Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: The CSPG4 gene is activated by Myocardin-Related Transcription Factors (MRTFs). To examine the transcriptional control of NG2/ CSPG4 we correlated the CSPG4 mRNA versus all other mRNAs ( www.GTExPortal.org ) and calculated the sum of correlation coefficients (R sum ) for all transcripts across 20 human tissues. Panel ( A ) shows the positive extreme of the resulting R sum distribution which has a theoretical maximum of 20 (seen only for CSPG4 itself, not included). Among the 600 (≈1%) most tightly correlating mRNAs we found classical smooth muscle cell (SMC) markers ( TAGLN , MYH11 ) and transcription factors ( SRF , MYOCD , MKL1 , all indicated by different green symbols). Two cell lineage markers ( MCAM and AOC3 , red symbols) were among the mRNAs in the absolute extreme (top 25). Examples of correlations between SRF , MYOCD and MKL1 versus CSPG4 in the transverse colon (N = 274) are shown in panels ( B – D ). P-values and Spearman Rho-coefficients are given in the respective panels. In panel ( E ), adenoviruses were used for overexpression of MRTFs (MYOCD, MRTF-A/ MKL1 , and MRTF-B/ MKL2 ) in primary human coronary artery smooth muscle cells in vitro. The CSPG4 mRNA level was determined by RT-qPCR and compared to that in cells treated with empty virus (ANOVA, followed by Dunnett's Multiple Comparison Test versus Null, N = 6 for all treatments). In this and the following figures showing RT-qPCR data, the relative mRNA level is represented by the official gene symbol in italics. Transcript levels were normalized to 18S as housekeeping gene (Pfaffl equation) and are given as fold change (FC). All statistical comparisons in panel ( E ) of figures 1 through 3 are versus Null as indicated by brackets. Panel ( F ) shows a western blot for NG2/CSPG4 following treatment with control (Null) virus and viruses encoding MRTFs. Membranes were cut horizontally in this, and the following, figures to allow for detection of multiple proteins on the same membrane. Full length blots (as full as possible) are found in Fig. . Panel ( G ) shows summarized data for the western blot experiments (N = 3). The bands migrating at ≥ 250 kDa were included in the analysis. In panel ( H ), cells were transduced with tagged MRTF-A/MKL1 (blue), fixed at 96 h, and stained for NG2/CSPG4 (red) and the intermediate filament synemin/SYNM (green), followed by confocal imaging. Red and green labels are shown in black and white below the colored panels for clarity. Summarized data from two independent experiments with three independent replicates each time (N = 6) is shown in panel ( I ). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, all versus Null.

Article Snippet: We used the following primary antibodies CSPG4 (MAB2029, clone 9.2.27, Millipore), MCAM (SAB5600062, Sigma Aldrich), AOC3 (MAB3957, R&D Systems, and SAB2501957, Sigma-Aldrich), KDM3A (12835-1-AP, Proteintech), H3K9Me2 (4658S, Cell Signaling Technology), Histone H3 (4499S, Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204, 9101S, Cell Signaling Technology), total ERK1/2 (9102S, Cell Signaling Technology), HSP90 (#610418, BD Biosciences), GAPDH (MAB374 from Sigma Aldrich).

Techniques: Over Expression, In Vitro, Quantitative RT-PCR, Virus, Comparison, Western Blot, Membrane, Transduction, Staining, Imaging

Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: AOC3 also resides in the CSPG4 co-expression module and is regulated by MRTFs. Panels ( A ) and ( B ) show correlations between MYOCD and AOC3 in the colon and prostate, respectively. Panels ( C ) and ( D ) show that AOC3 also correlates with CSPG4 and MCAM (ovary). Panel ( E ) shows mRNA levels for AOC3 following overexpression of MRTFs (all statistical comparisons versus Null). Panel ( F ) shows confocal imaging of AOC3 fluorescence in cells transduced with Null virus and with MRTF-A/MKL1-encoding virus. Panel ( G ) shows summarized data from the experiments in ( F ). Panels ( H ) through ( K ) examine if MYOCD is antagonistic with MRTF-A/ MKL1 . No antagonism was noted for the classical target gene ACTA2 ( H ), for CSPG4 ( I ), for MCAM ( J ) or for AOC3 ( K ). *P < 0.05, ***P < 0.001, versus null.

Article Snippet: We used the following primary antibodies CSPG4 (MAB2029, clone 9.2.27, Millipore), MCAM (SAB5600062, Sigma Aldrich), AOC3 (MAB3957, R&D Systems, and SAB2501957, Sigma-Aldrich), KDM3A (12835-1-AP, Proteintech), H3K9Me2 (4658S, Cell Signaling Technology), Histone H3 (4499S, Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204, 9101S, Cell Signaling Technology), total ERK1/2 (9102S, Cell Signaling Technology), HSP90 (#610418, BD Biosciences), GAPDH (MAB374 from Sigma Aldrich).

