi: 10.1093/cvr/cvab263

Figure Lengend Snippet: A small molecule TMA-lyase inhibitor reverses TMAO-stimulated increase in aortic TF and VCAM1. C57/BL6 mice were put on a choline diet with and without FMC in the drinking water. (A) Aortic tissue was subjected to immunohistochemistry using antibodies against TF, VCAM1, and the endothelial marker CD31. (B) Mean OD was quantified from three different anatomical sides and three data points are plotted for each animal. (B, lower panel) Levels of plasma TMAO, TMA, and choline were quantified via LC/MS/MS and (C) correlated with endothelial TF and VCAM1 expression. Results are presented as mean±SEM. Pairwise comparison was performed using a Mann–Whitney test. Correlation of TMA and TMAO levels with TF or VCAM1 mean endothelial OD was performed using non-parametric Spearman correlation.

Article Snippet: Gene expression was determined using TaqMan Fast Universal PCR Master Mix (Catalogue #4366072, Thermofisher, Waltham, MA, USA) or TaqManTM RNA-to-CTTM 1-Step Kit (Catalogue #4392653, Thermofisher, Waltham, MA, USA) using the following FAM- or VIC-tagged TaqMan ® gene expression assays (all from Life Technologies, Carlsbad, CA, USA): Human GAPDH (Assay ID Hs99999905_m1), human asTF and flTF (Custom probes, for details see Reference 57 ) human VCAM1 (Assay ID Hs01003372_m1), mouse GAPDH (Assay ID Mm99999915_g1), mouse flTF (Assay ID Mm00438856_m1), mouse asTF (custom probe, Clone ID AP7DRF4), mouse VCAM1 (Assay ID Mm00449197_m1).

Techniques: Immunohistochemistry, Marker, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Expressing, Comparison, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Vascular endothelial tissue factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis

doi: 10.1093/cvr/cvab263

Figure Lengend Snippet: The gut microbiota choline TMA-lyase inhibitor FMC shifts the choline diet-induced changes in caecal microbial community associated with vascular TF and VCAM1. (A) Shannon diversity indices distinguishing chow, choline, and choline+FMC samples. Statistical analysis was performed using ANOVA. (B) NMDS based on Bray–Curtis index between the caecal microbiota recovered from mice that were on indicated diets. Statistical analysis was performed using permutational multivariate ANOVA with R2 values for % variance explained by diet being the variable of interest. (C, upper panel) Statistically significant (Benjamini–Hochberg false discovery rate; P < 0.05) genera differentiating three groups (chow, choline, and choline+FMC). Plotted are interquartile ranges (IQRs) (boxes). The dark line in the box is the median, lower whiskers represent smallest observation (≥25% quantile—1.5×IQR), upper whiskers largest observation (≤75% quantile—1.5×IQR) with outliers as dots outside of the box. (C, second panel) Scatter plots based on linear regression showing correlation between abundance of indicated genera with plasma TMAO (μM) levels, (C, third panel) endothelial TF protein and (C, fourth panel) endothelial VCAM1 protein in mouse aortas on the indicated diets, expressed as OD within the annotated endothelial layer quantified by immunofluorescence (as described in Section 2). R2 and P-values are indicated in each panel. For all panels, the same colour scheme was used for data to indicate animal diet: chow (green), choline (purple), and choline+FMC (red). The grey area shows the 95% CI.

Article Snippet: Gene expression was determined using TaqMan Fast Universal PCR Master Mix (Catalogue #4366072, Thermofisher, Waltham, MA, USA) or TaqManTM RNA-to-CTTM 1-Step Kit (Catalogue #4392653, Thermofisher, Waltham, MA, USA) using the following FAM- or VIC-tagged TaqMan ® gene expression assays (all from Life Technologies, Carlsbad, CA, USA): Human GAPDH (Assay ID Hs99999905_m1), human asTF and flTF (Custom probes, for details see Reference 57 ) human VCAM1 (Assay ID Hs01003372_m1), mouse GAPDH (Assay ID Mm99999915_g1), mouse flTF (Assay ID Mm00438856_m1), mouse asTF (custom probe, Clone ID AP7DRF4), mouse VCAM1 (Assay ID Mm00449197_m1).

