Journal: Cancer Management and Research

Article Title: AOC1 Contributes to Tumor Progression by Promoting the AKT and EMT Pathways in Gastric Cancer

doi: 10.2147/CMAR.S225229

Figure Lengend Snippet: AOC1 is highly expressed in gastric cancer tissues compared with normal tissues. ( A ) The red and gray boxes indicate gastric cancer and normal tissues, respectively. Data were obtained from the GEPIA website, including 408 gastric cancer samples and 211 controls. The y-axis indicated the log2-transformed gene expression levels. qPCR ( B ) and western blot ( C ) were performed to detect AOC1 expression in the tumor and paracancerous tissues of 30 patients with gastric cancerthe. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: Primary antibodies against AOC1 (Cat#16338-1-AP, 1:1000), GAPDH (Cat# 60004-1-Ig, 1:10,000), Bax (Cat# 50599-2-Ig, 1:6000), Bcl2 (Cat# 12789-1-AP, 1:1000), Caspase-9 (Cat# 10380-1-AP, 1:300), Caspase-3 (Cat# 19677-1-AP, 1:10,000), AKT (Cat# 60203-2-Ig, 1:5000), p-AKT (Cat# 66444-1-Ig, 1:2000), Cyclin D1 (Cat# 60186-1-Ig, 1:10,000), p70S6K (Cat# 66638-1-Ig, 1:3000), E-cadherin (Cat# 60335-1-Ig, 1:5000), N-cadherin (Cat# 66219-1-Ig, 1:5000) and SNAIL (Cat# 26183-1-AP, 1:1000) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Transformation Assay, Gene Expression, Western Blot, Expressing

Journal: Cancer Management and Research

Article Title: AOC1 Contributes to Tumor Progression by Promoting the AKT and EMT Pathways in Gastric Cancer

doi: 10.2147/CMAR.S225229

Figure Lengend Snippet: AOC1 expression is effectively knocked down by siRNA transfection in human gastric cancer cells. qRT-PCR assay was used to detect the interference efficiencies of siRNAs targeting AOC1 on mRNA levels in human ( A ) AGS and ( B ) MKN45 cells. ( C ) Western blot validated the effectiveness of AOC1 knockdown on protein levels. A scrambled siRNA was used as the negative control (NC). All experiments were performed in triplicate, independently. *P<0.05.

Article Snippet: Primary antibodies against AOC1 (Cat#16338-1-AP, 1:1000), GAPDH (Cat# 60004-1-Ig, 1:10,000), Bax (Cat# 50599-2-Ig, 1:6000), Bcl2 (Cat# 12789-1-AP, 1:1000), Caspase-9 (Cat# 10380-1-AP, 1:300), Caspase-3 (Cat# 19677-1-AP, 1:10,000), AKT (Cat# 60203-2-Ig, 1:5000), p-AKT (Cat# 66444-1-Ig, 1:2000), Cyclin D1 (Cat# 60186-1-Ig, 1:10,000), p70S6K (Cat# 66638-1-Ig, 1:3000), E-cadherin (Cat# 60335-1-Ig, 1:5000), N-cadherin (Cat# 66219-1-Ig, 1:5000) and SNAIL (Cat# 26183-1-AP, 1:1000) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Knockdown, Negative Control

Journal: Cancer Management and Research

Article Title: AOC1 Contributes to Tumor Progression by Promoting the AKT and EMT Pathways in Gastric Cancer

doi: 10.2147/CMAR.S225229

Figure Lengend Snippet: AOC1 knockdown induces growth inhibition in human gastric cancer cells. ( A ) and ( B ) After transfection with siNC or si-AOC1, the viability of AGS and MKN45 cells was detected by using CCK-8 assay. ( C ) and ( D ) Clone formation ability of AOC1 silenced gastric cancer cells was detected. All experiments were performed in triplicate. *P<0.05.

Article Snippet: Primary antibodies against AOC1 (Cat#16338-1-AP, 1:1000), GAPDH (Cat# 60004-1-Ig, 1:10,000), Bax (Cat# 50599-2-Ig, 1:6000), Bcl2 (Cat# 12789-1-AP, 1:1000), Caspase-9 (Cat# 10380-1-AP, 1:300), Caspase-3 (Cat# 19677-1-AP, 1:10,000), AKT (Cat# 60203-2-Ig, 1:5000), p-AKT (Cat# 66444-1-Ig, 1:2000), Cyclin D1 (Cat# 60186-1-Ig, 1:10,000), p70S6K (Cat# 66638-1-Ig, 1:3000), E-cadherin (Cat# 60335-1-Ig, 1:5000), N-cadherin (Cat# 66219-1-Ig, 1:5000) and SNAIL (Cat# 26183-1-AP, 1:1000) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Knockdown, Inhibition, Transfection, CCK-8 Assay

Journal: Cancer Management and Research

Article Title: AOC1 Contributes to Tumor Progression by Promoting the AKT and EMT Pathways in Gastric Cancer

doi: 10.2147/CMAR.S225229

Figure Lengend Snippet: Knockdown of AOC1 inhibits cell invasion and migration in human gastric cancer cells. ( A ) and ( C ) Transwell assays detecting the invasion and migration of human AGS and MKN45 cells. ( B ) and ( D ) Invaded and migrated cells in si-AOC1 group or NC group were counted. All experiments were performed for three repeated times. *P<0.05.