Techniques: Expressing, Over Expression, Imaging, Fluorescence, Transduction, Virus

Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: The MCAM and AOC3 gene loci have SRF binding motifs that confer responsiveness to MRTF-SRF signaling. Panel ( A ) shows graphical representations of the CSPG4 , MCAM and AOC3 gene loci with known (red arrows, ENCODE data accessible via the UCSC genome browser) and predicted (lighter red/pink arrows) binding sites for MRTF-SRF . Gene loci and binding sites are not drawn to scale. Panel ( B ) shows effects of SRF silencing (by 43 ± 4%, P < 0.0001, N = 6), using a short hairpin construct, on the indicated mRNA levels measured by RT-qPCR. Panel ( C ) shows reporter assays for MCAM (S1-S3 plasmid and S2-S4 plasmid, see panel A ) and AOC3 . We used HEK 293 cells for transfection of the reporter plasmids in ( C ), because these cells were more readily transfected compared to human coronary artery smooth muscle cells used elsewhere. Panels ( D ) and ( E ) show time-courses of mRNA induction following overexpression of myocardin (N = 3 for all time points). CSPG4 , MCAM and AOC3 were all increased at least as fast as the direct target gene ACTA2 . Panel ( F ) shows that MRTF-A/MKL1 increases the mRNA level of KDM3A, and that short hairpin silencing of KDM3A (shKDM3A) antagonizes this effect (N = 5–6). Panel ( G ) shows RT-qPCR for MCAM , AOC3 , and CSPG4 in MRTF-A/MKL1-transduced cells in the absence and presence of shKDM3A (N = 5–6). Panel ( H ) shows western blots for cells transduced with null, MKL1, and MKL1 plus shKDM3A viruses. The bar graph at the bottom shows the quantitative analysis for MCAM (vs. HSP90). Quantitative analysis of the KDM3A protein level similarly showed it to be significantly increased by MRTF-A transduction and reduced by KDM3A silencing (not shown). Panel ( I ) shows the effect of MRTF-A/MKL1 on transcript levels of three transcription factors ( SOX10 , ASCL1 , and OLIG2 ) that control CSPG4 expression in brain glial cells. None of these transcription factors were significantly increased, while the positive controls ( ACTA2 , CSPG4 ) were increased in the same samples (N = 6).

Article Snippet: We used the following primary antibodies CSPG4 (MAB2029, clone 9.2.27, Millipore), MCAM (SAB5600062, Sigma Aldrich), AOC3 (MAB3957, R&D Systems, and SAB2501957, Sigma-Aldrich), KDM3A (12835-1-AP, Proteintech), H3K9Me2 (4658S, Cell Signaling Technology), Histone H3 (4499S, Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204, 9101S, Cell Signaling Technology), total ERK1/2 (9102S, Cell Signaling Technology), HSP90 (#610418, BD Biosciences), GAPDH (MAB374 from Sigma Aldrich).

Techniques: Binding Assay, Construct, Quantitative RT-PCR, Plasmid Preparation, Transfection, Over Expression, Western Blot, Transduction, Expressing

Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: SMC differentiation, actin dynamics, and loss of Ternary Complex Factors (TCFs) all affect AOC3 , MCAM, and CSPG4 expression. In panel ( A ), human coronary artery smooth muscle cells were incubated with either growth supplement (GS) or differentiation supplement (DS) for 72 h and the mRNA levels of AOC3 , MCAM , and CSPG4 were determined by RT-qPCR (N = 6). In panels ( B ) and ( C ), the effects of Latrunculin B (LatB), which depolymerizes actin, were tested using two different protocols ( B 24 h static, and C 20 h with drug + 4 h washout in cycles for 96 h, N > 6). Transcript levels were assayed using RT-qPCR. All cells in ( B ) and ( C ) were transduced with MRTF-A/ MKL1 . Panel ( D ) (72 h treatment) and panel ( E ) (time-course) show effects of the MRTF-SRF inhibitor CCG-1423. Panel ( F ) compares transcript levels in freshly isolated mouse caudal arteries with mouse caudal arteries that were organ cultured in the presence of vehicle (DMSO) or CCG-1423 (96 h). In panel ( G ), the levels of Aoc3 , Mcam , and Cspg4 were determined by RT-qPCR in mouse embryonic fibroblasts (MEFs) from wild type (WT) mice, and in MEFs from mice that lack three Ternary Complex Factors (Elk1, Elk3, and Elk4). *P < 0.05, **P < 0.01, ***P < 0.001, versus the respective controls.

Article Snippet: We used the following primary antibodies CSPG4 (MAB2029, clone 9.2.27, Millipore), MCAM (SAB5600062, Sigma Aldrich), AOC3 (MAB3957, R&D Systems, and SAB2501957, Sigma-Aldrich), KDM3A (12835-1-AP, Proteintech), H3K9Me2 (4658S, Cell Signaling Technology), Histone H3 (4499S, Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204, 9101S, Cell Signaling Technology), total ERK1/2 (9102S, Cell Signaling Technology), HSP90 (#610418, BD Biosciences), GAPDH (MAB374 from Sigma Aldrich).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Transduction, Isolation, Cell Culture

Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: Co-expression of CD146/ MCAM , NG2/ CSPG4 , and VAP1/ AOC3 in endothelial cells and pericytes in the human brain. Human brain specimen stained with antibodies versus MCAM ( A ) and CSPG4 ( B ) showed co-expression in pericytes and endothelial cells (overlay in C ). MCAM expression in the human brain was restricted to vascular structures ( D ). Panel ( E ) shows four examples of immunohistochemical staining for AOC3 (brown) in the human brain from the Human Protein Atlas (HPA) . Endothelial cells and pericytes (arrows) in capillaries and larger vessels were positive. Panel ( F ) shows a tentative model for regulation of MCAM , CSPG4 and AOC3 by MRTFs in pericytes (and endothelial cells) at the human blood–brain barrier. In the illustration, MRTF refers to the three myocardin-related transcription factors MYOCD, MRTF-A/MKL1, and MRTF-B/MKL2. Upstream activators of MRTFs were not examined herein, but some possibilities are given, such as sphingosine-1-phospate (S1P) and transforming growth factor β (TGFβ). Panel ( G ) shows RT-qPCR data for two validated markers of pericytes, RGS5 and PDGFRB (N = 6, 8 days of transduction) in control conditions and after overexpression of MYOCD. Panel ( H ) shows time-course data for the RGS5 transcript on overexpression of MYOCD and MRTF-A/MKL1, respectively (N = 4 for all times). Null data was generated for each time and used for statistical comparisons but was omitted from the graph for clarity. Panel ( I ) shows staining for TINAGL1 in human cerebral microvessels (from the HPA).

