Journal: International Journal of Biological Sciences

Article Title: Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level

doi: 10.7150/ijbs.49332

Figure Lengend Snippet: Pam3CSK4 induces inflammatory responses in AVIC and monocyte co-culture. A. AVICs and monocytes were cultured separately and treated with a low dose of Pam3CSK4 (0.03 µg/ml) for 24 h. Representative immunoblots and ELISA data of 4 separate experiments show that Pam3CSK4 elevates ICAM-1 level moderately and causes a slight increase in MCP-1 production in monocytes while Pam3CSK4 in this low dose has no effect on the levels of ICAM-1, VCAM-1 and MCP-1 in AVICs. B. AVIC and monocyte co-cultures were treated with Pam3CSK4 (0.03 µg/ml) for 24 hours or left untreated. Representative immunoblots and ELISA data show that low levels of ICAM-1, VCAM-1 and MCP-1 are present in untreated control. However, stimulation of TLR2 with a low dose of Pam3CSK4 markedly up-regulates the production of ICAM-1, VCAM-1 and MCP-1 in co-cultures. Data are presented as mean ± SEM. n=4 cell isolates from distinct donor valves, *P <0.05 vs. control. C. Representative images of ICAM-1 immunofluorescence staining from 3 experiments show that both AVICs and monocytes express ICAM-1 (red) following the exposure to a low dose of Pam3CSK4 (0.03 µg/ml). Immunofluorescence staining of vimentin (green) and DAPI counterstaining (blue) were applied to identify AVICs and to outline all nuclei in the co-culture. Original magnification 40× objective.

Article Snippet: Antibody against TLR2 (NB100-56728) was obtained from Novus Biologicals, LLC (Centennial, CO).

Techniques: Co-Culture Assay, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Immunofluorescence, Staining

Journal: International Journal of Biological Sciences

Article Title: Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level

doi: 10.7150/ijbs.49332

Figure Lengend Snippet: Conditioned medium from Pam3CSK4-treated monocytes induces inflammatory responses in human AVICs. Conditioned medium was collected from monocyte cultures treated with Pam3CSK4 (0.10 µg/ml, Pam3 CM) or untreated controls (control CM). Pam3 CM and control CM, 0.15 ml each, were added to AVIC culture in 0.35 ml growth medium [achieving a 30% (vol/vol) for CM in AVIC culture], and AVICs were treated with control CM or Pam3 CM for 24 hours. A. Representative immunoblots and densitometric data show that Pam3 CM markedly up-regulates ICAM-1 and VCAM-1 expression in AVICs while neither control CM nor 0.03 µg/ml of Pam3CSK4 (equivalent to the concentration in Pam3 CM-treated AVIC culture) demonstrated this effect. *P <0.05 vs. untreated control . B. ELISA data shows that Pam3 CM stimulates AVICs to produce and release MCP-1. *P <0.05 vs. untreated control. C. AVICs were treated with DMSO or CU CPT 22 for 1 hour before Pam3 CM treatment for 24 hours. Inhibition of TLR2 in AVICs markedly attenuated ICAM-1 and VCAM-1 expression induced by Pam3 CM. *P <0.05 vs. untreated control, #P <0.05 vs. Pam3 CM+DMSO. All data are presented as mean ± SEM. n=4 cell isolates from distinct donor valves.

Article Snippet: Antibody against TLR2 (NB100-56728) was obtained from Novus Biologicals, LLC (Centennial, CO).

Techniques: Control, Western Blot, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: International Journal of Biological Sciences

Article Title: Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level

doi: 10.7150/ijbs.49332

Figure Lengend Snippet: Pam3 CM up-regulates TLR2 expression in AVICs to elicit cellular inflammatory responses. A. AVICs were exposed to Pam3CSK4 (0.03 μg/ml), control CM (30%) or Pam3 CM (30%) for 24 hours. Representative immunoblots and densitometric data show that Pam3 CM elevates TLR2 levels, but not MyD88 levels in AVICs. *P <0.05 vs. untreated control, Pam3CKS4-treated and control CM-treated. B. AVICs were treated with control CM or Pam3 CM for 4 to 24 hours . A representative immunoblot and densitometric data show that cellular TLR2 levels increase at 8, 12 and 24 hours following the exposure to Pam3 CM. *P <0.05 vs. control CM treatment for the same time period. Representative images of immunofluorescence staining confirmed the up-regulation of TLR2 (red) at 8-24 hours of treatment with Pam3 CM. The nuclei were visualized with DAPI (blue) counterstaining. Original magnification 40× objective. C and D. AVICs were transduced with lentivirus expressing control (mock) shRNA or TLR2 shRNA. After expression for 5 days, cells were treated with Pam3 CM for 24 hours. Knockdown of TLR2 abolished TLR2 up-regulation induced by Pam3 CM, and reduced the production of adhesion molecules and MCP-1. *P <0.05 vs. mock transduction, #P <0.05 vs. Pam3 CM + mock transduction. All quantitative data are presented as mean ± SEM. n=4 cell isolates from distinct donor valves in each group.

