Journal: American Journal of Translational Research

Article Title: The embryonic transcription factor Brachyury confers chordoma chemoresistance via upregulating CA9

doi:

Figure Lengend Snippet: DNA primers used in the current study

Article Snippet: Immunoblotting was performed as previously described [ 18 ] and the following antibodies were used: rabbit anti-Brachyury antibody (Santa Cruz Biotechnology, cat. # sc-20109), rabbit anti-CA9 antibody (Santa Cruz Biotechnology, cat. # sc-25599), and mouse anti-β-actin antibody (Sigma Aldrich, cat. # A1978).

Techniques:

Journal: American Journal of Translational Research

Article Title: The embryonic transcription factor Brachyury confers chordoma chemoresistance via upregulating CA9

doi:

Figure Lengend Snippet: Brachyury-targeting genes in PCH1 cells. A. Validation of expression changes of representative genes detected in microarray analysis by RT-PCR in PCH1 negative control cells (shN) and Brachyury knockdown PCH1 cells (shT). B. Validation of expression changes of representative genes by RT-PCR in brachyury overexpression PCH1 cell. C. Validation of expression changes of representative genes by RT-PCR in U2OS cell with Brachyury overexpression. D. A graphic summary of the alteration of gene expression shows that CA9 and HHIP might be the best possibility to serve as the target regulated by T gene. The genes in the blue ellipse were down-regulated, while the genes in the red ellipse were up-regulated.

Article Snippet: Immunoblotting was performed as previously described [ 18 ] and the following antibodies were used: rabbit anti-Brachyury antibody (Santa Cruz Biotechnology, cat. # sc-20109), rabbit anti-CA9 antibody (Santa Cruz Biotechnology, cat. # sc-25599), and mouse anti-β-actin antibody (Sigma Aldrich, cat. # A1978).

Techniques: Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Negative Control, Over Expression

Journal: American Journal of Translational Research

Article Title: The embryonic transcription factor Brachyury confers chordoma chemoresistance via upregulating CA9

doi:

Figure Lengend Snippet: Transcriptional regulation of CA9 by T gene. ChIP assay was carried out using nuclear extracts from the H293T cells with the transfection of pcDNA3.1-Flag-Brachyury plasmid and an antibody against Flag, followed by RT-PCR analysis. Primers were designed for the 10 parts of the 5’ CA9 promoter sequence (-1/-2919 bp). IgG was taken as a negative control. DNA fragments corresponding to the 2, 4 and 10 segment of promoter sequence (the -2699 bp to -1700 bp and the -199 bp to -1 bp region) were specifically pulled down.

Article Snippet: Immunoblotting was performed as previously described [ 18 ] and the following antibodies were used: rabbit anti-Brachyury antibody (Santa Cruz Biotechnology, cat. # sc-20109), rabbit anti-CA9 antibody (Santa Cruz Biotechnology, cat. # sc-25599), and mouse anti-β-actin antibody (Sigma Aldrich, cat. # A1978).

Techniques: Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Sequencing, Negative Control

Journal: American Journal of Translational Research

Article Title: The embryonic transcription factor Brachyury confers chordoma chemoresistance via upregulating CA9

doi:

Figure Lengend Snippet: The immunostaining score of CA9 and Brachyury expression

Article Snippet: Immunoblotting was performed as previously described [ 18 ] and the following antibodies were used: rabbit anti-Brachyury antibody (Santa Cruz Biotechnology, cat. # sc-20109), rabbit anti-CA9 antibody (Santa Cruz Biotechnology, cat. # sc-25599), and mouse anti-β-actin antibody (Sigma Aldrich, cat. # A1978).