Article Snippet: We used the following primary antibodies CSPG4 (MAB2029, clone 9.2.27, Millipore), MCAM (SAB5600062, Sigma Aldrich), AOC3 (MAB3957, R&D Systems, and SAB2501957, Sigma-Aldrich), KDM3A (12835-1-AP, Proteintech), H3K9Me2 (4658S, Cell Signaling Technology), Histone H3 (4499S, Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204, 9101S, Cell Signaling Technology), total ERK1/2 (9102S, Cell Signaling Technology), HSP90 (#610418, BD Biosciences), GAPDH (MAB374 from Sigma Aldrich).

Techniques: Expressing, Staining, Immunohistochemical staining, Quantitative RT-PCR, Transduction, Over Expression, Generated

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: The MF marker mAb PR2D3 recognizes the AOC3 protein in human colonic lysates. (A) Immunofluorescence staining of cryostat sections of normal colon and CRC tissue using PR2D3 (green) for MFs and AUA-1 (red) to identify epithelial cells. (B) The immunoblot of human colonic smooth muscle (SM) lysate using mAb PR2D3 shows a band of about 150 kDa in a native (nonreduced) sample.

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Marker, Immunofluorescence, Staining, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: Mascot search result for protein identification of AOC3 and MYH11 using MALDI-TOF-MS/MS. (A) Electrophoretic analysis of proteins immunoprecipitated using PR2D3 and the IgG isoform under reduced and native conditions. The bands indicated by arrows were excised, digested with trypsin, and analyzed by MALDI-TOF/TOF. The marker lane is included on the left with sizes in kDa. The heavily stained lower-molecular-weight bands are Ig products. The mascot score histogram on the right showed AOC3 as the one significant match for the 100-kDa band. The only significant mascot score histogram match for the 250-kDa band was MYH11. The protein score on the x axis is −10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 56 are considered significant. (B) Protein immunoblot of human colonic smooth muscle lysate with anti-AOC3 (TK8-14) showing a band of about 150 kDa in the native (nonreduced) sample giving the same expression profile as PR2D3. (C) Western blot of immunoprecipitated smooth muscle lysate probed with PR2D3. The same antigen was recognized by anti-AOC3 (TK8-14) and PR2D3.

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Tandem Mass Spectroscopy, Immunoprecipitation, Marker, Staining, Molecular Weight, Western Blot, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: AOC3 is expressed on MFs in human normal and cancer tissues. (A) Confocal immunofluorescence of pericryptal MFs in a normal colon cryostat section. AOC3 (TK8-14) is shown in green and DAPI in blue in all cases where not otherwise mentioned. (Magnification: 100×.) The red arrow identifies the MFs. (B) Paraffin-embedded sections of normal colon were double-stained using AOC3 antibody (green) (393112; R&D Systems) and anti-αSMA (red) (1A4; Sigma). DAPI staining is shown in blue. The yellow staining in the merged image shows that the expression of AOC3 colocalizes with αSMA on MFs. (C) Anti-AOC3 (red) (393112; R&D Systems) is expressed on MFs in various FFPE normal and cancerous human tissues (colon, rectum, stomach, and prostate). (Magnification: 20×.) (D) AOC3 expression on MFs in FFPE CRC lymph node metastases. (i) Micrometastasis. (ii) Macrometastasis. (Magnification: 5×.) (iii) Macrometastasis. (Magnification: 20×.) AOC3 (393112; R&D) is shown in green, and AUA-1 is shown in red. (E) FFPE MFs in breast cancer are AOC3− but αSMA+. Several different cases were tested; one representative example is shown. (Magnification: 20×.) AOC3 (393112; R&D) is shown in red, and anti-αSMA (1A4; Sigma) is shown in green.

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Immunofluorescence, Staining, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: AOC3 expression in human tumor tissues. (A) The anti-AOC3 mAb (Clone 393112) staining shows AOC3 expression (red) on MFs in ovarian, endometrial, and liver cancers and in Hodgkin’s lymphoma lymph nodes. (B) Costaining with AOC3 (green) for MFs and AUA1 (red) to identify epithelial cells of prostate and rectum carcinomas. Blue staining is DAPI. (Magnification: 20×.)

