Journal: bioRxiv

Article Title: Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

doi: 10.1101/2020.03.24.006544

Figure Lengend Snippet: Coronavirus antigens on microarray.

Article Snippet: Coronavirus , 229E , 229E , S1+S2 , A0A1L7B942 , Insect Cells , Sino Biological , N-(AA16-1115)-His-C , 40605-V08B.

Techniques: Microarray, Expressing, Construct

Journal: Thoracic Cancer

Article Title: Tumor suppressor effect of an antibody on xenotransplanted sarcomatoid mesothelioma cells

doi: 10.1111/1759-7714.14591

Figure Lengend Snippet: Representative immunofluorescent staining of cultured mesothelioma cells using AX10 antibody (a), immunohistochemical staining (b), and secondary antibody‐drug conjugate assay in vitro (c). (a) AX10 immunoreactivity in MPM‐1, −2, and −3 cells, representing sarcomatoid, epithelioid, and biphasic type mesothelioma, respectively. All MPM‐1, −2, and −3 cells exhibited AX10 antibody immunoreactivity at the cell surface. The staining was analyzed using a Guava easyCyte cell analyzer and accompanying software to obtain a one‐parameter log histogram. (b) AX10 immunoreactivity in various mesothelioma tissue specimens. Weak or no AX10 immunoreactivity was detected in five out of 10 epithelioid mesothelioma tissues (a). One out of five biphasic mesotheliomas exhibited AX10 immunoreactivity in spindle sarcomatoid components (arrow) but weak immunoreactivity in epithelioid components (arrowhead) (b). Five out of six sarcomatoid mesothelioma tissues exhibited strong AX10 immunoreactivity (c). Little AX10 immunoreactivity was detected in normal human tissues. No significant AX10 immunoreactivity was detected in the lung (d) (pleural mesothelial cells; insert) tissue specimens. Weak AX10 immunoreactivity was detected in myofibrous cells in the uterus (e). We did not detect any significant AX10 immunoreactivity in the brain, liver, or kidney, whereas strong AX10 immunoreactivity was observed in a nonmelanocytic (hypomelanocytic) melanoma tissue sample that was supplementally included in the microarray (f) (staining without AX10 antibody; insert). (c) MPM‐1 sarcomatoid mesothelioma cells were incubated with AX10 at 10, 100, and 1000 ng/mL followed by incubation with anti‐murine IgG (Fc) antibody conjugated to duocarmycin. Representative staining with Annexin V‐PI is presented. Note the dose‐dependent Annexin V‐positive and PI‐negative apoptotic MPM‐1 cells in the presence of AX10 antibody

Article Snippet: Tissue microarrays composed of mesothelioma (Cat. No. MS801b) and Food and Drug Administration (FDA) normal organ tissue arrays (Cat. No. NBP2‐78057) were purchased from US Biomax and Novus Biologicals, respectively.

Techniques: Staining, Cell Culture, Immunohistochemical staining, In Vitro, Software, Microarray, Incubation

Journal: Thoracic Cancer

Article Title: Tumor suppressor effect of an antibody on xenotransplanted sarcomatoid mesothelioma cells

doi: 10.1111/1759-7714.14591

Figure Lengend Snippet: AX10 does not affect cell proliferation, but significantly decreases Matrigel invasion activity of MPM‐1 sarcomatoid mesothelioma cells in vitro. (a) Representative cell proliferation assay. At 24 h, the cell number was 1.80 ± 0.10 (mock) and 1.77 ± 0.06 (AX10). Respective numbers at 48 h were 2.40 ± 0.10 (mock) and 2.37 ± 0.12 (AX10), while at 72 h they were 3.90 ± 0.20 (mock) and 4.20 ± 0.61 (AX10). The data represent means ± SD from triplicate assays (Student's t ‐test, p > 0.5). (b) AX10 significantly reduced Matrigel invasion activity of MPM‐1 cells (Student's t ‐test, p < 0.01). The number of invading cells was 59.7 ± 7.02 (mock) and 10.3 ± 1.52 (AX10) at 24 h, and 210.7 ± 11.4 (mock) and 15.0 ± 3.00 (AX10) at 48 h. Data from triplicate assays are expressed as means ± SD ( n = 3). (c) Cells that migrated to the lower surface of the membrane are shown (48 h). Original magnification, ×100

Article Snippet: Tissue microarrays composed of mesothelioma (Cat. No. MS801b) and Food and Drug Administration (FDA) normal organ tissue arrays (Cat. No. NBP2‐78057) were purchased from US Biomax and Novus Biologicals, respectively.