Article Snippet: Primary antibodies against AOC1 (Cat#16338-1-AP, 1:1000), GAPDH (Cat# 60004-1-Ig, 1:10,000), Bax (Cat# 50599-2-Ig, 1:6000), Bcl2 (Cat# 12789-1-AP, 1:1000), Caspase-9 (Cat# 10380-1-AP, 1:300), Caspase-3 (Cat# 19677-1-AP, 1:10,000), AKT (Cat# 60203-2-Ig, 1:5000), p-AKT (Cat# 66444-1-Ig, 1:2000), Cyclin D1 (Cat# 60186-1-Ig, 1:10,000), p70S6K (Cat# 66638-1-Ig, 1:3000), E-cadherin (Cat# 60335-1-Ig, 1:5000), N-cadherin (Cat# 66219-1-Ig, 1:5000) and SNAIL (Cat# 26183-1-AP, 1:1000) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Knockdown, Migration

Journal: Cancer Management and Research

Article Title: AOC1 Contributes to Tumor Progression by Promoting the AKT and EMT Pathways in Gastric Cancer

doi: 10.2147/CMAR.S225229

Figure Lengend Snippet: Knockdown of AOC1 induces apoptosis in human gastric cancer cells. ( A ) and ( B ) The effect of AOC1 knockdown on the apoptosis of AGS cells was detected using flow cytometry. ( C ) and ( D ) The effect of AOC1 knockdown on the apoptosis of MKN45 cells was detected using flow cytometry. All experiments were performed in triplicate. *P<0.05.

Article Snippet: Primary antibodies against AOC1 (Cat#16338-1-AP, 1:1000), GAPDH (Cat# 60004-1-Ig, 1:10,000), Bax (Cat# 50599-2-Ig, 1:6000), Bcl2 (Cat# 12789-1-AP, 1:1000), Caspase-9 (Cat# 10380-1-AP, 1:300), Caspase-3 (Cat# 19677-1-AP, 1:10,000), AKT (Cat# 60203-2-Ig, 1:5000), p-AKT (Cat# 66444-1-Ig, 1:2000), Cyclin D1 (Cat# 60186-1-Ig, 1:10,000), p70S6K (Cat# 66638-1-Ig, 1:3000), E-cadherin (Cat# 60335-1-Ig, 1:5000), N-cadherin (Cat# 66219-1-Ig, 1:5000) and SNAIL (Cat# 26183-1-AP, 1:1000) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Knockdown, Flow Cytometry

Journal: Cancer Management and Research

Article Title: AOC1 Contributes to Tumor Progression by Promoting the AKT and EMT Pathways in Gastric Cancer

doi: 10.2147/CMAR.S225229

Figure Lengend Snippet: Knockdown of AOC1 induces activation of the mitochondrial apoptosis pathway, and also inhibits the AKT signaling pathway and EMT process. ( A and B ) The key members of the mitochondrial apoptosis pathway, including Bax, Bcl2, Caspase-9, and Caspase-3, were detected by Western blot. ( C and D ) AKT signaling pathway members, including AKT, p-AKT, Cyclin D1, and p70S6K, were detected by Western blot. ( E and F ) EMT-related proteins, including E-cadherin, N-cadherin, SNAIL and Slug, were detected by Western blot. All experiments were performed in triplicate. *P<0.05.

Article Snippet: Primary antibodies against AOC1 (Cat#16338-1-AP, 1:1000), GAPDH (Cat# 60004-1-Ig, 1:10,000), Bax (Cat# 50599-2-Ig, 1:6000), Bcl2 (Cat# 12789-1-AP, 1:1000), Caspase-9 (Cat# 10380-1-AP, 1:300), Caspase-3 (Cat# 19677-1-AP, 1:10,000), AKT (Cat# 60203-2-Ig, 1:5000), p-AKT (Cat# 66444-1-Ig, 1:2000), Cyclin D1 (Cat# 60186-1-Ig, 1:10,000), p70S6K (Cat# 66638-1-Ig, 1:3000), E-cadherin (Cat# 60335-1-Ig, 1:5000), N-cadherin (Cat# 66219-1-Ig, 1:5000) and SNAIL (Cat# 26183-1-AP, 1:1000) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Knockdown, Activation Assay, Western Blot

Journal: Cancer Management and Research

Article Title: AOC1 Contributes to Tumor Progression by Promoting the AKT and EMT Pathways in Gastric Cancer

doi: 10.2147/CMAR.S225229

Figure Lengend Snippet: IGF-1 blocked the inhibition of cell proliferation, migration and invasion caused by AOC1 knockdown. AOC1 low expressing cells were treated with IGF-1, an agonist of the AKT pathway. ( A ) CCK8 was performed to detected the proliferation of each group cells. ( B ) Transwell was performed to detected the migration and invasion of each group cells. *P<0.05 vs. NC group; #P<0.05 vs. si-AOC1 group.

Article Snippet: Primary antibodies against AOC1 (Cat#16338-1-AP, 1:1000), GAPDH (Cat# 60004-1-Ig, 1:10,000), Bax (Cat# 50599-2-Ig, 1:6000), Bcl2 (Cat# 12789-1-AP, 1:1000), Caspase-9 (Cat# 10380-1-AP, 1:300), Caspase-3 (Cat# 19677-1-AP, 1:10,000), AKT (Cat# 60203-2-Ig, 1:5000), p-AKT (Cat# 66444-1-Ig, 1:2000), Cyclin D1 (Cat# 60186-1-Ig, 1:10,000), p70S6K (Cat# 66638-1-Ig, 1:3000), E-cadherin (Cat# 60335-1-Ig, 1:5000), N-cadherin (Cat# 66219-1-Ig, 1:5000) and SNAIL (Cat# 26183-1-AP, 1:1000) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Inhibition, Migration, Knockdown, Expressing

Journal: Cell Death & Disease

Article Title: SOX15 transcriptionally increases the function of AOC1 to modulate ferroptosis and progression in prostate cancer

doi: 10.1038/s41419-022-05108-w

Figure Lengend Snippet: a The process of selecting the AOC1 gene. b , c Differentially expressed genes in prostate cancer tissues from four GEO datasets (GSE46602, GSE71016, GSE28204, and GSE27616), in the form of a Wayne diagram ( b ) and a volcano diagram ( c ), respectively. d AOC1 expression in prostate cancer based on TCGA database. e AOC1 expression in prostate cancer based on GEO datasets (GSE28204, GSE38241, and GSE46602). f – h RT-qPCR ( f ) and Western blot analysis ( g , h ) show the AOC1 expression in prostate cancer and normal tissues. GAPDH served as an internal reference. P < 0.0001, paired t -test.