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Expressing, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: Distribution of AOC3 in normal human tissue costained with anti-AOC3 mAb (Clone 393112) or anti-αSMA mAb (1A4; Sigma)

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: Tissue distribution of AOC3 in human cancers on a tissue microarray costained with anti-AOC3 mAb (Clone 393112) and anti-αSMA mAb (Sigma, 1A4)

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Microarray, In Situ, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: AOC3 is expressed in primary MFs but not in fibroblasts. (A) Immunofluorescent staining of CCD 18CO cells and foreskin fibroblast cells with AOC3 mAb (TK8-14) and PR2D3. (Magnification: 20×.) (B) Western blot using AOC3 mAb (TK8-14) detected a 150-kDa protein in CCD 18CO, myo2020, myo1998, and myo6544 cells but not in foreskin fibroblasts (F. Fibro) or skin fibroblasts (Skin Fibro). Tubulin was used as the loading control. (C) Flow cytometric analysis of primary cell cultures (myo6544 and myo1998) and CCD 18CO cells. Fibroblasts (foreskin fibroblasts and two sets of adult skin fibroblasts) and epithelial cells (SW1222 and LS174T) show that MFs are AOC3+, whereas fibroblasts and epithelial cells do not express AOC3. Isotype control is shown in red, and anti-AOC3 (TK8-14) is shown in green. (D) mRNA-expression levels of AOC3 and ACTA2 in MF and fibroblast cultures. AOC3 and ACTA2 mRNA expression in eight cell lines (four MF cultures and four fibroblast cultures) was measured by the Affymetrix Human Genome U133 Plus 2.0 microarray. The mRNA-expression levels along the y axis are actual ΔCt fluorescence intensities. (E) AOC3 functions as an SSAO enzyme in MFs. The enzyme activity of MFs was determined by SSAO-mediated H2O2 production. The siRNA for AOC3, the SSAO inhibitor semicarbazide (SEM) (1 mM), and the MAO-A inhibitor Clorgyline (1 mM) were added under serum-free conditions for 48 h. The enzyme activity in untreated CCD 18CO control cells was set to 100%. The P values are based on t tests for the difference between the treated and the serum-free control samples: **P < 0.001.

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Staining, Western Blot, Control, Expressing, Microarray, Fluorescence, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: Trypsin impaired the apparent expression of AOC3 protein on CCD 18CO cells. (A) Cell suspensions were prepared either by trypsin digestion (green) or EDTA solution (blue), and the cells were labeled with anti-AOC3 (TK 8-14)-APC. Isotype-APC (red) was used as the control. (B) Live cells from three primary MF cultures (myo6544, myo1998, and myo6526) and CCD 18CO cells were stained with anti-AOC3 (TK 8-14) and PR2D3. The primary antibodies were detected with a secondary antibody to mouse Ig conjugated to APC (green). The secondary antibody alone was used as a negative control (red). (C) RT-qPCR validation of AOC3 mRNA expression in 11 MF and two fibroblast cultures. Expression levels are determined by RT-qPCR as described in Materials and Methods and are given as linearized ΔCt values along the y axis. Red columns represent MF cultures; blue columns represent fibroblast cultures.

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Expressing, Labeling, Control, Staining, Negative Control, Quantitative RT-PCR, Biomarker Discovery

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: FACS isolation of MFs. (A) Flow cytometry-based separation of a 1:1 mixture of epithelial cells (SW1222) and primary MFs (myo6544). MFs were labeled with anti–AOC3-APC (TK8-14), and epithelial cells were labeled with AUA1-FITC. (B) FACS isolation of MFs from fresh tissue. Cells expressing AOC3 (mAb TK8-14) were gated using the Alexa-488 fluorescent signals (y axis) with a cutoff based on unstained samples. The x axis is forward scatter. (C) Immunofluorescent staining of AOC3 FACS-sorted cells with anti-vimentin (3B4; Dako) on fixed cells and anti-AOC3 (TK8-14) and PR2D3 on live cells. (Magnification: 20×.)

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Isolation, Flow Cytometry, Labeling, Expressing, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: Surface expression of AOC3 is sensitive to proteolytic digestion by collagenase and trypsin. Fresh patient-derived colorectal tissue was mechanically disrupted and then treated with either (A) collagenase type 4 for 3 h or (B) nonenzymatic EDTA-based cell dissociating solution (Sigma) for 1 h at 37 °C, as described in Materials and Methods. Cells were stained separately with anti-AOC3 (TK8-14) conjugated with PE (phycoerythrin), anti-EpCAM (AUA1, conjugated with PE), or PE IgG isotype control. R1-gated cells were negative to AOC3 staining with collagenase digestion, whereas the nonenzymatically isolated cells in the same gate contained anti-AOC3+ cells. When these positively staining cells were rerun on the FACS after trypsin treatment, the staining by anti-AOC3 was lost.

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Expressing, Derivative Assay, Staining, Control, Isolation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: Identification of candidate markers NKX2-3 for MFs and SHOX2 for fibroblasts. (A) NKX2-3, SHOX2, LRRC17, and TBX5 mRNA expression in MFs and fibroblasts measured by the Affymetrix Human Genome U133 Plus 2.0 microarray. NKX2-3 and LRRC17 are highly expressed in MFs; SHOX2 and TBX5 are highly expressed in fibroblasts. (B) RT-qPCR verification of NKX2-3 and SHOX2 mRNA expression in MF and fibroblast cultures. Expression levels are given as linearized ΔCt values generated as described in Materials and Methods. The NKX2-3 mRNA levels in skin fibroblasts and foreskin fibroblasts were too low to be determined (ND: no data). Columns in red represent MF cultures; columns in blue represent fibroblast cultures. (C) NKX2-3 protein is expressed in MF cultures (CCD 18CO, myo6769C, myo1998, and myo6526 cells) but not in fibroblast cultures. Western blot with anti NKX2-3 (polyclonal; LSBio). Anti-tubulin was used as a control. (D) The mRNA-expression levels of AOC3 and NKX2-3 in AOC3-sorted MFs. Expression levels were determined by RT-qPCR and are given as converted linearized ΔCt values. Red columns represent AOC3-sorted cells; the blue column represents skin fibroblast cultures. The NKX2-3 mRNA levels of skin fibroblasts and SHOX2 mRNA levels of AOC3-sorted cells were not determined, because there was no measurable Ct for the sample, indicating a very low or absent amount of mRNA.

