Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 2. Loss of ARG1 and AGMAT enhances liver tumor formation (A) Immunoblots of arginine-to-polyamine-converting enzymes (ARG1 and AGMAT) and polyamine metabolism enzymes (ODC, SRM, SMS, SAT1, PAOX, and SMOX) in Ctrl liver and L-dKO tumor tissues. Calnexin serves as loading control (same samples were used as in Figure 1E). n = 4 (Ctrl), n = 8 (L-dKO). (B) Total polyamine content in Ctrl liver and L-dKO tumor tissues. n = 6. (C) Relative 3H-putrescine uptake into Ctrl liver and L-dKO tumor tissues. n = 8. (D) Immunohistochemistry of Ctrl and L-dKO liver tissues stained for ARG1 or AGMAT. NT, adjacent non-tumor tissue; T, tumor. (E) Representative images of livers from L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. (F) Number of macroscopic tumors per liver of L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 9–10. (G) Arginine content in Ctrl liver and L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 4–10. *p < 0.05, **p < 0.01. ***p < 0.001, ****p < 0.0001 by unpaired t test (B and C) and one-way ANOVA (F and G).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2a (Cell Signaling, Cat# 2103), eIF2a-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Western Blot, Control, Immunohistochemistry, Staining, Injection

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 3. ARG1/AGMAT determine metabolic gene expression via arginine (A) Immunoblots of SNU-449 cells upon stable expression of ARG1 and/or AGMAT. Actin serves as loading control. (B) Representative clonogenic growth assay of control, ARG1-, and/or AGMAT-expressing SNU-449 cells grown in arginine-restricted medium. (C) Relative clonogenic growth of control, ARG1-, and/or AGMAT- expressing SNU-449 cells. N = 6. (D) Arginine content of control, ARG1-, and/or AGMAT-expressing SNU-449 cells. N = 4. (E) PCA analysis of RNA-seq data of control and ARG1/AGMAT-expressing SNU-449 cells. (F) Heatmap of a subset of differentially expressed metabolic genes in ARG1/AGMAT-expressing compared to control SNU-449 cells (log2 fold-change). (G) mRNA levels of ASNS, PSAT1, PSPH, GLSK, GLUT3, HK2, NNMT, and AOC3 in control and ARG1/AGMAT-expressing SNU-449 cells. N = 5–7. (H) Immunoblots of ASNS, PSAT, PSPH, and NNMT from two independent experiments of control and ARG1/AGMAT-expressing SNU-449 cells. Calnexin serves as loading control. (I) Immunoblots of ASNS, PSAT, PSPH, and NNMT of Ctrl liver and L-dKO tumor tissues. Calnexin serves as loading control. n = 4 (Ctrl), n = 8 (L-dKO). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA (C and D) and unpaired t test (G).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2a (Cell Signaling, Cat# 2103), eIF2a-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Gene Expression, Western Blot, Expressing, Control, Growth Assay, RNA Sequencing

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 4. ASNS promotes arginine uptake in liver cancer (A) Relative 3H-arginine uptake in control and ARG1/AGMAT-expressing SNU-449 cells with or without pre-loading with asparagine (Asn) or glutamine (Gln). N = 5–6. (B) Immunoblots of ARG1/AGMAT-expressing SNU-449 cells upon stable expression of ASNS or control. Calnexin serves as loading control. (C) Relative 3H-arginine uptake in control and ASNS-expressing SNU-449 ARG1/AGMAT-ex- pressing cells. N = 5. (D) Representative clonogenic growth assay of control and ASNS-expressing SNU-449 ARG1/ AGMAT-expressing cells grown in arginine- restricted medium. (E) mRNA levels of PSAT1, PSPH, GLSK, GLUT3, HK2, NNMT, and AOC3 in control and ASNS- expressing SNU-449 ARG1/AGMAT-expressing cells. N = 6–8. (F) Immunoblots of ASNS, PSAT, PSPH, and NNMT from two independent experiments of control and ASNS-expressing SNU-449 ARG1/ AGMAT-expressing cells. Calnexin serves as loading control. (G) mRNA levels of Asns in L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV- shCtrl or AAV-shAsns. n = 6–7. (H) Number of macroscopic tumors per liver in L-dKO mice injected with AAV-shCtrl or AAV- shAsns. n = 7. (I) Arginine content in L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-shCtrl or AAV-shAsns. n = 4–6. n.s. = not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired t test (A, C, E, G, and H) and one-way ANOVA (I).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2a (Cell Signaling, Cat# 2103), eIF2a-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Control, Expressing, Western Blot, Growth Assay, Injection