Article Snippet: The blotted nitrocellulose membranes were incubated with the primary antibody, anti-AOC1 antibody (1:1000; ab231558; Abcam, UK), anti-TFR antibody (1:1000; ab214039; Abcam), anti-TF antibody (1:1000; ab277635; Abcam,), anti-FTH1 antibody (1:1000; ab75973, Abcam), and anti-SOX15 antibody (1:100; sc-166964; Santa Cruz, Dallas, TX) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: SOX15 transcriptionally increases the function of AOC1 to modulate ferroptosis and progression in prostate cancer

doi: 10.1038/s41419-022-05108-w

Figure Lengend Snippet: a TCGA database showed AOC1 expression in prostate cancer and correlation with lymph nodes metastasis in. b TCGA database showed AOC1 expression in prostate cancer and correlation with Gleason score. c TCGA data showed prostate cancer patients' AOC1 expression correlation with prognosis. d GEO data (GSE6752) showed AOC1 expression in primary and metastatic prostate cancer. e , f Images ( e ) and dot plots ( f ) of AOC1 staining using tissue sections based on Gleason score (prostate cancer: n = 94, P < 0.001, unpaired t -test). g Overall survival analysis for patients with high and low expression of AOC1 using the cutoff value.

Article Snippet: The blotted nitrocellulose membranes were incubated with the primary antibody, anti-AOC1 antibody (1:1000; ab231558; Abcam, UK), anti-TFR antibody (1:1000; ab214039; Abcam), anti-TF antibody (1:1000; ab277635; Abcam,), anti-FTH1 antibody (1:1000; ab75973, Abcam), and anti-SOX15 antibody (1:100; sc-166964; Santa Cruz, Dallas, TX) overnight at 4 °C.

Techniques: Expressing, Staining

Journal: Cell Death & Disease

Article Title: SOX15 transcriptionally increases the function of AOC1 to modulate ferroptosis and progression in prostate cancer

doi: 10.1038/s41419-022-05108-w

Figure Lengend Snippet: Relationship between the expression of AOC1 in prostate cancer and clinic pathological parameters.

Article Snippet: The blotted nitrocellulose membranes were incubated with the primary antibody, anti-AOC1 antibody (1:1000; ab231558; Abcam, UK), anti-TFR antibody (1:1000; ab214039; Abcam), anti-TF antibody (1:1000; ab277635; Abcam,), anti-FTH1 antibody (1:1000; ab75973, Abcam), and anti-SOX15 antibody (1:100; sc-166964; Santa Cruz, Dallas, TX) overnight at 4 °C.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: SOX15 transcriptionally increases the function of AOC1 to modulate ferroptosis and progression in prostate cancer

doi: 10.1038/s41419-022-05108-w

Figure Lengend Snippet: a , b RT-qPCR ( a ) and Western blot analysis ( b ) showed the expression of AOC1 in 22Rv1 and DU145. GAPDH served as an internal reference. Unpaired t -test. c , d . RT-qPCR ( c ) and Western blot analysis ( d ) showed the efficiency of overexpressing AOC1 in 22Rv1 and DU145. GAPDH served as an internal reference. Unpaired t -test. e-g CCK8 ( f ), EdU ( e ) and Colony Formation assays ( g ) showed the proliferation ability of 22Rv1 and DU145 after overexpression of AOC1 . Unpaired t -test, ANOVA. h , i Wound-Healing ( i ) and Transwell Assay ( h ) showed the migration ability of 22Rv1 and DU145 after overexpression of AOC1 . Unpaired t -test. ( P < 0.05 as “*”; P < 0.01 as “**”; P < 0.001 as “***”).

Article Snippet: The blotted nitrocellulose membranes were incubated with the primary antibody, anti-AOC1 antibody (1:1000; ab231558; Abcam, UK), anti-TFR antibody (1:1000; ab214039; Abcam), anti-TF antibody (1:1000; ab277635; Abcam,), anti-FTH1 antibody (1:1000; ab75973, Abcam), and anti-SOX15 antibody (1:100; sc-166964; Santa Cruz, Dallas, TX) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Over Expression, Transwell Assay, Migration

Journal: Cell Death & Disease

Article Title: SOX15 transcriptionally increases the function of AOC1 to modulate ferroptosis and progression in prostate cancer

doi: 10.1038/s41419-022-05108-w

Figure Lengend Snippet: a CCK8 assay showed the spermidine optimal concentration. b–d CCK8 ( b ), and Colony Formation assays ( c , d ) showed the proliferation ability of 22Rv1 and DU145 after overexpressing AOC1 and adding spermidine. Unpaired t -test, ANOVA. e , f EdU showed the proliferation ability of 22Rv1 and DU145 after overexpressing AOC1 and adding spermidine. Unpaired t -test. g , h Transwell Assay showed the migration ability of 22Rv1 and DU145 after overexpressing AOC1 and adding spermidine. Unpaired t -test. ( P < 0.05 as “*”; P < 0.01 as “**”; P < 0.001 as “***”).

Article Snippet: The blotted nitrocellulose membranes were incubated with the primary antibody, anti-AOC1 antibody (1:1000; ab231558; Abcam, UK), anti-TFR antibody (1:1000; ab214039; Abcam), anti-TF antibody (1:1000; ab277635; Abcam,), anti-FTH1 antibody (1:1000; ab75973, Abcam), and anti-SOX15 antibody (1:100; sc-166964; Santa Cruz, Dallas, TX) overnight at 4 °C.