Techniques: Activity Assay, In Vitro, Proliferation Assay, Membrane

Journal: Thoracic Cancer

Article Title: Tumor suppressor effect of an antibody on xenotransplanted sarcomatoid mesothelioma cells

doi: 10.1111/1759-7714.14591

Figure Lengend Snippet: Inhibitory effect of AX10 on MPM‐1 xenotransplanted sarcomatoid mesothelioma cell proliferation. (a) Inoculation of AX10 antibody delayed the growth of xenotransplanted MPM‐1 sarcomatoid mesothelioma tumors. On day 0, SCID‐NOD mice were subcutaneously implanted with MPM‐1 cells. The following day, day 3, the mice were administered AX10 antibody or vehicle only by intraperitoneal injection and weekly thereafter as indicated by arrows. Values are represented as means ± standard error for n = 5 mice. Statistical significance was measured by a two‐sided unpaired Student's t ‐test (* p < 0.01). (b) On day 42, the xenotransplanted tumors were excised to determine their weight. Total tumor weights are represented as means ± standard error for n = 5 mice. Statistical significance was measured by a two‐sided unpaired Student's t ‐test ( p < 0.01). (c) Gross and histological appearance of a representative xenotransplanted tumor. Arrowhead indicates the tumor without AX10 antibody, while the arrow indicates the small tumor remaining following weekly AX10 injection. Note the elimination of tumor cells, which were histologically replaced by regenerative muscle in mice inoculated with AX10 antibody. Scale bar indicates 100 μm

Article Snippet: Tissue microarrays composed of mesothelioma (Cat. No. MS801b) and Food and Drug Administration (FDA) normal organ tissue arrays (Cat. No. NBP2‐78057) were purchased from US Biomax and Novus Biologicals, respectively.

Techniques: Injection

Journal: BMC Microbiology

Article Title: Separation of the bacterial species, Escherichia coli , from mixed-species microbial communities for transcriptome analysis

doi: 10.1186/1471-2180-11-59

Figure Lengend Snippet: Plot of gene expression of sorted/unsorted cells . Plot of one-sample T-test p-values with fold-change in gene expression for all ORFs in microarray study I. Vertical lines show the cutoff of fold-change of 2 (Log 2 ratio of ± 1), while the horizontal line shows the cutoff of p-value 0.05. Genes located in the left-bottom corner (Log 2 ratio <-1 and p-value <0.05) and in the right-bottom corner (Log 2 ratio >1 and p-value <0.05) were considered to have their expressions changed due to dispersion/homogenization and IMS (immuno-magnetic separation) cell sorting. A total of ten genes were selected using these criteria, eight of which also differentially expressed in the independent microarray study II.

Article Snippet: Hybridization was in a Corning Microarray Hybridization Chamber (Corning Inc.) in 42°C water bath.

Techniques: Expressing, Microarray, Homogenization, FACS

Journal: BMC Microbiology

Article Title: Separation of the bacterial species, Escherichia coli , from mixed-species microbial communities for transcriptome analysis

doi: 10.1186/1471-2180-11-59

Figure Lengend Snippet: Genes identified as differentially expressed # between IMS sorted E. coli cells versus unsorted E. coli cells* by the method of cDNA microarray and their differential expression confirmed with another method of qPCR

Article Snippet: Hybridization was in a Corning Microarray Hybridization Chamber (Corning Inc.) in 42°C water bath.

Techniques: Microarray, Expressing

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: 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 1 E). n = 4 (Ctrl), n = 8 (L-dKO). (B) Total polyamine content in Ctrl liver and L-dKO tumor tissues. n = 6. (C) Relative 3 H-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), eIF2α (Cell Signaling, Cat# 2103), eIF2α-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: Loss of ARG1 and AGMAT promote tumorgenicity by sustaining high levels of arginine, related to Figure 2 (A) Polyamine species in L-dKO tumors relative to Ctrl liver tissues (log 2 ratio). n = 5 (Ctrl), n = 6 (L-dKO). (B) Total polyamine content in Ctrl liver and L-dKO non-tumor (NT) and tumor (T) tissues of mice fed with arginine-modified diets. n = 3–9. (C) Immunohistochemistry of Ctrl and L-dKO liver tissues from 12- and 16-week-old mice stained for ARG1 or AGMAT proteins, respectively. NT, adjacent non-tumor tissue; T, tumor. (D) Immunoblots of ARG1 and AGMAT in paired L-dKO non-tumor (NT) and tumor (T) tissues from mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. AKT serves as loading control. n = 2 (AAV-Ctrl), n = 3 (AAV-ARG1), and n = 3 (AAV-AGMAT). (E) Liver-to-body-weight ratio of Ctrl and L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 4–10. (F) Total polyamine 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. n.s. = not significant; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by multiple t test (A) and one-way ANOVA (B, E, and F).