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 7. ARG1, AGMAT, arginine, and RBM39 in human HCC patients (A) Schematic representation of arginine and polyamine metabolism in HCC patients. Boxes below enzymes indicate changes in mRNA (left box) and protein (right box) levels in human HCC tumors (T) compared to paired non-tumor (NT) biopsies, respectively. Color coding according to level of log2 fold-change as indicated. ‘‘?’’ indicates unknown identity. Tumor aggressiveness is indicated by Edmondson-Steiner grade low (Edm. low, grade I and II) and high (Edm. high, grade III and IV). n = 73 (Edm. low) and n = 49 (Edm. high) for mRNA; n = 30 (Edm. low) and n = 21 (Edm. high) for protein. (B) Immunoblots of ARG1, AGMAT, RBM39, and ASNS in paired non-tumor (NT) and tumor (T) tissues of five HCC patients. Calnexin serves as loading control. (C) Tissue microarray for ARG1 and AGMAT. ARG1, normal liver n = 58, HCC n = 160; AGMAT, normal liver n = 49, HCC n = 142. (D) Representative IHC of ARG1 and AGMAT of an HCC patient (from C). Non-tumor, NT; tumor, T.

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2a (Cell Signaling, Cat# 2103), eIF2a-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Western Blot, Control, Microarray

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet:

Article Snippet: Rabbit anti-AGMAT , Novus Biological , Cat#1-82080.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, RNA Sequencing, Control, Mutagenesis, CRISPR, Plasmid Preparation, shRNA, Software

Journal: Journal of Animal Science and Biotechnology

Article Title: Pre-implantation exogenous progesterone and pregnancy in sheep: I. polyamines, nutrient transport, and progestamedins

doi: 10.1186/s40104-021-00554-6

Figure Lengend Snippet: Endometrial expression of mRNAs for progestamedins, enzymes involved in polyamine synthesis, and nutrient transporters. Expression of FGF10 ( d ), AZIN2 (H), SLC6A9 ( q ), and SLC1A4 ( r ) were affected by treatment. Expression of SLC2A1 ( n ), SLC5A1 ( o ), and SLC7A1 ( p ) were affected by day. Expression of FGF10 ( b ), ODC1 ( e ), SLC2A1 ( i ), SLC5A1 ( j ), SLC7A1 ( k ), and SLC6A9 ( l ) were affected by a day × treatment interaction. Expression of FGF7 ( a ), HGF ( c ), AZIN2 ( f ) , and AGMAT ( g ) were not affected by a day × treatment interaction. There was a tendency for the endometrial expression of SLC1A4 ( m ) to be affected by a day × treatment interaction. Data are presented as LSM ± SEM. Different means are indicated by different letters. Only endometria from ewes which were considered pregnant with normally developed conceptuse were utilized for these analyses

Article Snippet: AGMAT protein was detected using a primary rabbit polyclonal antibody to AGMAT (Abcam 231,894; Cambridge, UK) at a final dilution of 1:250.

Techniques: Expressing

Journal: Journal of Animal Science and Biotechnology

Article Title: Pre-implantation exogenous progesterone and pregnancy in sheep: I. polyamines, nutrient transport, and progestamedins

doi: 10.1186/s40104-021-00554-6

Figure Lengend Snippet: Localization of endometrial AGMAT. AGMAT proteins localized to uterine luminal epithelia (LE), superficial glandular epithelia (sGE), GE, and stromal cells in both P4- and CO-treated ewes on day 9 and 12 of pregnancy ( a ). Intensity of AGMAT staining in the GE was decreased in endometria at day 12 compared to day 9 ( P < 0.01) ( b ). A day × treatment interaction for the intensity of AGMAT staining in the LE was observed ( P < 0.001), with increased intensity of AGMAT staining observed in the LE from P4-treated ewes compared to CO-treated ewes at day 12. Scale bar represents 100 μm. Only endometria from ewes which were considered pregnant with normally developed conceptuses were utilized for these analyses

Article Snippet: AGMAT protein was detected using a primary rabbit polyclonal antibody to AGMAT (Abcam 231,894; Cambridge, UK) at a final dilution of 1:250.