Techniques: CCK-8 Assay, Concentration Assay, Transwell Assay, Migration

Journal: Cell Death & Disease

Article Title: SOX15 transcriptionally increases the function of AOC1 to modulate ferroptosis and progression in prostate cancer

doi: 10.1038/s41419-022-05108-w

Figure Lengend Snippet: a IC 50 curve showed that the sensitivity of prostate cancer cell lines to ferroptosis inducer, ML210 after overexpressing AOC1 . b H 2 O 2 assay showed that the content of H 2 O 2 significantly after overexpressing AOC1 and adding spermidine. Unpaired t -test. c ROS assay showed that the content of ROS after overexpressing AOC1 and adding spermidine. Unpaired t -test. d Liperfluo assay showed that the content of LPO after overexpressing AOC1 and adding spermidine. Unpaired t -test. e MDA assay showed MDA level after AOC1 overexpression with or without Spermidine treatment in prostate cancer cell lines. Unpaired t -test. f Western blot analysis showed that the ferroptosis-associated proteins changed (TFR, TF, FTH1) after transfecting with AOC1 plasmids and adding Spermidine. GAPDH served as an internal reference. Unpaired t -test. ( P < 0.05 as “*”; P < 0.01 as “**”; P < 0.001 as “***”).

Article Snippet: The blotted nitrocellulose membranes were incubated with the primary antibody, anti-AOC1 antibody (1:1000; ab231558; Abcam, UK), anti-TFR antibody (1:1000; ab214039; Abcam), anti-TF antibody (1:1000; ab277635; Abcam,), anti-FTH1 antibody (1:1000; ab75973, Abcam), and anti-SOX15 antibody (1:100; sc-166964; Santa Cruz, Dallas, TX) overnight at 4 °C.

Techniques: ROS Assay, Multiple Displacement Amplification, Over Expression, Western Blot

Journal: Cell Death & Disease

Article Title: SOX15 transcriptionally increases the function of AOC1 to modulate ferroptosis and progression in prostate cancer

doi: 10.1038/s41419-022-05108-w

Figure Lengend Snippet: a The process of selecting the transcription factor SOX15 upstream of AOC1 . b The TCGA database showed there was a correlation between SOX15 and AOC1 . c The TCGA database showed the correlation between ZNF410 and AOC1 . d The TCGA database showed SOX15 expression in prostate cancer. e , f The GEO datasets showed the correlation between SOX15 and AOC1 (GSE46602, GSE114740). g Location of AOC1 promoter on the chromosome. h Diagram shows the binding site of SOX15 in the promoter of AOC1 . ( P < 0.05 as “*”; P < 0.01 as “**”; P < 0.001 as “***”).

Article Snippet: The blotted nitrocellulose membranes were incubated with the primary antibody, anti-AOC1 antibody (1:1000; ab231558; Abcam, UK), anti-TFR antibody (1:1000; ab214039; Abcam), anti-TF antibody (1:1000; ab277635; Abcam,), anti-FTH1 antibody (1:1000; ab75973, Abcam), and anti-SOX15 antibody (1:100; sc-166964; Santa Cruz, Dallas, TX) overnight at 4 °C.

Techniques: Expressing, Binding Assay

Journal: Cell Death & Disease

Article Title: SOX15 transcriptionally increases the function of AOC1 to modulate ferroptosis and progression in prostate cancer

doi: 10.1038/s41419-022-05108-w

Figure Lengend Snippet: a , b RT-qPCR ( a ) and Western blot analysis ( b ) showed the expression of SOX15 and AOC1 in prostate cancer cell lines after overexpressing SOX15 . GAPDH served as an internal reference. Unpaired t -test. c IC 50 curve showed that the sensitivity of prostate cancer cells to ferroptosis inducer, ML210 after knocking down SOX15. d 22Rv1 and DU145 cells transfected with indicated shRNA or plasmid, and colony formation assay showed the proliferation ability of prostate cancer cells. e–g 22Rv1 cell transfected with indicated plasmid was injected into the right groin of SCID) mice, the tumors were collected after 33 days ( e ), the tumor volume ( f ) and tumor weight ( g ) were measured to show the tumor growth, Unpaired t-test, ANOVA. ( P < 0.05 as “*”; P < 0.01 as “**”; P < 0.001 as “***”). h Specific mechanistic diagram of the SOX15/ AOC1 /ROS axis.

Article Snippet: The blotted nitrocellulose membranes were incubated with the primary antibody, anti-AOC1 antibody (1:1000; ab231558; Abcam, UK), anti-TFR antibody (1:1000; ab214039; Abcam), anti-TF antibody (1:1000; ab277635; Abcam,), anti-FTH1 antibody (1:1000; ab75973, Abcam), and anti-SOX15 antibody (1:100; sc-166964; Santa Cruz, Dallas, TX) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, shRNA, Plasmid Preparation, Colony Assay, Injection

Journal: The Journal of Biological Chemistry

Article Title: Amine Oxidase Copper-containing 1 ( AOC1 ) Is a Downstream Target Gene of the Wilms Tumor Protein, WT1, during Kidney Development *

doi: 10.1074/jbc.M114.564336

Figure Lengend Snippet: AOC1 and WT1 protein expression in embryonic kidneys and gonads. Serial cryosections of a mouse embryo (13.5 d.p.c.) were stained with antibodies against AOC1 (a) and WT1 (b), respectively. Normal rabbit serum (NRbS) was used as a negative control (c). K, kidney; T, testis; M, mesonephros; A, adrenal gland. Representative double immunolabeling of AOC1 and WT1 in a 16.5 d.p.c. embryonic rat kidney (d–g) is shown. AOC1 (red) and WT1 (green) proteins were visualized with Cy3- and Alexa Fluor 488-conjugated secondary antibodies, respectively. Co-localization of AOC1 and WT1 in cells of the developing glomeruli is indicated by the yellow fluorescence signal in the merged image (g). Note that AOC1 protein is also present in cells that do not contain WT1 (d and g).