Techniques: Clinical Proteomics, Immunofluorescence

Journal: PLoS ONE

Article Title: IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

doi: 10.1371/journal.pone.0073024

Figure Lengend Snippet: ( A ) IKKα immunoblot of total cell lysates from two independent experiments using IKKα f/f ;CreERT2 immature articular chondrocytes (iMACs) treated with EtOH vehicle (WT control) or 4-OHT (IKKα-KO) for 72 hours. IKKβ and β-tubulin were used as 4-OHT specificity and protein loading controls, respectively. ( B ) (Left): Relative levels of IHC staining with COL2, C1,2C and COL10 antibodies in 3-week pellet cultures of IKKα f/f ;CreERT2 iMACs treated with EtOH vehicle (WT control) or 4-OHT (IKKα KO). Results are representative of 3 experiments. (Right): COL2 and COL10 immunopositive cells in 4-OHT (IKKα KO) treated IKKα f/f ;CreERT2 iMAC 3-week monolayer cultures vs. their matched WT (EtOH treated) controls. Results are representative of 2 (COL2) and 3 (COL10) independent experiments (Original magnification, 200X). Merged immunofluorescence images are shown. COL2 (monolayer cultures): green; COL10 (pellet and monolayer cultures): red; COL2–3/4C (pellet cultures): red; DAPI: blue. See for comparable results of other experiments. ( C ) qRT-PCR analysis of Runx2 (left) and Col10a1 (right) RNA in EtOH (WT) or 4-OHT (IKKα KO) treated IKKα f/f ;CreERT2 iMAC 1- and 3-week pellet cultures. Data are displayed as fold-change of 3-week vs. 1-week cultures, and shown as mean ± S.E.M. of 3 experiments. *p<0.05 by Student’s t-test. ( D ) qRT-PCR analysis of Runx2 and Col10a1 RNAs in IKKα f/f ;CreERT2 iMAC monolayer cultures treated with EtOH (WT) or 4-OHT (IKKα KO). Data are shown as fold change relative to 72 hour EtOH (WT) and 4-OHT (IKKα KO) treated cultures of at least 4 experiments. **p<0.01 by ANOVA. ( E ) (Left): Representative Alizarin red staining of EtOH (WT) and 4-OHT (IKKα KO) treated 3-week IKKα f/f ;CreERT2 iMAC high density monolayer cultures. (Right): Quantification of Alizarin red staining after dye solubilization of EtOH (WT) and 4-OHT (IKKα KO) monolayer cultures differentiated as indicated. Data are mean ± S.E.M. of 3 experiments, each performed in duplicate. ***p<0.001 by ANOVA.

Article Snippet: Total collagenase activity in the conditioned media of either human or murine chondrocytes cultures was quantified with an EnzChek®Gelatinase/Collagenase Assay Kit (Molecular Probes).

Techniques: Western Blot, Control, Immunohistochemistry, Immunofluorescence, Quantitative RT-PCR, Staining

Journal: PLoS ONE

Article Title: IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

doi: 10.1371/journal.pone.0073024

Figure Lengend Snippet: ( A ) qRT-PCR analysis of MMP10 RNA in 1-week differentiated pellet cultures of IKKα KD vs. WT (GL2 control) human OA chondrocytes. Results were obtained with primary chondrocytes derived from 8 OA patients. Data are shown as mean ± S.E.M. (error bars) and represented as fold-change vs. WT (set as 1.0). **p<0.01 by Wilcoxon matched pair test. ( B ) : MMP-10 protein secretion quantified by immunoassay of conditioned medium (performed as described in Methods) of 1-week differentiated pellet cultures (N = 5). Data are shown as mean ± S.E.M. *p<0.05 by Wilcoxon matched pair test. ( C ) MMP-10 immunostaining in 1-week differentiated pellet cultures of IKKα KD vs. WT (GL2 control) human OA chondrocytes (N = 3). Immunostaining expressed as percentage of signal per µm 2 unit area. **p<0.01 by Student’s t test. Representative images of MMP-10 immunostained micromasses are presented in . ( D ) qRT-PCR analysis of Mmp10 RNA in 4-OHT (IKKα KO) or EtOH vehicle-treated (WT) 1-week high density chondrocyte monolayer cultures of IKKα f/f ;CreERT2 iMACs. Data are represented as fold-change relative to the WT control (set as 1.0) and shown as mean ± S.E.M. of 3 independent experiments. **p<0.01 by Student’s t-test. ( E ) (Left): Representative MMP-10 immunoblot of whole cell lysates of 1-week EtOH (WT) and 4-OHT (IKKα KO) treated IKKα f/f ;CreERT2 high density iMAC monolayer cultures. (Right): Densitometric analysis of MMP-10 immunoblots shown as mean ± S.E.M. of 3 independent experiments. *p<0.05 by Student’s t test.

