Journal: Microorganisms

Article Title: Therapeutic Potential of Probiotic-Derived P8 Protein as an Anti-Metastatic Agent in Colorectal Cancer

doi: 10.3390/microorganisms13092175

Figure Lengend Snippet: Characteristics of P8 target proteins in DLD-1 cells. ( A ) Venn diagram illustrating the overlap of P8-bound proteins identified in DLD-1 cell lysates across three independent experiments. Shown are the distributions in each experiment compared with the distributions of shared and specific P8 targets. The numbers in parentheses reflect the number of detectable targets. ( B ) Evaluation of P8 interaction with Smad1 by immunoprecipitation. Aliquots containing 1 μg each of P8 and Smad1 in PBS were mixed for 12 h and applied to an anti-P8 IgG-coupled protein A column; lane 1, unbound Smad1; lane 2, final wash fraction; lane 3, eluted fraction; lane M, size markers. Immunoprecipitation results were visualized using anti-Smad1 IgG.

Article Snippet: Transfection was performed using Smad1 shRNA (Santa Cruz Biotechnology, Dallas, TX, USA) and Lipofectamine 3000 (Invitrogen, Paisley, UK) following the manufacturer’s recommendations [ ].

Techniques: Immunoprecipitation

Journal: Microorganisms

Article Title: Therapeutic Potential of Probiotic-Derived P8 Protein as an Anti-Metastatic Agent in Colorectal Cancer

doi: 10.3390/microorganisms13092175

Figure Lengend Snippet: P8-associated anti-metastasis cell signaling in CRC. Inhibition of the BMP pathway strongly inhibited Smad1-induced EMT and cell adhesion in DLD-1 cells. ( A ) Schematic diagram of the BMP signal pathway with P8. After interacting with Smad1, P8 strongly interfered with Smad1-associated EMT signal transduction by inhibiting the phosphorylation of Smad1 and its complexes (Smad1/5 and Smad1/5/8). ( B ) Western blot analysis of whole-cell lysates from DLD-1 cells left untreated or treated with P8 (40 µM, 72 h). Protein identities are indicated on the left; GAPDH served as a loading control. Quantification of protein expression related to EMT and cell–cell adhesion. Band intensities were measured using ImageJ software (Version 1.54p) and normalized to GAPDH.

Article Snippet: Transfection was performed using Smad1 shRNA (Santa Cruz Biotechnology, Dallas, TX, USA) and Lipofectamine 3000 (Invitrogen, Paisley, UK) following the manufacturer’s recommendations [ ].

Techniques: Inhibition, Transduction, Phospho-proteomics, Western Blot, Control, Expressing, Software

Journal: Microorganisms

Article Title: Therapeutic Potential of Probiotic-Derived P8 Protein as an Anti-Metastatic Agent in Colorectal Cancer

doi: 10.3390/microorganisms13092175

Figure Lengend Snippet: Verification of Smad1 as being associated with the inhibition of migration activity of P8 in DLD-1 cells. To determine whether Smad1 was directly targeted by P8, the P8 sensitivity of ∆Smad1 cells was assessed using the wound healing method. Cells were transfected with Smad1 shRNA or nonspecific shRNA (wild type), and their sensitivity to P8 was compared. ( A ) Assessment of Smad1 knockout by Smad1 shRNA. ( B ) Wound healing assay, showing that ∆Smad1 cells were less sensitive to P8 treatment than wild-type DLD-1 cells. Wound recovery was analyzed using Image J software (Version 1.54p).

Article Snippet: Transfection was performed using Smad1 shRNA (Santa Cruz Biotechnology, Dallas, TX, USA) and Lipofectamine 3000 (Invitrogen, Paisley, UK) following the manufacturer’s recommendations [ ].

Techniques: Inhibition, Migration, Activity Assay, Transfection, shRNA, Knock-Out, Wound Healing Assay, Software

Journal: PLoS ONE

Article Title: Endoglin Requirement for BMP9 Signaling in Endothelial Cells Reveals New Mechanism of Action for Selective Anti-Endoglin Antibodies

doi: 10.1371/journal.pone.0050920

Figure Lengend Snippet: A–C) HUVECs were serum-starved for 3 hours and stimulated with increasing amounts of TGFβ1 or BMP9 for 30 minutes. Levels of SMAD3, pSMAD3, pSMAD1/5/8 and β-Actin in the total cell extracts were assessed by western blotting. (D) Total RNA extract from HUVECs was subjected to reverse transcription and quantitative PCR assessment of the SMAD1, 2, 3, 5 and 8 transcripts relative to the levels of the β-Actin transcript. (E) HUVECs were serum-starved for 3 hours and treated with increasing amounts of BMP9 and levels of phospho-SMAD1 normalized for levels of total SMAD1 were monitored using a SMAD1/pSMAD1 MSD assay. (F-G) Primary human ECs (HUVEC, HMVECd, HAEC, HPAEC –2500 cells/well) and immortalized mouse EC lines (MS1, EOMA, bEnd.3, SVR, C166–1250 cells/well) were serum-starved for 3 hours and monitored for pSMAD1 induction with a SMAD1/pSMAD1 MSD assay upon treatment with BMP9 or increasing amounts of TGFβ1 for 30 minutes. Results are the mean +/− standard deviation of technical triplicates.

Article Snippet: The following primary antibodies were used for western blot detection: ENG (anti-human CD105, BD Biosciences, Cat# 611315), pSMAD1/5/8 (Cell Signaling Technology #9511), SMAD1 (R&D Systems, Cat# AF2039), SMAD3 (Abcam #ab75512), pSMAD3 (Abcam #ab51451), beta-Actin (Sigma #A2228).

