Journal: Cell

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

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 1. Arginine is elevated in liver tumors and promotes tumor formation (A) Hierarchical clustering of significantly altered metabolites from control (Ctrl) liver and tumor tissues (T) from liver-specific Tsc1 and Pten double-knockout (hereafter, L-dKO) mice. n = 5 (Ctrl), n = 6 (L-dKO). (B) Up- and downregulated metabolic pathways in L-dKO tumors compared to Ctrl liver tissues, summarized from MPWEA (see Table S1). (C) Amino acid profile of L-dKO tumor relative to Ctrl liver tissues (log2 ratio). n = 5. (D) Schematic representation of arginine and polyamine metabolism. Boxes below enzymes indicate changes in mRNA (left box) and protein (right box) levels in L-dKO tumors compared to Ctrl livers, respectively. Color coding according to level of log2-fold change as indicated. SMOX, spermine oxidase; SAT1, sper- midine/spermine N-acetyltransferase 1; PAOX, polyamine oxidase; ‘‘?’’ indicates unknown identity. n = 6 (Ctrl), n = 12 (L-dKO). (E) Immunoblots of arginine-synthesizing enzymes (CPS1, OTC, ASS1, and ASL) and arginine transporters (SLC7A1, SLC7A6, and SLC7A7) in Ctrl liver and L-dKO tumor tissues. Calnexin serves as loading control. n = 4 (Ctrl), n = 8 (L-dKO). (F) Relative 3H-arginine uptake into Ctrl liver and L-dKO tumor tissues. n = 8. (G) Number of macroscopic tumors per liver of L-dKO mice fed diets containing standard content (100%), 10%, or 1% of arginine for 8–20 weeks of age. n = 6–9. (H) Arginine content in Ctrl liver and L-dKO non-tumor (NT) and tumor (T) tissues of mice fed with arginine-modified diets. n = 3–9. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired t test (C and F) and one-way ANOVA (G and H).

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, Double Knockout, Western Blot

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: Nature communications

Article Title: Promotion of homology-directed DNA repair by polyamines.

doi: 10.1038/s41467-018-08011-1

Figure Lengend Snippet: Fig. 2 Polyamines affect DSB repair by homologous recombination. a Schematic of the DR-GFP reporter assay to assess HR activity. b, c U2OS cells were treated with 500 μM DFMO for the indicated times. b Levels of three polyamines were quantified and normalized to that in untreated cells. Note that, in agreement with previous studies7,14, treatment with DFMO significantly attenuated levels of putrescine and spermidine but had a lesser effect on that of spermine. c Determination of HR efficiency using the DR-GFP reporter assay. GFP+ cells were quantified by flow cytometry 48 h after I-SceI transfection. Relative HR frequency was normalized to the percentage of untreated cells. Expression levels of I-SceI and tubulin were examined by immunoblotting. d, e U2OS cells were transfected without (Ctrl) or with scrambled siRNA (SCR), ODC siRNA, or expression vector of SAT1 for 24 h. The cells were harvested for analyses 48 h after I-SceI transfection. d The level of individual polyamine was quantified and normalized to that in untransfected cells. e HR efficiency was determined using the DR-GFP reporter assay. The percentage of GFP+ cells was normalized to untransfected cells. Expression levels of ODC and tubulin were examined by immunoblotting. f, g The effects of DFMO (500 µM) treatment and ODC overexpression on HR efficiency were examined in U2OS cells. Untreated cells without any treatment or transfection were included as the control (Ctrl). f The relative level of individual polyamine was determined 2 days after overexpression of ODC. g HR efficiency was quantified by flow cytometry and normalized to control cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant (P > 0.05). Data are the mean ± s.e.m. from three independent experiments (n = 3). Statistics was performed by one-way ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file

Article Snippet: The human SAT1 (RefSeq NM_002970.1) expression plasmid was purchased from OriGene.

Techniques: Homologous Recombination, Reporter Assay, Activity Assay, Cytometry, Transfection, Expressing, Western Blot, Plasmid Preparation, Over Expression, Control

Journal: International Journal of Molecular Sciences

Article Title: Effects of Spermine Synthase Deficiency in Mesenchymal Stromal Cells Are Rescued by Upstream Inhibition of Ornithine Decarboxylase

doi: 10.3390/ijms25052463

Figure Lengend Snippet: Schematic overview of the polyamine synthesis pathway. Abbreviations: AMD1: Adenosylmethionine decarboxylase 1, ODC1: Ornithine decarboxylase, SRM: Spermidine synthase, SMS: Spermine synthase, SMOX: Spermine oxidase, SAT1: Spermidine/spermine N1-acetyltransferase, PAOX: Polyamine oxidase, DFMO: Difluoromethylornithine, CDAP: N-cyclohexyl-1,3-propanediamine.

Article Snippet: Reverse transcription was performed using TaqMan Reverse Transcription Reagents (Life Technologies, Grand Island, NY, USA), and mRNA was measured by real-time PCR using TaqMan Gene Expression Master Mix (Life Technologies, Carlsbad, CA, USA) with TaqMan probes for the following genes: ALPL (Hs01029144_m1), SPP1 (Hs00959010_m1), IBSP (Hs00913377_m1), ODC1 (Hs00159739_m1), SMS (Hs04998877_g1), SRM (Hs01027696_g1), AMD1 (Hs00750876_s1), SAT1 (Hs00971739_g1), SMOX (Hs00602494_m1), PAOX (Hs00382210_m1), ATF4 (Hs00909569_g1), ATF5 (Hs01119208_m1), CHOP (Hs00358796_g1), and GAPDH (Hs02786624_g1).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Effects of Spermine Synthase Deficiency in Mesenchymal Stromal Cells Are Rescued by Upstream Inhibition of Ornithine Decarboxylase

doi: 10.3390/ijms25052463

Figure Lengend Snippet: Polyamine regulation during osteogenesis. MSCs were cultured for 21 days in osteogenic media. ( A ) mRNA levels of osteogenic markers (ALPL, IBSP, and SPP1) and polyamine-associated enzymes (n = 5). ( B ) Representative image of a Western blot showing increased SAT1 (24 kDa) expression during differentiation (n = 3). ( C ) HPLC measurements of spermidine (SPD) and spermine (SPM) (n = 6). ( D ) Putrescine levels measured by mass spectrometry (n = 4). * p < 0.05 calculated using a non-paired Student’s t -test comparing the given time point to day 0 (baseline).

Article Snippet: Reverse transcription was performed using TaqMan Reverse Transcription Reagents (Life Technologies, Grand Island, NY, USA), and mRNA was measured by real-time PCR using TaqMan Gene Expression Master Mix (Life Technologies, Carlsbad, CA, USA) with TaqMan probes for the following genes: ALPL (Hs01029144_m1), SPP1 (Hs00959010_m1), IBSP (Hs00913377_m1), ODC1 (Hs00159739_m1), SMS (Hs04998877_g1), SRM (Hs01027696_g1), AMD1 (Hs00750876_s1), SAT1 (Hs00971739_g1), SMOX (Hs00602494_m1), PAOX (Hs00382210_m1), ATF4 (Hs00909569_g1), ATF5 (Hs01119208_m1), CHOP (Hs00358796_g1), and GAPDH (Hs02786624_g1).

Techniques: Cell Culture, Western Blot, Expressing, Mass Spectrometry