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Expressing, Microarray, Quantitative RT-PCR, Generated, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: Regulation of AOC3, NKX2-3, and SHOX2 expression in MFs. (A) Serum starvation increased AOC3 expression in CCD 18CO cells. Cells were cultured with 10% FBS or without serum for 24, 48, 72, or 96 h. AOC3 protein expression was measured using Western blots with anti-AOC3 (TK8-14) and with anti-tubulin as the control. (B) AOC3 expression is down-regulated by TGFβ treatment. Cells were incubated with or without 10 ng/mL TGFβ in serum-free medium or in medium containing 10% normal serum for 24, 48, 72, or 96 h, and AOC3 levels were determined by Western blots as previously described. (C) The inhibitory effect of TGFβ on AOC3 expression in CCD 18CO cells. The AOC3 mRNA levels were determined using RT-qPCR. Cells were serum starved and then were treated with TGFβ (10 ng/mL) at the indicated times. The blue line indicates serum-starved levels; the red line indicates the levels after the addition of TGFβ. The Ct values were normalized to UBC, and the fold-change was calculated relative to cells in serum-free conditions for 24 h (y axis). (D) AOC3 expression is not inducible in foreskin fibroblast cells. Foreskin fibroblasts were serum starved (SF) and treated with TGFβ for 72 h. Protein expression was measured using Western blots with anti-AOC3 (TK8-14) and anti-tubulin. CON, control. (E) Skin fibroblast mRNA expression of AOC3, ACTA2, and SHOX2 following TGFβ treatment. Cells were grown in normal 10% serum (NS) or under serum-free conditions with (SF/TGFβ) or without TGFβ (10 ng/mL) treatment for 48 h. Expression levels were determined using RT-qPCR and are given as linearized ΔCt values. The NKX2-3 mRNA level was undetectable. The ΔCt values for NKX2-3, AOC3, and SHOX2 are given on the left y axis, and the ΔCt values for ACTA2 are given on the right y axis. (F) The expression of ACTA2, MYH11, and SHOX2 is regulated by NKX2-3 in MFs. Relative mRNA-expression levels were determined using RT-qPCR following transfection of CCD 18CO and Myo6526 cells with siNKX2-3 or with the scrambled sequence (siCON) as control. Raw Ct values were normalized to UBC, and the fold-changes relative to siCON were calculated using the ΔΔCt method. (G) AOC3 silencing decreases NKX2-3 expression in CCD 18CO cells. The expression levels of NKX2-3, SHOX2, MYH11, and ACTA2 in siAOC3-transfected relative to siCON-transfected CCD 18CO cells were determined using RT-qPCR. Raw Ct values were normalized to UBC, and the fold-changes relative to siCON were calculated using the ΔΔCt method. The P values are based on t tests for the difference between the treated and the serum-free control samples: **P < 0.001. (H) Schematic model for feedback gene regulation in MFs and fibroblasts.

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Expressing, Cell Culture, Western Blot, Control, Incubation, Quantitative RT-PCR, Transfection, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: Serum starvation increased AOC3 expression in CCD 18CO cells. (A) CCD 18CO cells were cultured with 10% FBS or without serum for 24, 48, 72, or 96 h. The AOC3 mRNA level was determined using RT-qPCR. Ct values were normalized to UBC, and fold-change was calculated relative to cells cultured for 24 h with 10% FBS. *P < 0.01; **P < 0.001 compared with the 10% FBS condition. (B) The inhibitory effect of TGFβ was assessed by RT-qPCR. CCD 18CO cells were incubated with or without TGFβ (10 ng/mL) in serum-free or normal serum-containing medium for 48 or 72 h. Ct values were normalized to UBC, and fold-change was calculated relative to cells cultured for 48 h with 10% FBS. **P < 0.001 compared with both the 10% FBS condition and with TGFβ in the serum-free condition. (C) The inhibitory effect of TGFβ on AOC3 protein expression. The AOC3 protein-expression level was measured by Western blot using anti-AOC3 (TK8-14) and with anti-tubulin as a loading control. Cells were cultured under serum-free conditions.

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Incubation, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

doi: 10.1073/pnas.1603534113

Figure Lengend Snippet: Heterogeneous expression of αSMA and MYH11 in AOC3-sorted MFs. Immunofluorescent staining with αSMA (1A4; Sigma) and MYH11 [EPR5336(B); Abcam] in fixed AOC3-sorted MFs. (Magnification: 20×.)

Article Snippet: Comparative real-time PCR was performed in triplicate. cDNA samples were used as input for TaqMan qPCR using predesigned, manufacturer-validated primers and probes for AOC3 (Hs02560271_s1), ubiquitin C (Hs01871556_s1), NKX2-3 (Hs00414553_g1), SHOX2 (Hs00243203_m1), MYH11 (Hs00224610-m1), and ACTA2 (Hs00426835_g1), all from Life Technologies.