Techniques: Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: PLoS ONE

Article Title: Endoglin Requirement for BMP9 Signaling in Endothelial Cells Reveals New Mechanism of Action for Selective Anti-Endoglin Antibodies

doi: 10.1371/journal.pone.0050920

Figure Lengend Snippet: A) HUVECs were reversed-transfected in a 96-well plate with Mock, Random or ENG siRNAs for 48 hours then serum-starved for 3 hours prior to stimulation with BMP9 (0.1 ng/ml) (+) or PBS (−). Levels of ENG, pSMAD1/5/8, SMAD1 and β-Actin in the total cell extracts were measured by western blotting. (B–D) ECs were dispensed in each well of a 96-well plate (HUVEC, HPAEC, HAEC –2500 cells/well, MS1–1250 cells/well) in serum free media. Cells were incubated with antibodies at various concentrations for 1 hour and stimulated with BMP9 (0.1 ng/ml) (+) or PBS (−) for 30 min prior to lysis. Levels of pSMAD1 normalized for levels of total SMAD1 were monitored using a SMAD1/pSMAD1 MSD assay. The following antibodies were used: rIgG2a (rat IgG2a isotype control), M999 (rat IgG2a anti-human ENG), hIgG2 (human IgG2 isotype control), cSN6j (human IgG2 anti-human ENG), Ab3209 (mouse IgG2b anti-human BMP9), M1041-43 (rat IgG2a anti-mouse ENG). * Antibody concentrations of Ab3209 used were 10 times lower than other antibodies. Results are the mean +/− standard deviation of technical triplicates (** = P Value <10E-04; *** = P value <10E-03).

Article Snippet: The following primary antibodies were used for western blot detection: ENG (anti-human CD105, BD Biosciences, Cat# 611315), pSMAD1/5/8 (Cell Signaling Technology #9511), SMAD1 (R&D Systems, Cat# AF2039), SMAD3 (Abcam #ab75512), pSMAD3 (Abcam #ab51451), beta-Actin (Sigma #A2228).

Techniques: Transfection, Western Blot, Incubation, Lysis, Control, Standard Deviation

Journal: PLoS ONE

Article Title: Endoglin Requirement for BMP9 Signaling in Endothelial Cells Reveals New Mechanism of Action for Selective Anti-Endoglin Antibodies

doi: 10.1371/journal.pone.0050920

Figure Lengend Snippet: A–B) HUVECs were dispensed in a 96 well plate (3000 cells/well) and grown in 0.1% FBS and incubated with the following antibodies at various concentrations for 72 hours, with a daily change of media: (A) mIgG2b (mouse IgG2b isotype control) and Ab3209 (mouse IgG2b anti-BMP9); (B) rIgG2a (rat IgG2a isotype control), M999 (rat IgG2a anti-human ENG), hIgG2 (human IgG2 isotype control) and cSN6j (human IgG2 anti-human ENG). After 72 hours, cell growth in each well was assessed using Cell Titer-Glo®. (C–D) GFP-labeled HUVECs admixed with human dermal fibroblasts dispensed in 96 well plates were treated on day 0 with angiogenic factors: VEGF (1 ng/ml) or bFGF (2 ng/ml). Negative controls wells (No AF) contained no angiogenic factors. Measurement of Branch Points were performed at day 13.5 following treatment with PBS (no Ab), Suramin (20 μM – positive control that inhibits RTK-mediated tube formation), or the following antibodies: hIgG2 (isotype control), cSN6j (hIgG2 anti-human ENG), rIgG2a (isotype control), M999 (rIgG2a anti-human ENG), mIgG2 (isotype control) or Ab3209 (mIgG2 anti-human BMP9). Antibody concentrations used in (C): 10 μg/ml; in (D): 20 μg/ml. (E) Representative image of individual wells from the HUVEC tube formation assay following treatment with bFGF (2 ng/ml) and the indicated antibody (20 μg/ml). (F) HUVECs were dispensed in each well of a 96-well plate (2500 cells/well) in serum free media. Cells were incubated with antibodies at various concentrations for 1 hour and stimulated with BMP9 (0.1 ng/ml) (+) or PBS (−) for 30 min prior to lysis. Levels of pSMAD1 normalized for levels of total SMAD1 were monitored using a SMAD1/pSMAD1 MSD assay. The following antibodies were used: mIgG1k (mouse IgG1k isotype control), SN6h (mouse IgG1k anti-human ENG), hIgG1 (human IgG1 isotype control), TRC105 (human IgG1 anti-human ENG). (G) GFP-labeled HUVECs admixed with human dermal fibroblasts dispensed in 96-well plates were treated on day 0 with bFGF (2 ng/ml); No Angiogenic Factor (No AF) wells did not receive bFGF. Measurement of Branch Points were performed at day 13.5 following treatment with the following antibodies: mIgG1k (mouse IgG1k isotype control), SN6h (mouse IgG1k anti-human ENG), hIgG1 (human IgG1 isotype control), TRC105 (human IgG1 anti-human ENG). Antibody concentration used in (G): 10 μg/ml. Results are the mean +/− standard deviation of technical triplicates. Results are the mean +/− standard deviation of technical triplicates (Δ = P value >0.05; * = P value <0.05; ** = P value <0.005; *** P = value <0.001).

Article Snippet: The following primary antibodies were used for western blot detection: ENG (anti-human CD105, BD Biosciences, Cat# 611315), pSMAD1/5/8 (Cell Signaling Technology #9511), SMAD1 (R&D Systems, Cat# AF2039), SMAD3 (Abcam #ab75512), pSMAD3 (Abcam #ab51451), beta-Actin (Sigma #A2228).

Techniques: Incubation, Control, Labeling, Positive Control, HUVEC Tube Formation Assay, Lysis, Concentration Assay, Standard Deviation