Techniques: Staining

Journal: Cell Death & Disease

Article Title: Agmatinase promotes the lung adenocarcinoma tumorigenesis by activating the NO-MAPKs-PI3K/Akt pathway

doi: 10.1038/s41419-019-2082-3

Figure Lengend Snippet: a Analysis of AGMAT mRNA expression in paired LUAD tissues from the three LUAD mRNA expression profiling datasets (GSE10072, GSE21933, and GSE32863). b Analysis of AGMAT mRNA expression in LUAD and normal lung tissues from the Selamat lung dataset based on the Oncomine database. c Analysis of the TCGA database for the levels of AGMAT mRNA expression in normal lung tissues and LUAD tissues. d – f Compared with the LUAD patients with a low level of AGMAT expression (the lower 30%), the patients with high AGMAT mRNA expression had higher death rates, shorter DFS, and OS from the TCGA LUAD specimen cohorts. g Representative IHC images of AGMAT expression in LUAD tissues and the corresponding adjacent normal lung tissues. h Differences in the AGMAT expression scores between LUAD tissues ( n = 50) and adjacent normal lung tissues ( n = 50) are shown as a box plot

Article Snippet: After incubating the sections with an anti-human monoclonal AGMAT antibody (1:100; sc-166414, Santa Cruz, TX, USA), they were incubated with a biotin-labeled mouse anti-goat antibody.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Agmatinase promotes the lung adenocarcinoma tumorigenesis by activating the NO-MAPKs-PI3K/Akt pathway

doi: 10.1038/s41419-019-2082-3

Figure Lengend Snippet: Comparison of clinical features between LUAD patients with low and high AGMAT levels in TCGA database

Article Snippet: After incubating the sections with an anti-human monoclonal AGMAT antibody (1:100; sc-166414, Santa Cruz, TX, USA), they were incubated with a biotin-labeled mouse anti-goat antibody.

Techniques: Comparison, Translocation Assay

Journal: Cell Death & Disease

Article Title: Agmatinase promotes the lung adenocarcinoma tumorigenesis by activating the NO-MAPKs-PI3K/Akt pathway

doi: 10.1038/s41419-019-2082-3

Figure Lengend Snippet: a , b The indicated constructs were stably expressed in Hela cells, and Flag and AGMAT were immuno-stained using anti-Flag and AGMAT antibodies, respectively. c , d The overexpression of AGMAT was detected by RT-q-PCR and Western blot in NCI-H1975 and A549 cells. e GSEA plot indicating between DNA replication signatures and the level of AGMAT mRNA expression and between the cell cycle signatures and the level of AGMAT mRNA expression are significantly correlated in the TCGA adenocarcinoma specimen cohorts. f , g The effects of AGMAT transient overexpression on cell proliferation ability f and cell cycle g were detected in NCI-H1975 and A549 cells. h – j The effects of AGMAT stable overexpression on cell growth h , colony formation i , migration, and invasion j were detected in NCI-H1975 and A549 cells. Data are represented as mean ± SEM. * p < 0.05, ** p < 0.01 or *** p < 0.001

Article Snippet: After incubating the sections with an anti-human monoclonal AGMAT antibody (1:100; sc-166414, Santa Cruz, TX, USA), they were incubated with a biotin-labeled mouse anti-goat antibody.

Techniques: Construct, Stable Transfection, Staining, Over Expression, Western Blot, Expressing, Migration

Journal: Cell Death & Disease

Article Title: Agmatinase promotes the lung adenocarcinoma tumorigenesis by activating the NO-MAPKs-PI3K/Akt pathway

doi: 10.1038/s41419-019-2082-3

Figure Lengend Snippet: a , b AGMAT expression was stably silenced in NCI-H1975 and A549 cells, which was detected by RT-q-PCR and Western blot. c – g The effects of stable AGMAT silencing on cell growth c , cell proliferation d , colony formation e , cell cycle progression f , migration, and invasion g were detected in NCI-H1975 and A549 cells. Data are represented as mean ± SEM. * p < 0.05, ** p < 0.01 or *** p < 0.001

Article Snippet: After incubating the sections with an anti-human monoclonal AGMAT antibody (1:100; sc-166414, Santa Cruz, TX, USA), they were incubated with a biotin-labeled mouse anti-goat antibody.