Techniques: Expressing, Staining

Journal: Frontiers in Physiology

Article Title: Perivascular Adipose Tissue's Impact on Norepinephrine-Induced Contraction of Mesenteric Resistance Arteries

doi: 10.3389/fphys.2017.00037

Figure Lengend Snippet: Maoa and Aoc3 are highly expressed in rat mesenteric PVAT . Amine oxidase gene expression of (A) Maoa , (B) Maob , and (C) Aoc3 (gene for SSAO) in mesenteric resistance vessels (MRV) and in mesenteric PVAT (MPVAT) normalized to β-actin as the reference gene. Bars represent means ± SEM. Means were compared with a one-way ANOVA followed by the Holm-Sidak's multiple comparisons test for parametric data sets ( Maob ) or the Kruskal-Wallis followed by the Dunn's test for multiple comparisons for the non-parametric data sets ( Maoa and Aoc3 ). * p < 0.05, ** p < 0.01, **** p < 0.0001. The number above each bar indicates the number of animals used.

Article Snippet: Thus, we have listed the catalog numbers which are as follows: Aoc3 (cat# 4448892, assay ID: Rn01452826_m1), Comt (cat#4448892, assay ID: Rn01404927_g1: Actb (cat#4448892, assay ID: Rn00667869_m1), Maoa (cat#4448892, assay ID: Rn01430950_m1), Maob (cat#4448892, assay ID: Rn00566203_m1).

Techniques: Gene Expression

Journal: Frontiers in Physiology

Article Title: Perivascular Adipose Tissue's Impact on Norepinephrine-Induced Contraction of Mesenteric Resistance Arteries

doi: 10.3389/fphys.2017.00037

Figure Lengend Snippet: MAO-A, MAO-B, and SSAO but not COMT are present in rat MRV and MPVAT. (A) Western blot analysis of monoamine metabolism enzymes MAO-A, MAO-B, COMT, and SSAO in mesenteric resistance vessels (MRV) and in mesenteric PVAT (MPVAT) from eight animals. Positive controls were gut mucosa for MAO-A, stomach fundus for MAO-B, Jurkat cells for COMT and aorta for SSAO. Densitometry analysis of Western blot bands for (B) MAO-A, (C) MAO-B, (D) SSAO. Bars represent the means ± SEM. Western blot densitometry was statistically analyzed with a paired Student's t -test. * p < 0.05, ** p < 0.01.

Article Snippet: Thus, we have listed the catalog numbers which are as follows: Aoc3 (cat# 4448892, assay ID: Rn01452826_m1), Comt (cat#4448892, assay ID: Rn01404927_g1: Actb (cat#4448892, assay ID: Rn00667869_m1), Maoa (cat#4448892, assay ID: Rn01430950_m1), Maob (cat#4448892, assay ID: Rn00566203_m1).

Techniques: Western Blot

Journal: Frontiers in Physiology

Article Title: Perivascular Adipose Tissue's Impact on Norepinephrine-Induced Contraction of Mesenteric Resistance Arteries

doi: 10.3389/fphys.2017.00037

Figure Lengend Snippet: SSAO mediates tyramine-driven amine oxidase activity in the MPVAT and its fractions, the AF and SVF, but not the MRV . Percent inhibition of tyramine driven oxidase activity by pharmacological inhibitors of MAO-A (1 μM clorgyline), MAO-B (1 μM pargyline), both MAO-A and MAO-B (10 μM pargyline) or SSAO (1 mM semicarbazide) in the (A) mesenteric resistance vessels (MRV), (B) mesenteric PVAT (MPVAT), (C) adipocyte fraction (AF), (D) stromal vascular fraction (SVF), and the positive controls (E) brain (positive control for MAO-A/B), and (F) aorta (positive control for SSAO). Bars represent the means ± SEM for the number of animals indicated near each bar. Means were analyzed by a Kruskal-Wallis test followed by the Dunn's test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. vehicle control (no inhibition).

Article Snippet: Thus, we have listed the catalog numbers which are as follows: Aoc3 (cat# 4448892, assay ID: Rn01452826_m1), Comt (cat#4448892, assay ID: Rn01404927_g1: Actb (cat#4448892, assay ID: Rn00667869_m1), Maoa (cat#4448892, assay ID: Rn01430950_m1), Maob (cat#4448892, assay ID: Rn00566203_m1).

Techniques: Activity Assay, Inhibition, Positive Control, Control

Journal: Frontiers in Physiology

Article Title: Perivascular Adipose Tissue's Impact on Norepinephrine-Induced Contraction of Mesenteric Resistance Arteries

doi: 10.3389/fphys.2017.00037

Figure Lengend Snippet: SSAO mediates benzylamine-driven amine oxidase activity in the MRV, MPVAT and its fractions, the AF and SVF . Percent inhibition of benzylamine driven oxidase activity by pharmacological inhibitors of MAO-B (1 μM pargyline), both MAO-A and MAO-B (10 μM pargyline) or SSAO (1 mM semicarbazide) in the (A) mesenteric resistance vessels (MRV), (B) mesenteric PVAT (MPVAT), (C) adipocyte fraction (AF), (D) stromal vascular fraction (SVF), and the positive controls (E) brain (positive control for MAO-A/B), and (F) aorta (positive control for SSAO). Bars represent the means ± SEM for the number of animals indicated above each bar. Means were analyzed by a Kruskal-Wallis test followed by the Dunn's test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. vehicle control (no inhibition).