Article Snippet: The organ rudiments were treated with 1 μg/μl AOC1 protein isolated from pig kidneys (Sigma-Aldrich), 100 μ m putrescine (Sigma-Aldrich), or 1 μ m AOC1 inhibitor aminoguanidine (Sigma-Aldrich).

Techniques: Expressing, Staining, Negative Control, Immunolabeling, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Amine Oxidase Copper-containing 1 ( AOC1 ) Is a Downstream Target Gene of the Wilms Tumor Protein, WT1, during Kidney Development *

doi: 10.1074/jbc.M114.564336

Figure Lengend Snippet: Aoc1 and Odc1 mRNA in cultured murine embryonic kidneys. Both kidneys were isolated from murine embryos at 11.5, 12.5, and 13.5 d.p.c. and cultured for 72 h in the presence of either Wt1 antisense (Wt1 morpholino) or mismatch vivo-morpholino (mismatch). Aoc1 and Odc1 transcripts were measured by real time RT-PCR and normalized to Gapdh mRNA. A pairwise comparison was performed between kidneys incubated with Wt1 morpholino and mismatch morpholino. Values of the mismatch morpholino-treated kidneys were set to 1. Significant differences in Aoc1 (A) and Odc1 (B) mRNA levels are indicated (*, p < 0.05; **, p < 0.005; Student's paired t test). Knockdown efficiencies were assessed by immunoblotting with anti-WT1 antibody using actin as a loading control (C). n.s., not significant. D, analysis of ureter branching in kidneys (11.5, 12.5, and 13.5 d.p.c.) treated with either Wt1 or mismatch vivo-morpholino (10 μm each). After 48 h of culture in the presence of vivo-morpholinos, the whole mount preparations were stained with a FITC-conjugated anti-pancytokeratin antibody (dark field images), and a skeleton of the ureter was extracted manually (schematic images). Branching of the ureter was analyzed from the schematic images. Data are presented as means ± S.D. (error bars). * (p < 0.05) and ** (p < 0.005) indicate significant differences (Student's paired t test).

Article Snippet: The organ rudiments were treated with 1 μg/μl AOC1 protein isolated from pig kidneys (Sigma-Aldrich), 100 μ m putrescine (Sigma-Aldrich), or 1 μ m AOC1 inhibitor aminoguanidine (Sigma-Aldrich).

Techniques: Cell Culture, Isolation, Quantitative RT-PCR, Incubation, Western Blot, Staining

Journal: The Journal of Biological Chemistry

Article Title: Amine Oxidase Copper-containing 1 ( AOC1 ) Is a Downstream Target Gene of the Wilms Tumor Protein, WT1, during Kidney Development *

doi: 10.1074/jbc.M114.564336

Figure Lengend Snippet: Aoc1 mRNA in cultured murine embryonic testis and ovary. The gonads were isolated from male (A) and female (B) murine embryos at the indicated developmental stages and cultured for 72 h in the presence of either Wt1 antisense (Wt1 morpholino) or mismatch vivo-morpholino (mismatch). Aoc1 transcripts were quantified by real time RT-PCR and normalized to Gapdh mRNA. A pairwise comparison was performed between Wt1 morpholino-treated gonads and the mismatch morpholino controls. Significant differences are indicated (*, p < 0.05; **, p < 0.005; Student's paired t test). Representative WT1 immunoblots to assess knockdown efficiencies are shown below the bar graphs.

Article Snippet: The organ rudiments were treated with 1 μg/μl AOC1 protein isolated from pig kidneys (Sigma-Aldrich), 100 μ m putrescine (Sigma-Aldrich), or 1 μ m AOC1 inhibitor aminoguanidine (Sigma-Aldrich).

Techniques: Cell Culture, Isolation, Quantitative RT-PCR, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Amine Oxidase Copper-containing 1 ( AOC1 ) Is a Downstream Target Gene of the Wilms Tumor Protein, WT1, during Kidney Development *

doi: 10.1074/jbc.M114.564336

Figure Lengend Snippet: Aoc1 transcripts and WT1 protein in permanent cell lines. A, murine mesonephros-derived M15 cells were transfected either with a pool of four different siRNAs targeting the Wt1 gene (siWt1) or with non-targeting siRNAs (sicontrol). Aoc1 and Gapdh transcripts in siRNA-transfected M15 cells were quantified by real time RT-PCR. Down-regulation of WT1 protein in response to RNA interference was assessed by immunoblot analysis using anti-WT1 antibody. Detection of actin protein served as a loading control (D). B and C, Aoc1 mRNA in osteosarcoma-derived UB27 and UD28 cells, which express the WT1(−KTS) and WT1(+KTS) isoforms, respectively. AOC1 and GAPDH transcripts were measured by real time RT-PCR. Temporal changes of WT1 protein in response to tetracycline depletion were detected by immunoblot analysis. Values are shown as means ± S.D. (M15 cells, n = 5; UB27 cells, n = 4; UD28 cells, n = 3). Statistical significance is indicated by an asterisk (t test, p < 0.05) or hash tag (analysis of variance, F(3,10) = 8.195, p < 0.05).

Article Snippet: The organ rudiments were treated with 1 μg/μl AOC1 protein isolated from pig kidneys (Sigma-Aldrich), 100 μ m putrescine (Sigma-Aldrich), or 1 μ m AOC1 inhibitor aminoguanidine (Sigma-Aldrich).