Techniques: Immunostaining, Expressing

Journal: American Journal of Translational Research

Article Title: The embryonic transcription factor Brachyury confers chordoma chemoresistance via upregulating CA9

doi:

Figure Lengend Snippet: IHC staining of Brachyury and CA9. IHC staining was performed on the adjacent sections of the same sample to evaluate the correlation between Brachyury and CA9. The strong staining of Brachyury in one chordoma case which also exhibited strong staining of CA9 (the upper panel); reduced staining of Brachyury in one chordoma case, which also displayed weak staining of CA9 (the lower panel). In addition, non-parametric Spearman correlation tests were used to statistically analyze the correlation of Brachyury and CA9 expression score, with results showed in Table 2.

Article Snippet: Immunoblotting was performed as previously described [ 18 ] and the following antibodies were used: rabbit anti-Brachyury antibody (Santa Cruz Biotechnology, cat. # sc-20109), rabbit anti-CA9 antibody (Santa Cruz Biotechnology, cat. # sc-25599), and mouse anti-β-actin antibody (Sigma Aldrich, cat. # A1978).

Techniques: Immunohistochemistry, Staining, Expressing

Journal: American Journal of Translational Research

Article Title: The embryonic transcription factor Brachyury confers chordoma chemoresistance via upregulating CA9

doi:

Figure Lengend Snippet: CA9 protects PCH1 from paclitaxel induced apoptosis. A. Western blot analysis of Brachyury, CA9 and β-actin in the transfected PCH1 cells, which were used for the cell viability assay. B. CA9 protects PCH1 from Paclitaxel induced apoptosis. Cell viability was measure by CCK-8 assay. Each experiment was repeated three times. *indicates P-value <0.05 and **indicates P-value <0.01.

Article Snippet: Immunoblotting was performed as previously described [ 18 ] and the following antibodies were used: rabbit anti-Brachyury antibody (Santa Cruz Biotechnology, cat. # sc-20109), rabbit anti-CA9 antibody (Santa Cruz Biotechnology, cat. # sc-25599), and mouse anti-β-actin antibody (Sigma Aldrich, cat. # A1978).

Techniques: Western Blot, Transfection, Viability Assay, CCK-8 Assay

Journal: bioRxiv

Article Title: Glutamine Deprivation Regulates the Origin and Function of Cancer Cell Exosomes

doi: 10.1101/859447

Figure Lengend Snippet: HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System. (A) Sections immunostained for CD31, which labels endothelial cells and blood vessels, with blood vessel number (upper) and total area (lower) represented in bar charts. (B) Sections immunostained for Ki67, which stains proliferative cells. Proportion of tumour cells with Ki69 staining is represented in bar chart. (C) Sections stained with haematoxylin and eosin, which highlights necrotic regions. Proportion of tumour area that is necrotic is represented in bar chart. (D) Sections immunostained for CA9, which is expressed in hypoxic regions. Proportion of tumour cells with CA9 staining is represented in bar chart. (E) Schematic model showing how in cancer cells, regulation of endosomal trafficking by depletion of exogenous glutamine or reduced Akt/mTORC1 signalling can induce a switch in exosome production from the established Rab7-late endosomal mutivesicular bodies (MVBs; producing ‘CD63-exosomes’), to Rab11a-labelled recycling endosomal MVBs (generating ‘Rab11a-exosomes’). In recipient cells, the resulting vesicles can increase ERK signalling and cell growth in tumour cells, and enhance growth and stability of endothelial network formation. Thickness of arrows indicates levels of membrane flux through the two exosome-generating endosomal routes. Scale bar is 250 µm, except for (C), which is 500 µm. Data were analysed by one-way ANOVA; n = 7; * P < 0.05, ** P < 0.01, *** P < 0.001. See also .

Article Snippet: Endogenous peroxidase activity was blocked before slides were stained with antibodies diluted 1:100, namely rabbit anti-Ki67 (M7240; Dako, Glostrup, Denmark), rabbit anti-CA9 (M75; BD Biosciences), and mouse anti-CD31 (JC70, Dako) for 1 h at 22°C.

Techniques: Produced, Injection, Isolation, Staining