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), eIF2α (Cell Signaling, Cat# 2103), eIF2α-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: Modification, Immunohistochemistry, Staining, Western Blot, Injection, Control

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: ARG1 and AGMAT expression determine metabolism and growth of liver cancer cells, related to Figure 3 (A) Immunoblots of ARG1, AGMAT, CPS1, OTC, ASS1, and ASL expression in human liver cancer cell lines. Actin serves as loading control. (B) Representative clonogenic growth assay of control, ARG1-, and/or AGMAT-expressing SNU-449 cells grown in standard, arginine-rich DMEM (i.e., 400 μM) medium. (C) Relative clonogenic growth of control, ARG1-, and/or AGMAT- expressing SNU-449 cells grown in standard, arginine-rich DMEM medium. N = 3. (D) Arginine content in plasma and TME of L-dKO mice. n = 8 (plasma), n = 6 (TME). (E) Representative clonogenic growth assay of control and ARG1/AGMAT-expressing SNU-449 cells grown in medium containing 100 μM arginine (“plasma-like”) or 20 μM arginine (“TME-like”). (F) Relative polyamine content of control, ARG1-, and/or AGMAT-expressing SNU-449 cells. N = 4. (G) Immunoblots of SNU-449 cells upon stable overexpression of ASS1-FLAG. Huh1 cells serve as control for expression of arginine synthesis enzymes. Calnexin serves as loading control. (H) Arginine content of control or ASS1-FLAG-overexpressing SNU-449 cells. (I) Representative clonogenic growth assay of control or ASS1-FLAG-overexpressing SNU-449 cells grown under arginine-restricted conditions. (J) Immunoblots of ARG1/AGMAT-expressing SNU-449 cells upon stable overexpression of ASS1 or 3xHA-ASS1. Huh1 cells serve as control for expression of arginine synthesis enzymes. Calnexin serves as loading control. (K) Arginine content of control, ASS1-, or 3xHA-ASS1-overexpressing SNU-449 ARG1/AGMAT cells. (L) Clonogenic growth assay of control, ASS1-, or 3xHA-ASS1-overexpressing SNU-449 ARG1/AGMAT cells grown under arginine-rich (400 μM) or arginine-restricted (4 μM) conditions. (M) Representative images of hepatospheres of control and ARG1/AGMAT-expressing SNU-449 cells grown in arginine-restricted medium in ultra-low attachment plates. Scale bar, 100 μm. (N) Number of hepatospheres (as in G). N = 6. (O) Immunoblot analyses of ARG1 and AGMAT in sgCtrl, sgARG1, and sgAGMAT Huh7 cells. Calnexin serves as loading control. (P) Representative clonogenic growth assay of sgCtrl, sgARG1, and sgAGMAT Huh7 cells. (Q) Relative clonogenic growth of sgCtrl, sgARG1, and sgAGMAT Huh7 cells. N = 3. (R) Clonogenic growth of ARG1/AGMAT-expressing SNU-449 cells grown in arginine-restricted medium in the presence of 400 μM of indicated metabolites. (S) Volcano plot of the −log 10 (adjusted p value) against the log 2 fold-change of the differentially expressed genes in ARG1/AGMAT-expressing compared to control SNU-449 cells. Blue and red dots indicate significantly decreased and increased gene expression, respectively. (T) Deregulated metabolic pathways (within top 25 of all deregulated pathways; see Table S2 ) in ARG1/AGMAT-expressing compared to control SNU-449 cells after PWEA (using KEGG pathways, presented by enrichment factor) of differentially expressed genes from RNA-seq. (U) mRNA levels of ASNS , PSAT1 , PSPH , GLSK , GLUT3 , and HK2 in ARG1/AGMAT-expressing SNU-449 cells grown in arginine-restricted medium with or without supplementation of excess arginine (i.e., 4 mM equal to 10× compared to standard DMEM medium) for 16 h. N = 4–8. n.s. = not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by one-way ANOVA (C, F, K, and Q) and unpaired t test (D, H, N, and U).