Article Snippet: Total collagenase activity in the conditioned media of either human or murine chondrocytes cultures was quantified with an EnzChek®Gelatinase/Collagenase Assay Kit (Molecular Probes).

Techniques: Quantitative RT-PCR, Control, Derivative Assay, Immunostaining, Western Blot

Journal: PLoS ONE

Article Title: IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

doi: 10.1371/journal.pone.0073024

Figure Lengend Snippet: MMP-10 immunostaining in 1-week differentiated pellet cultures of IKKα KD vs. matched WT (GL2 control) human OA chondrocytes. Global views of micromass sphere sections (100Xmagnification) are shown on the left; and three random fields at 400Xmagnification, which were submitted to quantitative image analysis appear to the right. Antibody stained images are shown in the right side upper rows (labeled as 1, 2 & 3), with each 400Xfield submitted to analysis by Nikon Imaging Software in the lower rows (1.1, 2.1 & 3.1). For each sample an IHC staining threshold was established based on an antibody isotype control. Antibody staining of MMP-10 is in red in conjunction with hematoxylin (blue) nuclear counterstaining. Data are representative examples of multiple sections of micromasses prepared with ACs of 3 OA patients; and results of all such experiments are presented as statistically analyzed bar graphs in .

Article Snippet: Total collagenase activity in the conditioned media of either human or murine chondrocytes cultures was quantified with an EnzChek®Gelatinase/Collagenase Assay Kit (Molecular Probes).

Techniques: Immunostaining, Control, Staining, Labeling, Imaging, Software, Immunohistochemistry

Journal: PLoS ONE

Article Title: IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

doi: 10.1371/journal.pone.0073024

Figure Lengend Snippet: ( A ) Quantitative image analysis for TIMP-3 immunostaining of 1-week IKKα KD human pellet cultures vs. matched WT (GL2 control) for 3 OA patients. Results are shown as mean ± S.E.M. *p<0.05 by Student’s t-test. Representative images of TIMP-3 immunostained micromasses are presented in . ( B ) TIMP-3 immunoblot of whole cell lysates of IKKα KD and matched WT (GL2 control) of 1-week human chondrocyte pellet cultures of one representative OA patient, with β-tubulin as protein loading control. ( C ) qRT-PCR analysis of TIMP3 mRNA in 1-week pellet cultures of IKKαKD human chondrocytes vs. matched WT (GL2) controls (N = 6). Results are shown as mean ± S.E.M. ns = not significant.

Article Snippet: Total collagenase activity in the conditioned media of either human or murine chondrocytes cultures was quantified with an EnzChek®Gelatinase/Collagenase Assay Kit (Molecular Probes).

Techniques: Immunostaining, Control, Western Blot, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

doi: 10.1371/journal.pone.0073024

Figure Lengend Snippet: TIMP-3 immunostaining in 1-week differentiated pellet cultures of IKKα KD vs. matched WT (GL2 control) human OA chondrocytes. Global views of micromass sphere sections (100Xmagnification) are shown on the left; and three random fields at 400Xmagnification, which were submitted to quantitative image analysis, appear to the right. Antibody stained images are shown in the right side upper rows (labeled as 1, 2 & 3), with each 400Xfield submitted to analysis by Nikon Imaging Software in the lower rows (1.1, 2.1 & 3.1). For each sample an IHC staining threshold was established based on an antibody isotype control. Antibody staining of TIMP-3 is in red in conjunction with hematoxylin (blue) nuclear counterstaining. Data are representative examples of multiple sections of micromasses prepared with ACs of 3 OA patients; and results of all such experiments are presented as statistically analyzed bar graphs in .

Article Snippet: Total collagenase activity in the conditioned media of either human or murine chondrocytes cultures was quantified with an EnzChek®Gelatinase/Collagenase Assay Kit (Molecular Probes).