Techniques: Expressing, Stable Transfection, Western Blot, Migration

Journal: Cell Death & Disease

Article Title: Agmatinase promotes the lung adenocarcinoma tumorigenesis by activating the NO-MAPKs-PI3K/Akt pathway

doi: 10.1038/s41419-019-2082-3

Figure Lengend Snippet: a GSEA plot indicating differences between the arginine and proline metabolism signatures and the level of AGMAT mRNA expression is significantly correlated in the TCGA adenocarcinoma specimen cohorts. b , c Following AGMAT overexpression for 36 h, the level of intracellular and extracellular NO was determined using DAF-FM DA staining and a nitrite assay kit, respectively. d Following AGMAT overexpression for 36 h, the level of phosphorylated Erk1/2, P38, and Akt, as well as the total level of Erk1/2, P38, Akt, AGMAT, c-myc, and cyclinD1 were determined by Western blot. d – f Following AGMAT overexpression for 36 h, the cells were treated with the NO scavenger, Carboxy-PTIO (100 μM) for 1 h. The level of intracellular d and extracellular e NO were determined. f The level of phosphorylated Erk1/2, P38, and Akt, and the total level of Erk1/2, P38, Akt, AGMAT, c-myc, and cyclinD1 were determined. Data are represented as mean ± SEM. * p < 0.05, ** p < 0.01 or *** p < 0.001

Article Snippet: After incubating the sections with an anti-human monoclonal AGMAT antibody (1:100; sc-166414, Santa Cruz, TX, USA), they were incubated with a biotin-labeled mouse anti-goat antibody.

Techniques: Expressing, Over Expression, Staining, Nitration, Western Blot

Journal: Cell Death & Disease

Article Title: Agmatinase promotes the lung adenocarcinoma tumorigenesis by activating the NO-MAPKs-PI3K/Akt pathway

doi: 10.1038/s41419-019-2082-3

Figure Lengend Snippet: a , b After AGMAT overexpression for 24 h, the cells were treated with L-NAME (100 μM), or SMT (1 mM), or Spermidine (1 mM) for 24 h. The level of intracellular a and extracellular b NO was determined. c , d After AGMAT overexpression for 36 h or stably silencing, the level of nNOS, eNOS, and iNOS expression were detected by Western blot. e After AGMAT overexpression for 24 h, the cells were treated with L-NAME (100 μM), SMT (1 mM), or Spermidine (1 mM) for 24 h. The level of phosphorylated Erk1/2, P38, and Akt, and total level of Erk1/2, P38, Akt, AGMAT, c-myc, and cyclinD1 were determined. f – h The indicated synonymous mutant sAGMAT-Flag plasmids or its control plasmid was transfected into NCI-H1975 and A549 cells with stable silencing of AGMAT expression or into cells of the control group. After plasmid transfection for 24 h, the cells were treated SMT (1 mM), or for 24 h. The level of intracellular f and extracellular g NO was determined. h The level of phosphorylated Erk1/2, P38, and Akt, and total level of Erk1/2, P38, Akt, AGMAT, c-myc, and cyclinD1 were determined. Data are represented as mean ± SEM. * p < 0.05, ** p < 0.01 or *** p < 0.001

Article Snippet: After incubating the sections with an anti-human monoclonal AGMAT antibody (1:100; sc-166414, Santa Cruz, TX, USA), they were incubated with a biotin-labeled mouse anti-goat antibody.

Techniques: Over Expression, Stable Transfection, Expressing, Western Blot, Mutagenesis, Control, Plasmid Preparation, Transfection

Journal: Cell Death & Disease

Article Title: Agmatinase promotes the lung adenocarcinoma tumorigenesis by activating the NO-MAPKs-PI3K/Akt pathway

doi: 10.1038/s41419-019-2082-3

Figure Lengend Snippet: Silencing AGMAT inhibited the tumorigenicity of LUAD cells in vivo. a The in vivo growth of the NCI-H1975 and A549 cells lines stably silencing AGMAT were examined. Representative images of tumors in the indicated groups in nude mice were shown. b The weight of the xenograft tumors are presented ( n = 6). c Representative images of Ki67 staining in tumors formed by the indicated cells are presented. d Tumors were lysed and the level of phosphorylated Erk1/2, P38, and Akt, and total level of Erk1/2, P38, Akt, AGMAT, iNOS, c-myc, and cyclinD1 were detected

Article Snippet: After incubating the sections with an anti-human monoclonal AGMAT antibody (1:100; sc-166414, Santa Cruz, TX, USA), they were incubated with a biotin-labeled mouse anti-goat antibody.

Techniques: In Vivo, Stable Transfection, Staining