Article Snippet: Thus, we have listed the catalog numbers which are as follows: Aoc3 (cat# 4448892, assay ID: Rn01452826_m1), Comt (cat#4448892, assay ID: Rn01404927_g1: Actb (cat#4448892, assay ID: Rn00667869_m1), Maoa (cat#4448892, assay ID: Rn01430950_m1), Maob (cat#4448892, assay ID: Rn00566203_m1).

Techniques: Activity Assay, Inhibition, Positive Control, Control

Journal: Frontiers in Physiology

Article Title: Perivascular Adipose Tissue's Impact on Norepinephrine-Induced Contraction of Mesenteric Resistance Arteries

doi: 10.3389/fphys.2017.00037

Figure Lengend Snippet: Metabolism of NE and H 2 O 2 release by PVAT inhibits contraction of rat mesenteric resistance arteries . NE-induced contraction of the rat mesenteric resistance arteries with or without PVAT and with or without (vehicle) the following inhibitors of NE metabolism. (A) pargyline (10 μM; inhibits MAO-A and B), (B) semicarbazide (1 mM; inhibits SSAO), (C) SP = semicarbazide (1 mM) and pargyline (10 μM inhibits both MAO-A/B and SSAO). Arteries +PVAT were incubated with (D) SP and/or catalase (2000 U/ml). Force of contraction was normalized to the percent of the 60 mM KCl contraction. Bars represent means ± SEM. N = the number of animals used in each group.

Article Snippet: Thus, we have listed the catalog numbers which are as follows: Aoc3 (cat# 4448892, assay ID: Rn01452826_m1), Comt (cat#4448892, assay ID: Rn01404927_g1: Actb (cat#4448892, assay ID: Rn00667869_m1), Maoa (cat#4448892, assay ID: Rn01430950_m1), Maob (cat#4448892, assay ID: Rn00566203_m1).

Techniques: Incubation

Journal: Frontiers in Physiology

Article Title: Perivascular Adipose Tissue's Impact on Norepinephrine-Induced Contraction of Mesenteric Resistance Arteries

doi: 10.3389/fphys.2017.00037

Figure Lengend Snippet: NE-induced contraction of mesenteric resistance arteries with and without PVAT incubated with NE uptake inhibitors. (A) NE-induced contraction of the rat mesenteric resistance arteries + or −PVAT, with corticosterone (100 μM; inhibits OCT3) or vehicle (H 2 O). (B) NE-induced contraction of the rat mesenteric resistance arteries + or −PVAT, incubated with nisoxetine (1 μM; inhibits NET) or vehicle (H 2 O). (C) SPC = semicarbazide (1 mM), pargyline (10 μM), and corticosterone (100 μM; used to inhibit SSAO, MAO-A/B and OCT3), and (D) SPN = semicarbazide (1 mM), pargyline (10 μM) and nisoxetine (1 μM; used to inhibit SSAO, MAO-A/B, and NET). The force of contraction was normalized to the percent of the 60 mM KCl contraction. Bars represent means ± SEM. N = the number of animals used in each group.

Article Snippet: Thus, we have listed the catalog numbers which are as follows: Aoc3 (cat# 4448892, assay ID: Rn01452826_m1), Comt (cat#4448892, assay ID: Rn01404927_g1: Actb (cat#4448892, assay ID: Rn00667869_m1), Maoa (cat#4448892, assay ID: Rn01430950_m1), Maob (cat#4448892, assay ID: Rn00566203_m1).

Techniques: Incubation

Journal: Frontiers in Immunology

Article Title: A novel immune cell signature for predicting osteosarcoma prognosis and guiding therapy

doi: 10.3389/fimmu.2022.1017120

Figure Lengend Snippet: Construction of a risk model. (A) Univariate COX regression analysis for the 94 genes associated with the infiltration of resting dendritic cells. (B, C) LASSO analysis for other important genes associated with the survival rate of OS. (D) The HR and p value of genes (including AOC3, CDK6, COL22A1 and RNASE6) under multivariate COX analysis are shown.

Article Snippet: The primary antibodies used were AOC3 (1:500; Cat no. 66834-1-Ig, Proteintech, Wuhan, China), CDK6 (1:200; Cat no. 14052-1-AP, Proteintech, Wuhan, China), COL22A1 (1:250; Cat no. ab121846; Abcam, USA), and RNASE6 (1:100; Cat. ab121111; Abcam, USA), for 14 hours at 4°C.

Techniques:

Journal: Frontiers in Immunology

Article Title: A novel immune cell signature for predicting osteosarcoma prognosis and guiding therapy

doi: 10.3389/fimmu.2022.1017120

Figure Lengend Snippet: The risk model exhibits high prognostic value in the TARGET discovery cohort. (A) OS tissues in TARGET were divided into low- and high-risk groups, according to median risk scores. (B) Kaplan survival analysis indicated the difference between low- and high-risk group OS tissues in TARGET. (C–E) ROC analyses of the risk model for the 1-year, 3-year, and 5-year survival rates for OS patients in TARGET. (F) Alive and death cases between low- and high-risk group OS tissues in TARGET. (G) Expression of COL22A1, CDK6, RNASE6 and AOC3 between low- and high-risk group OS tissues in TARGET.