Techniques: Derivative Assay, Transfection, Quantitative RT-PCR, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Amine Oxidase Copper-containing 1 ( AOC1 ) Is a Downstream Target Gene of the Wilms Tumor Protein, WT1, during Kidney Development *

doi: 10.1074/jbc.M114.564336

Figure Lengend Snippet: WT1(−KTS) activates the promoter of the AOC1 gene. A, HEK293 cells were transiently transfected with reporter constructs expressing a firefly luciferase (Lucif.) under control of AOC1 promoter pieces of various lengths. The reporter constructs were co-transfected with either a WT1(−KTS) expression plasmid or empty vector. A Renilla luciferase plasmid was utilized for normalization of transfection efficiencies. Data shown are means ± S.E. (error bars) (n = 4). Statistical significance versus transfection with empty expression vector is indicated by an asterisk (p < 0.05, t test). mut., mutation; B, binding of WT1(−KTS) to the predicted element (lane 2 versus lane 1) in the human AOC1 promoter was proven by electrophoretic mobility shift assay. Interaction of WT1(−KTS) protein with the AOC1 promoter oligonucleotide could be competed with excess amounts of unlabeled DNA (lanes 3–5) containing the previously identified WT1 binding element of the murine Adamts16 promoter (43). Mutation of the WT1 consensus motif abrogated WT1(−KTS) binding (lanes 6 and 7). Interaction of WT1(−KTS) protein with the Adamts16 promoter element (43) served as an internal quality control (lanes 8 and 9). C, ChIP was performed to detect WT1 protein bound to the promoter region of the murine Aoc1 gene in its native chromosomal configuration. A specific antibody against WT1 was used for immunoprecipitation of chromatin prepared from M15 cells. Incubation with normal rabbit IgG (NRb-IgG) served as a negative control. Amplicons encompassing the 5′-flanking region of the Aoc1 gene were generated by real time PCR. The gene encoding anti-Müllerian hormone receptor 2 (Amhr2), a previously identified WT1 target (52), was used as a positive control. Data shown are means ± S.D. (error bars). Statistical significances between normal rabbit IgG and anti-WT1 are indicated by an asterisk (p < 0.05, paired t test, n = 7).

Article Snippet: The organ rudiments were treated with 1 μg/μl AOC1 protein isolated from pig kidneys (Sigma-Aldrich), 100 μ m putrescine (Sigma-Aldrich), or 1 μ m AOC1 inhibitor aminoguanidine (Sigma-Aldrich).

Techniques: Transfection, Construct, Expressing, Luciferase, Plasmid Preparation, Mutagenesis, Binding Assay, Electrophoretic Mobility Shift Assay, Immunoprecipitation, Incubation, Negative Control, Generated, Real-time Polymerase Chain Reaction, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Amine Oxidase Copper-containing 1 ( AOC1 ) Is a Downstream Target Gene of the Wilms Tumor Protein, WT1, during Kidney Development *

doi: 10.1074/jbc.M114.564336

Figure Lengend Snippet: Effect of AOC1, given either alone or in combination with putrescine and vitamin C, respectively, on branching morphogenesis in embryonic kidney explants. Kidneys were excised from murine embryos at the indicated gestational stages and incubated for 48 h in the presence of AOC1 (50 milliunits/ml) alone (A) or together with 100 μm putrescine (B) and 200 μm vitamin C (VitC) (C), respectively. As a control, the contralateral kidney of each embryo was treated with BSA. After 48 h of culture, the ureter was stained with a FITC-conjugated anti-pancytokeratin antibody (dark field images), and a skeleton of the ureteric bud branches was drawn manually (schematic images). Branching of the ureter was analyzed based on the schematic sketches. The absolute numbers of branches are indicated in the columns. Data are presented as means ± S.D. (error bars). * (p < 0.05) and ** (p < 0.005) indicate significant differences (paired t test).

Article Snippet: The organ rudiments were treated with 1 μg/μl AOC1 protein isolated from pig kidneys (Sigma-Aldrich), 100 μ m putrescine (Sigma-Aldrich), or 1 μ m AOC1 inhibitor aminoguanidine (Sigma-Aldrich).

Techniques: Incubation, Staining

Journal: The Journal of Biological Chemistry

Article Title: Amine Oxidase Copper-containing 1 ( AOC1 ) Is a Downstream Target Gene of the Wilms Tumor Protein, WT1, during Kidney Development *

doi: 10.1074/jbc.M114.564336

Figure Lengend Snippet: Effect of putrescine and AOC1 inhibition by aminoguanidine on branching morphogenesis in embryonic kidney explants. Embryonic kidney organ cultures (11.5, 12.5, and 13.5 d.p.c.) were treated for 48 h with 100 μm putrescine (A) or 1 μm aminoguanidine to inhibit AOC1 enzyme activity (B). The contralateral kidney of each embryo was cultured in the presence of BSA. The preparations were processed and analyzed as described in the legend of Fig. 6. The absolute numbers of branches are given in the columns. Data are means ± S.D. (error bars). * (p < 0.05) and ** (p < 0.005) indicate significant differences (paired t test).

Article Snippet: The organ rudiments were treated with 1 μg/μl AOC1 protein isolated from pig kidneys (Sigma-Aldrich), 100 μ m putrescine (Sigma-Aldrich), or 1 μ m AOC1 inhibitor aminoguanidine (Sigma-Aldrich).

Techniques: Inhibition, Activity Assay, Cell Culture

Journal: Oncology Letters

Article Title: Downregulation of amine oxidase copper containing 1 inhibits tumor progression by suppressing IL-6/JAK/STAT3 pathway activation in hepatocellular carcinoma

doi: 10.3892/ol.2021.13118

Figure Lengend Snippet: Quantitative PCR primer sequences.

Article Snippet: Huh-7 and Hep3B2.1–7 cells were transfected with 20 nM small interfering (si)RNAs targeting AOC1, the corresponding negative control (siNC), pcDNA3.1-AOC1 or the empty pcDNA3.1 vector (Guangzhou RiboBio Co., Ltd.), using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 6 h. Huh-7 and Hep3B2.1–7 cells were harvested 48 h after transfection.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Oncology Letters

Article Title: Downregulation of amine oxidase copper containing 1 inhibits tumor progression by suppressing IL-6/JAK/STAT3 pathway activation in hepatocellular carcinoma

doi: 10.3892/ol.2021.13118

Figure Lengend Snippet: AOC1 expression levels in HCC tissues. (A) AOC1 mRNA expression in 85 HCC and adjacent-normal liver tissues. (B) Overall survival of 234 patients with HCC based on data from The Cancer Genome Atlas. ***P<0.001. AOC1, amine oxidase copper containing 1; HCC, hepatocellular carcinoma.