Techniques: Immunostaining, Control, Staining, Labeling, Imaging, Software, Immunohistochemistry

Journal: PLoS ONE

Article Title: IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

doi: 10.1371/journal.pone.0073024

Figure Lengend Snippet: ( A ) MMP-13 activity in conditioned medium of 1-week (N = 9) and 3-week (N = 7) IKKα KD pellet cultures of human OA chondrocytes. Data are represented as mean ± S.E.M. (error bars). *p<0.05, **p<0.01 by Wilcoxon matched pair test. ( B ) MMP-13 (Left) and total collagenase (Right) activities in serum-free conditioned media of 1-week IKKα KD and matched WT (GL2 control) pellet cultures of ACs from 2 OA patients. Data are represented as mean ± S.E.M. (error bars). ( C ) qRT-PCR analysis of Mmp13 expression in 1-week high density monolayer cultures of IKKα f/f ;CreERT2 iMACs treated with EtOH (WT control) or 4-OHT (IKKα KO). Data are from 3 independent experiments and shown as mean ± S.E.M. (error bars). ns = not significant by Student’s t test. ( D ) Total collagenase activity quantified in the conditioned medium of 1-week EtOH (WT control) and 4-OHT (IKKα KO) treated IKKα f/f ;CreERT2 iMACs. ***p<0.001 by Student’s t test.

Article Snippet: Total collagenase activity in the conditioned media of either human or murine chondrocytes cultures was quantified with an EnzChek®Gelatinase/Collagenase Assay Kit (Molecular Probes).

Techniques: Activity Assay, Control, Quantitative RT-PCR, Expressing

Journal: PLoS ONE

Article Title: IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

doi: 10.1371/journal.pone.0073024

Figure Lengend Snippet: ( A ) Immunoblots of whole cell lysates of monolayer cultures of primary human (Hu) IKKα KD chondrocytes, which were stably transduced with a neomycin-resistant retroviral vector (BIN), or with BIN retroviral vectors co-expressing WT murine IKKα (Mu IKKα-BIN) or a kinase-dead murine IKKα mutant {Mu IKKα(K444M-BIN)}. Blots were first probed with an anti-IKKα antibody and then sequentially re-probed with anti-HA and β-tubulin antibodies. ( B ) Quantitative image analysis of COL2 immunostaining in 1-week pellet cultures of primary human chondrocytes of 3 OA patients: WT (GL2 control), IKKα KD cells with BIN, IKKα KD cells with Mu IKKα-BIN or IKKα KD cells with Mu IKKα(K44M)-BIN. Data are shown as mean ± S.E.M (error bars). *p<0.05 and **p<0.01 by Student’s t-test. ( C ) Quantitative image analysis of C1,2C immunostaining in the same WT (GL2 control), Hu-IKKα KD+BIN and Hu-IKKα KD+Mu IKKα(K44M)-BIN samples as in panel B. Data are shown as mean ± S.E.M. (error bars). *p<0.05 and ***p<0.001 by Student’s t-test. ( D ) Quantitative image analysis of TIMP-3 immunostaining of WT (GL2 control), Hu-IKKα KD+BIN and Hu-IKKα KD+Mu IKKα(K44M)-BIN of 3-week pellet cultures prepared with primary ACs derived from 3 OA patients. *p<0.05 and **p<0.01 by Student’s t test. ( E ) Quantitative image analysis of Runx2 and COL10 immunostaining of the same WT (GL2 control), Hu-IKKα KD+BIN and Hu-IKKα KD+Mu IKKα(K44M)-BIN samples in panels B and C. Data are shown as mean ± S.E.M. (error bars). *p<0.05 and **p<0.01 by Student’s t-test. Representative examples of COL2, COL2–3/4, TIMP-3, Runx2 and COL10 immunostained micromasses, which were submitted to quantitative imaging analysis, are respectively shown in – .

Article Snippet: Total collagenase activity in the conditioned media of either human or murine chondrocytes cultures was quantified with an EnzChek®Gelatinase/Collagenase Assay Kit (Molecular Probes).