Article Snippet: Antibody against TLR2 (NB100-56728) was obtained from Novus Biologicals, LLC (Centennial, CO).

Techniques: Expressing, Control, Western Blot, Immunofluorescence, Staining, Transduction, shRNA, Knockdown

Journal: International Journal of Biological Sciences

Article Title: Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level

doi: 10.7150/ijbs.49332

Figure Lengend Snippet: TNF-α up-regulates TLR2 and contributes to the mechanism underlying the inflammatory responses in AVICs, but recombinant TNF-α is inadequate to induce the inflammatory responses. A. ELISA assay confirmed that Pam3 CM has greater levels of TNF-α in comparison to control CM. Data are presented as mean ± SEM. n=5, *P <0.05 vs. control CM. B. AVICs were treated for 24 hours with Pam3 CM pre-incubated with TNF-α neutralizing antibody (10 µg/ml) or non-immune mouse IgG (10 µg/ml). Neutralization of TNF-α abolished the effect of Pam3 CM on TLR2 up-regulation and attenuated the capacity of Pam3 CM to induce ICAM-1 and VCAM-1 expression in AVICs. *P <0.05 vs. untreated control, #P <0.05 vs. Pam3 CM and Pam3 CM + IgG. C. AVICs were treated with recombinant TNF-α (1.0 or 2.0 ng/ml) or Pam3 CM for 24 hours. TNF-α in the tested doses increased TLR2 levels in AVICs, but had no effect on the expression of ICAM-1 and VCAM-1. Data are presented as mean ± SEM. n=4 cell isolates from distinct donor valves in each group. *P <0.05 vs. untreated control.

Article Snippet: Antibody against TLR2 (NB100-56728) was obtained from Novus Biologicals, LLC (Centennial, CO).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Comparison, Control, Incubation, Neutralization, Expressing

Journal: International Journal of Biological Sciences

Article Title: Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level

doi: 10.7150/ijbs.49332

Figure Lengend Snippet: Schematic diagram depicting the mechanism underlying the interaction between monocytes and AVICs. Monocytes activated by TLR2 agonist release pro-inflammatory cytokines. Among these cytokines, TNF-α is responsible for up-regulating AVIC expression of TLR2. AVICs with greater levels of TLR2 become hypersensitive to TLR2 stimulation by a low level of activator.

Article Snippet: Antibody against TLR2 (NB100-56728) was obtained from Novus Biologicals, LLC (Centennial, CO).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: TFII-I/GTF2I regulates globin gene expression and stress response in erythroid cells

doi: 10.1016/j.jbc.2025.108227

Figure Lengend Snippet: Gene expression analysis in control and TFII-I deficient TF-1 cells. A and B , expression analysis of stress-response, erythroid-specific, and cell-cycle-specific gene in TF-1 cells harboring the control pLKO.1 vector or expressing TFII-I shRNA-2 or -3. Cells were subjected to RT-qPCR using primers specific for the genes listed on the x-axis; mRNA expression was normalized to that of GAPDH, and expression in empty vector (PLKO.1) harboring cells was set to 1 (∗∗∗: p < 0.0001; ∗∗∗: p < 0.001; ∗∗: p < 0.01; ∗: p < 0.05; two-tailed Students t test). C , Western blot analysis of Elongin A, TAF15, E2F6, CTCF, and β-actin in TF-1 cells harboring the pLKO.1 vector or expressing TFII-I shRNA-2 or-3. D , expression analysis of globin genes in TF-1 cells harboring the control pLKO.1 vector or expressing TFII-I shRNA-2. Cells were subjected to RT-qPCR using primers specific for the genes listed on the x-axis; mRNA expression was normalized to that of GAPDH, and expression in cells harboring the empty vector (PLKO.1) was set to 1. The error bars reflect the SEM from three independent experiments (∗∗∗∗: p < 0.001; ∗∗: p < 0.01; ns: nonsignificant, p > 0.05; two-tailed Students t test).