Article Snippet: The primary antibodies used were AOC3 (1:500; Cat no. 66834-1-Ig, Proteintech, Wuhan, China), CDK6 (1:200; Cat no. 14052-1-AP, Proteintech, Wuhan, China), COL22A1 (1:250; Cat no. ab121846; Abcam, USA), and RNASE6 (1:100; Cat. ab121111; Abcam, USA), for 14 hours at 4°C.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: A novel immune cell signature for predicting osteosarcoma prognosis and guiding therapy

doi: 10.3389/fimmu.2022.1017120

Figure Lengend Snippet: The risk model exhibits high prognostic value in the GSE21257 verification cohort. (A) OS tissues in GSE21257 were divided into low- and high-risk groups according to median risk scores. (B) Kaplan survival analysis indicated the difference between low- and high-risk group OS tissues in GSE21257. (C–E) ROC analysis of the risk model for the 1-year, 3-year, and 5-year survival rates for OS patients in GSE21257. (F) Alive and death cases between low- and high-risk group OS tissues in GSE21257. (G) Expression of COL22A1, CDK6, RNASE6 and AOC3 between low- and high-risk group OS tissues in GSE21257.

Article Snippet: The primary antibodies used were AOC3 (1:500; Cat no. 66834-1-Ig, Proteintech, Wuhan, China), CDK6 (1:200; Cat no. 14052-1-AP, Proteintech, Wuhan, China), COL22A1 (1:250; Cat no. ab121846; Abcam, USA), and RNASE6 (1:100; Cat. ab121111; Abcam, USA), for 14 hours at 4°C.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: A novel immune cell signature for predicting osteosarcoma prognosis and guiding therapy

doi: 10.3389/fimmu.2022.1017120

Figure Lengend Snippet: The risk model has the potential to predict metastasis in patients with OS. (A) Non-metastasis and metastasis cases between low- and high-risk group OS tissues in the TARGET and GSE21257 cohorts. (B) ROC analysis for the diagnostic value of the risk model in the prediction of OS tissue metastasis. (C, D) IHC was performed to detect the expression of COL22A1, CDK6, RNASE6 and AOC3 in non-metastasis and metastasis OS tissues (magnification 200× and 400×). *p < 0.05; **p < 0.01; ns, no significant.

Article Snippet: The primary antibodies used were AOC3 (1:500; Cat no. 66834-1-Ig, Proteintech, Wuhan, China), CDK6 (1:200; Cat no. 14052-1-AP, Proteintech, Wuhan, China), COL22A1 (1:250; Cat no. ab121846; Abcam, USA), and RNASE6 (1:100; Cat. ab121111; Abcam, USA), for 14 hours at 4°C.

Techniques: Diagnostic Assay, Expressing

Journal: Frontiers in Oncology

Article Title: Screening and identification of novel protein markers of early-stage lung cancer and construction and application of screening models

doi: 10.3389/fonc.2025.1567673

Figure Lengend Snippet: Survival analysis of the candidate protein markers. (A) The survival curve of CLEC3B. (B) The survival curve of AOC3. (C) The survival curve of HBB. (D) The survival curve of CAT. (E) The survival curve of SEPP1. (F) The survival curve of FGA. (G) The survival curve of ORM1. HR, hazard ratio; CLEC3B, C-type lectin domain family 3 member B; AOC3, membrane primary amine oxidase; HBB, hemoglobin subunit beta; CAT, catalase; SEPP1, selenoprotein P; FGA, Fibrinogen alpha chain; ORM1, Alpha-1-acid glycoprotein 1.

Article Snippet: The levels of AOC3, CAT, CLEC3B, SEPP1, and HBB in the plasma and alveolar lavage fluid of mice were measured using ELISA (Cusabio Biotechnology, Wuhan, China).

Techniques: Membrane

Journal: Frontiers in Oncology

Article Title: Screening and identification of novel protein markers of early-stage lung cancer and construction and application of screening models

doi: 10.3389/fonc.2025.1567673

Figure Lengend Snippet: Expression of candidate protein markers in cells of different passages. Expression and comparison of AOC3, CAT, CLEC3B, and SEPP1 among saline group, DMSO group and CTPE exposure group (A) in passage 10 (* CTPE vs . Saline group, P < 0.05). (B) in passage 20 (* CTPE vs . Saline group, P < 0.05). (C) in passage 30 (* CTPE vs . Saline group, P < 0.05). (D) in passage 40 (* CTPE vs . Saline group, P < 0.05). (E) Expression and comparison of AOC3, CAT, CLEC3B, and SEPP1 between BEAS-2B and A549 (*: A549 vs . BEAS-2B, P < 0.05).

Article Snippet: The levels of AOC3, CAT, CLEC3B, SEPP1, and HBB in the plasma and alveolar lavage fluid of mice were measured using ELISA (Cusabio Biotechnology, Wuhan, China).

Techniques: Expressing, Comparison, Saline

Journal: Frontiers in Oncology

Article Title: Screening and identification of novel protein markers of early-stage lung cancer and construction and application of screening models

doi: 10.3389/fonc.2025.1567673

Figure Lengend Snippet: Expression of candidate protein markers in lung tissue of mice in different months. Expressions and comparisons of AOC3, CAT, CLEC3B, SEPP1, and HBB among normal control (NC) group, vehicle control (VC) group and CTPE exposure group (A) in the 3rd month. (B) in the 6th month. (C) in the 9th month. (D) in the 12th month (* CTPE vs . NC, P < 0.05).

Article Snippet: The levels of AOC3, CAT, CLEC3B, SEPP1, and HBB in the plasma and alveolar lavage fluid of mice were measured using ELISA (Cusabio Biotechnology, Wuhan, China).

Techniques: Expressing, Control