Techniques: Western Blot, Stable Transfection, Transduction, Retroviral, Plasmid Preparation, Expressing, Mutagenesis, Immunostaining, Control, Derivative Assay, Imaging

Journal: PLoS ONE

Article Title: IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

doi: 10.1371/journal.pone.0073024

Figure Lengend Snippet: COL2 immunostaining in 1-week pellet cultures of primary human OA chondrocytes: WT (GL2 control), IKKα KD cells with empty BIN retroviral vector, IKKα KD cells with WT murine IKKα-BIN and IKKα KD cells with kinase-dead murine IKKα(K44M)-BIN. Global views of micromass sphere sections (100Xmagnification) are shown on the left; and three random fields at 400Xmagnification submitted to quantitative image analysis are on the right. Antibody stained images are shown in the right side upper rows (labeled 1, 2 & 3), with each 400Xfield analyzed by Nikon Imaging Software in the lower rows (labeled 1.1, 2.1 & 3.1). For each sample an IHC staining threshold was established based on an antibody isotype control. Antibody staining is in red in conjunction with hematoxylin (blue) nuclear counterstaining. Data are representative examples of multiple sections of micromasses prepared with ACs of 3 OA patients; and results of all such experiments are presented as statistically analyzed bar graphs in .

Article Snippet: Total collagenase activity in the conditioned media of either human or murine chondrocytes cultures was quantified with an EnzChek®Gelatinase/Collagenase Assay Kit (Molecular Probes).

Techniques: Immunostaining, Control, Retroviral, Plasmid Preparation, Staining, Labeling, Imaging, Software, Immunohistochemistry

Journal: PLoS ONE

Article Title: IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

doi: 10.1371/journal.pone.0073024

Figure Lengend Snippet: C1,2C antibody detection of COL2–3/4C neoepitopes in the same 1-week pellet cultures of primary human chondrocytes employed above in . Global views of micromass sphere sections (100Xmagnification) are shown on the left; and three random fields at 400Xmagnification submitted to quantitative image analysis are on the right. Antibody stained images are shown in the right side upper rows (labeled 1, 2 & 3), with each 400Xfield analyzed by Nikon Imaging Software in the lower rows (labeled 1.1, 2.1 & 3.1). For each sample an IHC staining threshold was established based on an antibody isotype control. Antibody staining is in red in conjunction with hematoxylin (blue) nuclear counterstaining. Data are representative examples of multiple sections of micromasses prepared with ACs of 3 OA patients; and results of all such experiments are presented as statistically analyzed bar graphs in .

Article Snippet: Total collagenase activity in the conditioned media of either human or murine chondrocytes cultures was quantified with an EnzChek®Gelatinase/Collagenase Assay Kit (Molecular Probes).

Techniques: Staining, Labeling, Imaging, Software, Immunohistochemistry, Control

Journal: PLoS ONE

Article Title: IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

doi: 10.1371/journal.pone.0073024

Figure Lengend Snippet: TIMP-3 immunostaining in 1-week pellet cultures of primary human OA chondrocytes: WT (GL2 control), IKKα KD cells with empty BIN retrovector and IKKα KD cells with kinase-dead murine IKKα(K44M)-BIN. Global views of micromass sphere sections (100Xmagnification) are shown on the left, and three random fields at 400Xmagnification submitted to quantitative image analysis are on the right. Antibody stained images are shown in the right side upper rows (labeled 1, 2 & 3), with each 400Xfield analyzed by Nikon Imaging Software in the lower rows (labeled 1.1, 2.1 & 3.1). For each sample an IHC staining threshold was established based on an antibody isotype control. Antibody staining is in red in conjunction with hematoxylin (blue) nuclear counterstaining. Data are representative examples of multiple sections of micromasses prepared with ACs of 3 OA patients; and results of all such experiments are presented as statistically analyzed bar graphs in .

Article Snippet: Total collagenase activity in the conditioned media of either human or murine chondrocytes cultures was quantified with an EnzChek®Gelatinase/Collagenase Assay Kit (Molecular Probes).

Techniques: Immunostaining, Control, Staining, Labeling, Imaging, Software, Immunohistochemistry

Journal: PLoS ONE

Article Title: IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

doi: 10.1371/journal.pone.0073024

Figure Lengend Snippet: Runx2 immunostaining in 3-week pellet cultures of primary human OA chondrocytes: WT (GL2 control), IKKα KD cells with empty BIN retroviral vector and IKKα KD cells with kinase-dead murine IKKα(K44M)-BIN. Global views of micromass sphere sections (100Xmagnification) are shown on the left, and three random fields at 400Xmagnification submitted to quantitative image analysis are on the right. Antibody stained images are shown in the right side upper rows (labeled 1, 2 & 3), with each 400Xfield analyzed by Nikon Imaging Software in the lower rows (labeled 1.1, 2.1 & 3.1). For each sample an IHC staining threshold was established based on an antibody isotype control. Antibody staining is in red in conjunction with hematoxylin (blue) nuclear counterstaining. Data are representative examples of multiple sections of micromasses prepared with ACs of 3 OA patients; and results of all such experiments are presented as statistically analyzed bar graphs in .