Article Snippet: The following primary antibodies were used: Rabbit polyclonal anti-CTCF (D31H2, Cell Signaling Technology Inc., Beverly, MA, USA), TAF15 (25521-1-AP, Proteintech Group, Inc, USA), TCEB3 (Elongin A) (A300–942A, Bethyl Laboratories, Inc), TFII- I (sc-9943, Santa Cruz Biotechnologies), αE2F6 (sc-390022, Santa Cruz Biotechnologies), GRP78 (ab21685, Abcam) and β-actin (15G5A11/E2, Thermo Fisher Scientific Inc).

Techniques: Gene Expression, Control, Expressing, Plasmid Preparation, shRNA, Quantitative RT-PCR, Two Tailed Test, Western Blot

Journal: Science Advances

Article Title: Coactivator condensation drives cardiovascular cell lineage specification

doi: 10.1126/sciadv.adk7160

Figure Lengend Snippet: ( A ) Representative micrographs (max projections) of COS-7 cells expressing MYOCD-mEGFP WT or ΔTAD below or above the critical concentration threshold. Scale bar, 5 μm. ( B ) Bar chart of the fraction of cells with condensates. Data are mean ± SEM. P values from Mann-Whitney test, **** P ≤ 0.0001. Three biological replicates total, n = 292 (WT) and 106 (ΔTAD). ( C ) Violin plot of MYOCD concentration and condensate formation in COS-7 cells expressing MYOCD WT or ΔTAD, where each dot represents one nucleus. ( D ) Representative micrographs (max projections) of COS-7 cells expressing MYOCD-mEGFP WT or ΔTAD above the critical concentration threshold. Scale bar, 5 μm. ( E ) Bar chart of the fraction of cells with condensates. Data are mean ± SEM. P values from Mann-Whitney test, **** P ≤ 0.0001. n ~ 40. ( F ) Schematic of MYOCD WT, ΔTAD, aromatic substitution to alanine (FWYtoA) mutants, and TAD domain chimeras with indicated disordered region (FUS and TAF15) with transcription factor (TF)–binding domains (blue box). See fig. S5A for detailed domain architecture of MYOCD. ( G and K ) Representative micrographs of COS-7 cells expressing indicated MYOCD variants above the critical concentration threshold. ( H and L ) Bar chart of the fraction of cells with condensates. Data are mean ± SEM. P values represent results of one-way analysis of variance (ANOVA) with Dunn’s multiple comparison test ( P values: ns P ≥ 0.05, **** P ≤ 0.0001). (H) n ~ 20. (l) n ~ 15. ( I and M ) Representative micrographs (max projections) of COS-7 cells expressing the fluorescent reporter and the indicated MYOCD variant above the critical concentration threshold. Scale bar, 5 μm. ( J and N ) Bar chart of the fraction of cells expressing reporter. Data are mean ± SEM. P values from one-way ANOVA with Dunn’s multiple comparison test ( P values: ns P ≥ 0.05, **** P ≤ 0.0001). (I) n ~ 40. (N) n ~ 20.

Article Snippet: For the chimeras, the DNA encoding the IDR regions from TAF15 (Addgene, no. 26379), FUS (Addgene, no. 26374), DDX4 (Addgene, no. 101225), NPHS1 (a gift from the Rosen laboratory), and CDT1 (a gift from the Parker laboratory) were PCR-amplified and inserted in-frame with the N-terminal (ΔTAD) region of MYOCD.

Techniques: Expressing, Concentration Assay, MANN-WHITNEY, Binding Assay, Comparison, Variant Assay

Journal: Cancer Research

Article Title: Diagnostic and Therapeutic Potential of a Human Antibody Cloned from a Cancer Patient That Binds to a Tumor-Specific Variant of Transcription Factor TAF15

doi: 10.1158/0008-5472.can-09-2186

Figure Lengend Snippet: Figure 2. Purification and identification of PAT-BA4 antigen by affinity chromatography and MALDI mass spectroscopy. A, protein purification of PAT-BA4 antigen from pooled membrane extracts of pancreas carcinoma cell line BxPC-3. Purified protein eluate was gained by affinity chromatography with crude membrane extracts. Western blot analysis was done with only secondary antibody, control IgG, and PAT-BA4 antibody. A Coomassie gel of purified protein eluate was performed. The protein band marked with an arrow was excised from Coomassie-stained gel. B, identification of the excised 78-kDa protein band by MALDI peptide mass mapping. The peaks with asterisks matched the calculated masses of tryptic peptides of human TAF15 (NP_631961.1 isoform 1/NP_003478.1 isoform 2). Thereby, the peptide mass error was <30 ppm and the minimum sequence coverage of the corresponding amino acids equates 22%.

Article Snippet: On both blots, the 78-kDa band was detected with anti-TAF15 antibody (3 μg/ mL; AVIVA Systems Biology).