Article Snippet: Huh-7 and Hep3B2.1–7 cells were transfected with 20 nM small interfering (si)RNAs targeting AOC1, the corresponding negative control (siNC), pcDNA3.1-AOC1 or the empty pcDNA3.1 vector (Guangzhou RiboBio Co., Ltd.), using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 6 h. Huh-7 and Hep3B2.1–7 cells were harvested 48 h after transfection.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Downregulation of amine oxidase copper containing 1 inhibits tumor progression by suppressing IL-6/JAK/STAT3 pathway activation in hepatocellular carcinoma

doi: 10.3892/ol.2021.13118

Figure Lengend Snippet: Association between AOC1 expression and clinical characteristics of patients with hepatocellular carcinoma.

Article Snippet: Huh-7 and Hep3B2.1–7 cells were transfected with 20 nM small interfering (si)RNAs targeting AOC1, the corresponding negative control (siNC), pcDNA3.1-AOC1 or the empty pcDNA3.1 vector (Guangzhou RiboBio Co., Ltd.), using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 6 h. Huh-7 and Hep3B2.1–7 cells were harvested 48 h after transfection.

Techniques: Expressing, Virus, Infection

Journal: Oncology Letters

Article Title: Downregulation of amine oxidase copper containing 1 inhibits tumor progression by suppressing IL-6/JAK/STAT3 pathway activation in hepatocellular carcinoma

doi: 10.3892/ol.2021.13118

Figure Lengend Snippet: AOC1-knockdown suppresses HCC cell proliferation. (A and B) AOC1-knockdown and overexpression efficiencies were validated by reverse transcription-quantitative PCR and western blotting in Huh-7 and Hep3B2.1–7 cells. (C) Following transfection, cell viability was determined by Cell Counting Kit 8 analysis, and (D) cellular proliferation was measured using a colony formation assay. *P<0.05 vs. the siNC or Vector group. AOC1, amine oxidase copper containing 1; HCC, hepatocellular carcinoma; si, small interfering (RNA); NC, negative control.

Article Snippet: Huh-7 and Hep3B2.1–7 cells were transfected with 20 nM small interfering (si)RNAs targeting AOC1, the corresponding negative control (siNC), pcDNA3.1-AOC1 or the empty pcDNA3.1 vector (Guangzhou RiboBio Co., Ltd.), using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 6 h. Huh-7 and Hep3B2.1–7 cells were harvested 48 h after transfection.

Techniques: Knockdown, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Cell Counting, Colony Assay, Plasmid Preparation, Small Interfering RNA, Negative Control

Journal: Oncology Letters

Article Title: Downregulation of amine oxidase copper containing 1 inhibits tumor progression by suppressing IL-6/JAK/STAT3 pathway activation in hepatocellular carcinoma

doi: 10.3892/ol.2021.13118

Figure Lengend Snippet: AOC1-knockdown suppresses HCC cell migration and invasiveness. Cellular migration and invasion ability were analyzed using (A) wound-healing and (B) Transwell assays, respectively. (C) Effect of AOC1 on HCC epithelial-mesenchymal transition-related protein levels was determined by western blotting. *P<0.05 vs. the siNC or Vector group. AOC1, amine oxidase copper containing 1; HCC, hepatocellular carcinoma; si, small interfering (RNA); NC, negative control.

Article Snippet: Huh-7 and Hep3B2.1–7 cells were transfected with 20 nM small interfering (si)RNAs targeting AOC1, the corresponding negative control (siNC), pcDNA3.1-AOC1 or the empty pcDNA3.1 vector (Guangzhou RiboBio Co., Ltd.), using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 6 h. Huh-7 and Hep3B2.1–7 cells were harvested 48 h after transfection.

Techniques: Knockdown, Migration, Western Blot, Plasmid Preparation, Small Interfering RNA, Negative Control

Journal: Oncology Letters

Article Title: Downregulation of amine oxidase copper containing 1 inhibits tumor progression by suppressing IL-6/JAK/STAT3 pathway activation in hepatocellular carcinoma

doi: 10.3892/ol.2021.13118

Figure Lengend Snippet: AOC1-knockdown inhibits IL-6/JAK/STAT3 pathway activation in HCC cells. (A) Effect of AOC1-knockdown on HCC cell reactive oxygen species generation. (B) Gene set enrichment analysis results revealed that the IL-6/JAK/STAT3 pathway was enriched in AOC1-related HCC. (C) IL-6 levels in HCC cell lines were analyzed by ELISA. (D) Phosphorylation levels of JAK2 and STAT3 were analyzed by western blotting. *P<0.05 vs. siNC group. AOC1, amine oxidase copper containing 1; HCC, hepatocellular carcinoma; si, small interfering (RNA); NC, negative control.

Article Snippet: Huh-7 and Hep3B2.1–7 cells were transfected with 20 nM small interfering (si)RNAs targeting AOC1, the corresponding negative control (siNC), pcDNA3.1-AOC1 or the empty pcDNA3.1 vector (Guangzhou RiboBio Co., Ltd.), using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 6 h. Huh-7 and Hep3B2.1–7 cells were harvested 48 h after transfection.

Techniques: Knockdown, Activation Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Small Interfering RNA, Negative Control

Journal: Oncology Letters

Article Title: Downregulation of amine oxidase copper containing 1 inhibits tumor progression by suppressing IL-6/JAK/STAT3 pathway activation in hepatocellular carcinoma

doi: 10.3892/ol.2021.13118

Figure Lengend Snippet: AOC1-knockdown blocks the IL-6-induced proliferation, migration and invasiveness in HCC cell lines. HCC cell (A) viability, (B) proliferation, (C) migration and (D) invasiveness were analyzed following IL-6 treatment using Cell Counting Kit 8, colony formation, wound-healing and Transwell assays, respectively. *P<0.05 vs. siNC group; # P <0.05 vs. siNC + IL-6 group. AOC1, amine oxidase copper containing 1; HCC, hepatocellular carcinoma; si, small interfering (RNA); NC, negative control.