Article Snippet: Total collagenase activity in the conditioned media of either human or murine chondrocytes cultures was quantified with an EnzChek®Gelatinase/Collagenase Assay Kit (Molecular Probes).

Techniques: Immunostaining, Control, Retroviral, Plasmid Preparation, Staining, Labeling, Imaging, Software, Immunohistochemistry

Journal: PLoS ONE

Article Title: IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

doi: 10.1371/journal.pone.0073024

Figure Lengend Snippet: COL10 immunostaining in 3-week pellet cultures of primary human OA chondrocytes: WT (GL2 control), IKKα KD cells with empty BIN retroviral vector and IKKα KD cells with kinase-dead murine IKKα(K44M)-BIN. Global views of micromass sphere sections (100Xmagnification) are shown on the left, and three random fields at 400Xmagnification submitted to quantitative image analysis are on the right. Antibody stained images are shown in the right side upper rows (labeled 1, 2 & 3), with each 400Xfield analyzed by Nikon Imaging Software in the lower rows (labeled 1.1, 2.1 & 3.1). For each sample an IHC staining threshold was established based on an antibody isotype control. Antibody staining is in red in conjunction with hematoxylin (blue) nuclear counterstaining. Data are representative examples of multiple sections of micromasses prepared with ACs of 3 OA patients; and results of all such experiments are presented as statistically analyzed bar graphs in .

Article Snippet: Total collagenase activity in the conditioned media of either human or murine chondrocytes cultures was quantified with an EnzChek®Gelatinase/Collagenase Assay Kit (Molecular Probes).

Techniques: Immunostaining, Control, Retroviral, Plasmid Preparation, Staining, Labeling, Imaging, Software, Immunohistochemistry

Journal: iScience

Article Title: Pharmacological targeting of Sam68 functions in colorectal cancer stem cells

doi: 10.1016/j.isci.2021.103442

Figure Lengend Snippet: Reverse/β-turn peptidomimetic compounds are direct interactors of Sam68 (A) Chemical structure of HAT inhibitor C646 and bromodomain ligand I-CBP112, as well as β-turn peptidomimetics ICG-001 and CWP232228. (B) Western blot analysis of CBP-catalyzed H3K14ac and H3K18ac histone acetylation marks in C646 (0.25 μM), I-CBP112 (0.25 μM), and CWP232228 (0.1 μM) t-hESCs versus control DMSO. Total histone H3 and GAPDH were used as loading control. Relative OD signal quantification versus H3 intensity is presented (C646: n = 3, I-CBP112: n = 5, CWP232228: n ≥ 3, ∗: p = 0.0183, ∗∗: p = 0.0078, ∗∗∗: p ≤ 0.00033, two-tailed t test). Data are represented as mean ± SEM (error bars). (C) Dose-response experiment assessing the impact of bromodomain ligand-based (C646 and I-CBP112), and peptidomimetic (CWP232228) inhibition of CBP on t-hESC growth (C646, I-CPB112: n = 4; CWP232228: n = 3). (D) Early endoderm differentiation assay performed in t-hESCs in the presence of CWP232228 (0.1 μM, n = 6), I-CBP112 (0.25 μM, n = 3), or C646 (0.25 μM, n = 3) versus control DMSO (n = 6) and basal culture media (n = 3). Bar graph represents relative counts of FOXA2-positive (early endoderm marker)/OCT4-negative cells in DMSO, CWP232228, I-CBP112, and C646-treated t-hESCs versus basal culture media (one-way ANOVA, ∗∗∗: p < 0.0001). Data are represented as mean ± SEM (error bars). Scale bar: 100 μm. (E) Pro-drug CWP232228 is converted into its active form CWP231904 via hydrolysis of the phosphate group by serum/cellular alkaline phosphatase. (F) Affinity pull-down experiments using CWP231904-conjugated magnetic beads performed on whole hESC lysates. Physical interaction between CBP, Sam68, beta-Catenin, ETS, MYB, GATA2, and PTMA with immobilized CWP231904 was assessed by immunoblotting. Each protein was previously shown as a member of the CBP interactome showing selective enrichment in human primary AML versus healthy blood ( Benoit et al., 2017 ). Excess of soluble compounds (CWP and ICG001, 100 μM) were used to compete with immobilized CWP231904. Whole-cell lysate was used as input, and amine-functionalized beads were used as negative control (n = 2). The heatmap presents mean background-corrected OD signal for each putative interactor tested (gray: not tested).