Techniques: Purification, Affinity Chromatography, Mass Spectrometry, Protein Purification, Membrane, Western Blot, Control, Staining, Sequencing

Journal: Cancer Research

Article Title: Diagnostic and Therapeutic Potential of a Human Antibody Cloned from a Cancer Patient That Binds to a Tumor-Specific Variant of Transcription Factor TAF15

doi: 10.1158/0008-5472.can-09-2186

Figure Lengend Snippet: Figure 3. Validation of TAF15 as target for PAT-BA4 antibody by siRNA technology. Pancreas carcinoma cell line BxPC-3 was transfected with siRNA against human TAF15. Forty-eight hours after transfection, protein level of TAF15 was monitored by FACS analysis. Nontransfected cells and cells transfected with scrambled siRNA showed a positive binding of (A) PAT-BA4 antibody and (B) anti-CD55 antibody. TAF15 siRNA-transfected cells showed a reduced PAT-BA4 antibody binding but an unchanged binding of anti-CD55 antibody. Percentages of cell surface antigens of (C) PAT-BA4 and (D) anti-CD55 antibody are depicted. Columns, mean; bars, SD. *, P < 0.05.

Article Snippet: On both blots, the 78-kDa band was detected with anti-TAF15 antibody (3 μg/ mL; AVIVA Systems Biology).

Techniques: Biomarker Discovery, Transfection, Binding Assay

Journal: Cancer Research

Article Title: Diagnostic and Therapeutic Potential of a Human Antibody Cloned from a Cancer Patient That Binds to a Tumor-Specific Variant of Transcription Factor TAF15

doi: 10.1158/0008-5472.can-09-2186

Figure Lengend Snippet: Figure 4. Comparison of TAF15PAT-BA4 and wild-type TAF15 by Western blotting, FACS analysis, and immunohistochemical staining. A, Western blot inhibition assay with PAT-BA4 (300 μg/mL) and anti-TAF15 (3 μg/mL) antibody. One assay of each antibody was preincubated with the opponent antibody to check blocking effects; the other one was performed without the opponent antibody. B, FACS analyses with either PAT-BA4 (300 μg/mL) or commercial anti-TAF15 antibody (30 μg/mL; Santa Cruz Biotechnology) were performed on BxPC-3 cells and lymphocytes. Both assays were done with the same cell amount (2 × 105) and the same antibody concentrations. C, immunohistochemical staining of pancreas normal tissue and pancreas adenocarcinoma tissue. The paraffin-embedded sections were incubated with either PAT-BA4 (100 μg/mL) or anti-TAF15 antibody (1:25).

Article Snippet: On both blots, the 78-kDa band was detected with anti-TAF15 antibody (3 μg/ mL; AVIVA Systems Biology).

Techniques: Comparison, Western Blot, Immunohistochemical staining, Staining, Inhibition, Blocking Assay, Incubation

Journal: Cancer Research

Article Title: Diagnostic and Therapeutic Potential of a Human Antibody Cloned from a Cancer Patient That Binds to a Tumor-Specific Variant of Transcription Factor TAF15

doi: 10.1158/0008-5472.can-09-2186

Figure Lengend Snippet: Figure 5. Important characteristics of TAF15 protein and PAT-BA4 antibody. Expression of TAF15 on (A) protein and (B) mRNA level. A, Western blot analysis with anti-TAF15 (3 μg/mL) antibody on cell lysates of BxPC-3 cells and lymphocytes made of the same cell amount (1.2 × 106). B, semiquantitative PCR with mRNA from malignant and nonmalignant lung tissue. GAPDH mRNA level was used as internal standard. Functional assays with PAT-BA4 on BxPC-3 cells. C, histogram of adhesion assay with BxPC-3 cells. For tumor cell engraftment, the BxPC-3 cells were incubated with PAT-BA4 antibody. Chrompure human IgG served as isotype control. The amount of adherent cells was determined after different periods of time. Untreated cells grown in complete RPMI 1640 were adjusted to 100%. D, motility assay with BxPC-3 cells. The tumor cells were grown in 24-well plates to a confluence of 80%. A cross was then scratched into the cell monolayer and PAT-BA4 antibody was added. Complete RPMI 1640 and unrelated human IgG served as negative controls. Columns, mean; bars, SD. *, P < 0.05.

Article Snippet: On both blots, the 78-kDa band was detected with anti-TAF15 antibody (3 μg/ mL; AVIVA Systems Biology).

Techniques: Expressing, Western Blot, Functional Assay, Cell Adhesion Assay, Incubation, Control, Motility Assay