Article Snippet: Huh-7 and Hep3B2.1–7 cells were transfected with 20 nM small interfering (si)RNAs targeting AOC1, the corresponding negative control (siNC), pcDNA3.1-AOC1 or the empty pcDNA3.1 vector (Guangzhou RiboBio Co., Ltd.), using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 6 h. Huh-7 and Hep3B2.1–7 cells were harvested 48 h after transfection.

Techniques: Knockdown, Migration, Cell Counting, Small Interfering RNA, Negative Control

Journal: Frontiers in Pharmacology

Article Title: Photodynamic Therapy of Novel Photosensitizer Ameliorates TNBS-Induced Ulcerative Colitis via Inhibition of AOC 1

doi: 10.3389/fphar.2021.746725

Figure Lengend Snippet: LD 4 -PDT suppressed the expression of AOC 1 . (A,B) Protein expression of AOC 1 in colon tissue and in HCoEpiC cells were determined by western blot analysis. (C) Gene expression of AOC 1 in HCoEpiC cells was examined by qRT-PCR. (D) Protein expression of AOC 1 was detected by immunofluorescence. Bars, 20 μm ** p < 0.01 , *** p < 0.001 vs control group; # p < 0.05 , ## p < 0.01 , ### p < 0.001 vs TNBS model group. Data were representative of three independent experiments, expressed as mean ± SD.

Article Snippet: Electrophoresis was performed at 150 V constant pressure for 50 min. A constant pressure of 100 V was set using the wet rotation method, and the film was transferred by ice bath for 2 h. The membrane was immersed in western blot blocking solution (232100; BD) and shaken gently at room temperature for 2 h. The following rabbit primary antibodies were diluted with TBST and prepared according to the manufacturer’s instructions; AOC 1 (16338-1-AP; Proteintech), AOC 1 (A6249; ABclonal), p-NF-κB (3033; CST), NF-κB (8242; CST), p- IκB (2859S; CST), IκB (4812; CST), IKK (2697; CST), AKT (4691; CST), p-AKT (4060; CST), p-IKK (ab178870; Abcam), IL-6 (WL02841; Wanleibio) and TNF-α (WL01581; Wanleibio).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Photodynamic Therapy of Novel Photosensitizer Ameliorates TNBS-Induced Ulcerative Colitis via Inhibition of AOC 1

doi: 10.3389/fphar.2021.746725

Figure Lengend Snippet: LD 4 -PDT inhibited the expression of AOC 1 / AKT / IKK / NF-κB . (A–D) Protein expression of (A) p-AKT , (B) p-IKK, (C) p-IκB and (D) p-p65 in colon tissue were determined by western blot. (E–I) Protein expression of (E) p-AKT , (F) p-IKK , (G) p-IκB , (H) p-p65 (in nuclear), and (I) p-p65 (in cytoplasm) in HCoEpiC cells were determined by western blot ** p < 0.01 , *** p < 0.001 vs control group; # p < 0.05 , ## p < 0.01 , ### p < 0.001 vs TNBS model or LPS model group. Data are representative of three independent experiments, expressed as mean ± SD.

Article Snippet: Electrophoresis was performed at 150 V constant pressure for 50 min. A constant pressure of 100 V was set using the wet rotation method, and the film was transferred by ice bath for 2 h. The membrane was immersed in western blot blocking solution (232100; BD) and shaken gently at room temperature for 2 h. The following rabbit primary antibodies were diluted with TBST and prepared according to the manufacturer’s instructions; AOC 1 (16338-1-AP; Proteintech), AOC 1 (A6249; ABclonal), p-NF-κB (3033; CST), NF-κB (8242; CST), p- IκB (2859S; CST), IκB (4812; CST), IKK (2697; CST), AKT (4691; CST), p-AKT (4060; CST), p-IKK (ab178870; Abcam), IL-6 (WL02841; Wanleibio) and TNF-α (WL01581; Wanleibio).

Techniques: Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Photodynamic Therapy of Novel Photosensitizer Ameliorates TNBS-Induced Ulcerative Colitis via Inhibition of AOC 1

doi: 10.3389/fphar.2021.746725

Figure Lengend Snippet: LD 4 -PDT protected intestine via inhibition of AOC 1 . (A,B) LD 4 -PDT effect on TNF-α , IL-6 and IL-1 expression in AOC 1 knockdown HCoEpiC cells. (C,D) LD 4 -PDT effect on TNF-α , IL-6 and IL-1 expression in AOC 1 over-expression cells. ** p < 0.01, *** p < 0.001 vs control group; # p < 0.05 , ## p < 0.01 , ### p < 0.001 vs LPS groups. Data are representative of three independent experiments, expressed as mean ± SD.

Article Snippet: Electrophoresis was performed at 150 V constant pressure for 50 min. A constant pressure of 100 V was set using the wet rotation method, and the film was transferred by ice bath for 2 h. The membrane was immersed in western blot blocking solution (232100; BD) and shaken gently at room temperature for 2 h. The following rabbit primary antibodies were diluted with TBST and prepared according to the manufacturer’s instructions; AOC 1 (16338-1-AP; Proteintech), AOC 1 (A6249; ABclonal), p-NF-κB (3033; CST), NF-κB (8242; CST), p- IκB (2859S; CST), IκB (4812; CST), IKK (2697; CST), AKT (4691; CST), p-AKT (4060; CST), p-IKK (ab178870; Abcam), IL-6 (WL02841; Wanleibio) and TNF-α (WL01581; Wanleibio).

Techniques: Inhibition, Expressing, Over Expression