Article Snippet: Custom mutagenesis of human KHDRBS1 cDNA and cloning into pLenti-mGFP-P2A-Puro vector was performed by OriGene Technologies, and lentiviral particles for control, wild-type Sam68/ KHDRBS1 , and G305N Sam68/ KHDRBS1 overexpression were generated as above-described.

Techniques: Western Blot, Two Tailed Test, Inhibition, Differentiation Assay, Marker, Magnetic Beads, Negative Control

Journal: iScience

Article Title: Pharmacological targeting of Sam68 functions in colorectal cancer stem cells

doi: 10.1016/j.isci.2021.103442

Figure Lengend Snippet: In silico screening of peptidomimetics with enhanced binding affinity for Sam68 (A) Representation of Sam68 interacting with SH3 domain in Src kinase family proteins via proline-rich motifs located in N-terminal P1-P2 and between residues 275 and 374 (P3, P4, P5). Small molecule UCS15A is known to disrupt SH3-mediated interaction of Src with Sam68 P3-5 domains ( Sharma et al., 2001 ). (B) 2D representation of UCS15A and the peptidomimetic ICG-001 in silico predicted binding pocket in Sam68 275-374 peptide (red, oxygen; blue, nitrogen). Common residues involved in both small-molecule-binding pockets are highlighted in red. Predicted hydrogen bond length is represented by dashed lines (Å). (C) Schematic representation of the in silico structure-activity relationship analysis pipeline (PyRx) used to identify β-turn peptidomimetic molecules with enhanced binding affinity for Sam68 275-374 domain. “A” and “B” represent the positions of distinct substituents added to reverse-turn mimetic cores. (D) Dose-response curves assessing selective toxicity of peptidomimetics ICG-001, CWP232228, and PRI-724 in HT29 human colorectal cancer cell line versus normal intestinal progenitor cells HIEC (n ≥ 4, 48-h treatments). (E) Compound ranking based on predicted Keq for each β-turn analog (black dots). Only molecules presenting a standard deviation below 0.1 for a minimum of three analysis runs, with an exhaustiveness (“E”) level of “8” were plotted. Dots corresponding to ICG-001, CWP231904, PRI-724-OH, and YB-0159 were highlighted in red. Random structure ranking is represented by green dots. See also . (F) Structure of YB-0158, a phosphate-stabilized prodrug of YB-0159. (G) Docked poses of CWP231904 (left) and YB-0159 (right) in human Sam68 257-374 fragment (red, oxygen; blue, nitrogen). Glycine 305 is highlighted in red, where distinct hydrogen bond (gray dashed line) was predicted between YB-0159 and Sam68. The inset in the right pose represents a higher magnification view of the predicted hydrogen bond formation between YB-0158 and Gly305. See also .

Article Snippet: Custom mutagenesis of human KHDRBS1 cDNA and cloning into pLenti-mGFP-P2A-Puro vector was performed by OriGene Technologies, and lentiviral particles for control, wild-type Sam68/ KHDRBS1 , and G305N Sam68/ KHDRBS1 overexpression were generated as above-described.

Techniques: In Silico, Binding Assay, Activity Assay, Standard Deviation

Journal: iScience

Article Title: Pharmacological targeting of Sam68 functions in colorectal cancer stem cells

doi: 10.1016/j.isci.2021.103442

Figure Lengend Snippet: YB-0158 alters Sam68 biology in human cancer cells (A) Dose-response experiment assessing growth inhibition caused by peptidomimetics analogs CWP232228 and YB-0158 in t-hESCs (n = 3, 48-h treatments). Calculated EC 50 for each small molecule is presented in the inset table. See also . (B) Dose-response experiment assessing growth inhibition caused by peptidomimetic analogs CWP232228 and YB-0158 in HT29 colorectal cancer cells (n = 2, 48-h treatments). Calculated EC 50 for each small molecule is presented in the inset table. (C) Co-immunoprecipitation (IP) assessing changes in interaction levels between Src and Sam68 in response to CWP232228 (1.5 μM) and YB-0158 (0.3 μM) in HT29 cells (48 h) (n = 3, ∗: p = 0.021, ∗∗: p = 0.0088, two-tailed t test). Data are represented as mean ± SEM (error bars). Mouse IgGs were used as negative control for pull down. (D) Immunofluorescence staining of Sam68 in DMSO, CWP232228, and YB-0158-treated t-hESCs (48 h, n = 9). Quantification of nuclear Sam68 was performed by high-content imaging and presented as relative levels versus DMSO (∗∗: p < 0.01, ∗∗∗: p < 0.0001, two-tailed t test). Data are represented as mean ± SEM (error bars). Scale bar: 100 μm. (E) Quantification of nuclear Sam68 in HT29 cells treated with increasing doses of YB-0158, in the presence (n = 4) or absence (n = 3) of a PRMT1 inhibitor (Furamidine, 10 μM). Cells were treated for 48 h and nuclear Sam68 immunostaining was quantified by high-content imaging. Data are represented as mean ± SEM (error bars). (F) Western blot analysis of Sam68 levels in normal human intestinal progenitor cells HIEC; human colorectal cancer SW480, HT29, and HCT116 lines; mouse colon adenocarcinoma MC38 cells; t-hESCs; as well as patient-derived CSC-enriched spheroids and 3D organoids from colorectal tumor samples (n ≥ 3). Relative OD signal quantification for Sam68 versus loading control (GAPDH) is presented. (G) Dose-response experiment monitoring growth of normal intestinal cells HIEC, as well as HT29, SW480, and HCT116 colorectal cancer lines treated with YB-0158 (n ≥ 3, 48 h). A significant correlation was established between calculated EC 50 and Sam68 expression (R 2 = 0.8510, p < 0.0001, simple linear regression). (H) Cell growth experiment in HCT116 cells transduced with control/empty-mGFP (pLenti Control) or KHDRBS1 -mGFP (pLenti Sam68) overexpression vectors and treated with YB-0158 (0.3 μM, 48 h) or vehicle control (DMSO). GFP-positive cell counts upon treatments are presented versus their corresponding DMSO-treated group (n = 7, ∗∗: p = 0.003, two-tailed t test). Data are represented as mean ± SEM (error bars). (I) Cell growth experiment using HCT116 cells overexpressing wild-type Sam68 ( KHDRBS1 ) (wt Sam68) or with a mutated G305 motif (G305N Sam68) and subjected to increasing doses of YB-0158 (0.08–10 μM versus DMSO control) for 48 h. Residual transduced cells (GFP reporter) were counted for each dose and presented versus DMSO control (n ≤ 5, ∗∗∗: p < 0.001, two-tailed t test). Data are represented as mean ± SEM (error bars).

Article Snippet: Custom mutagenesis of human KHDRBS1 cDNA and cloning into pLenti-mGFP-P2A-Puro vector was performed by OriGene Technologies, and lentiviral particles for control, wild-type Sam68/ KHDRBS1 , and G305N Sam68/ KHDRBS1 overexpression were generated as above-described.

Techniques: Inhibition, Immunoprecipitation, Two Tailed Test, Negative Control, Immunofluorescence, Staining, Imaging, Immunostaining, Western Blot, Derivative Assay, Expressing, Transduction, Over Expression

Journal: iScience

Article Title: Pharmacological targeting of Sam68 functions in colorectal cancer stem cells

doi: 10.1016/j.isci.2021.103442

Figure Lengend Snippet:

Article Snippet: Custom mutagenesis of human KHDRBS1 cDNA and cloning into pLenti-mGFP-P2A-Puro vector was performed by OriGene Technologies, and lentiviral particles for control, wild-type Sam68/ KHDRBS1 , and G305N Sam68/ KHDRBS1 overexpression were generated as above-described.

Techniques: Recombinant, Derivative Assay, Immunoprecipitation, Staining, Chromatin Immunoprecipitation, DNA Purification, SYBR Green Assay, Purification, Western Blot, RNA Sequencing Assay, Sequencing, Expressing, Transformation Assay, shRNA, Software