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), eIF2α (Cell Signaling, Cat# 2103), eIF2α-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: Expressing, Western Blot, Control, Growth Assay, Over Expression, RNA Sequencing Assay

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: 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 (log 2 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), eIF2α (Cell Signaling, Cat# 2103), eIF2α-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: Expressing, Western Blot, Control, Growth Assay, RNA Sequencing Assay

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: ARG1/AGMAT-regulated ASNS enhances arginine uptake required for tumorigenicity, related to Figure 4 (A) Top ten differentially expressed genes in ARG1/AGMAT-expressing compared to control SNU-449 cells by log 2 fold-change (left) and −log 10 (adjusted p value) (right). (B) Clonogenic growth of control and ARG1/AGMAT-expressing SNU-449 cells grown in arginine-restricted medium supplemented with asparagine as indicated. (C) Clonogenic growth of ARG1/AGMAT+control or ARG1/AGMAT+ASNS-expressing SNU-449 cells grown in arginine-restricted or arginine-deficient medium. (D) mRNA levels of ATF4 and ATF4 target genes SESN2 , GPT2 , MTHFD2 , VEGFA , and SLC1A5 in control and ARG1/AGMAT-expressing SNU-449 cells grown under arginine-restricted conditions. Unpaired t test; n.s. = not significant. N = 7. (E) Representative images of livers from L-dKO mice injected with AAV-shCtrl or AAV-sh Asns . (F) Immunoblot of ASNS in non-tumor (NT) and tumor (T) tissues of L-dKO mice injected with AAV-shCtrl or AAV-sh Asns . n = 3. Calnexin serves as loading control. ∗ indicates a cross-reaction.

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), eIF2α (Cell Signaling, Cat# 2103), eIF2α-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: Expressing, Control, Injection, 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: ASNS promotes arginine uptake in liver cancer (A) Relative 3 H-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 3 H-arginine uptake in control and ASNS-expressing SNU-449 ARG1/AGMAT-expressing 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-sh Asns . n = 6–7. (H) Number of macroscopic tumors per liver in L-dKO mice injected with AAV-shCtrl or AAV-sh Asns . n = 7. (I) Arginine content in L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-shCtrl or AAV-sh Asns . 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), eIF2α (Cell Signaling, Cat# 2103), eIF2α-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: RBM39 requires arginine binding to transcriptionally control metabolic gene expression and tumorigenicity, related to Figure 6 (A) mRNA levels of ASNS , PSAT1 , PSPH , GLSK , GLUT3 , HK2 , NNMT , AOC3 , and RBM39 upon si RBM39 and siCtrl in SNU-449 cells. N = 5–7. (B) mRNA levels of ASNS , PSAT1 , HK2 , NNMT , and RBM39 upon stable knockdown of RBM39 (sh RBM39_1 and sh RBM39_2) and shCtrl in SNU-449 cells. N = 5–6. (C) mRNA levels of ATF4 in indisulam- or DMSO-treated SNU-449 cells. N = 6. (D) mRNA levels of ASNS , PSAT1 , PSPH , GLUT3 , and NNMT in indisulam- or DMSO-treated ARG1/AGMAT-expressing SNU-449 cells. N = 5–6. (E) mRNA levels of PSAT1 , PSPH , GLUT3 , and NNMT in indisulam- or DMSO-treated ARG1/AGMAT+ASNS-expressing SNU-449 cells. N = 4. (F) Representative clonogenic growth assay of SNU-449 shCtrl, sh RBM39_1 , and sh RBM39_2 cells grown under arginine-restricted conditions in the absence or presence of 100 μM asparagine. (G) Immunoblot of 3xHA-RBM39 expressed in ARG1/AGMAT-expressing SNU-449 cells. Calnexin serves as loading control. (H) mRNA levels of ASNS , PSAT1 , PSPH , GLSK , NNMT, HK2 , and RBM39 in control and 3xHA-RBM39-expressing SNU-449 ARG1/AGMAT cells. N = 3. (I) mRNA levels of RBM39 in indisulam- or DMSO-treated SNU-449 cells. N = 4. (J) PCA analysis of RNA-seq data of control and RBM39-depleted SNU-449 cells. (K) Volcano plot of the −log 10 (adjusted p value) against the log 2 fold-change of differentially expressed genes in RBM39-depleted compared to control SNU-449 cells. Blue and red dots indicate significantly decreased and increased gene expression, respectively. (L) Clustering of the top 2,500 differentially expressed genes in ARG1/AGMAT-expressing compared to control SNU-449 cells with the differentially expressed genes in RBM39-depleted compared to control SNU-449 cells. Values of differentially expressed genes were binarized prior to clustering. (M) Table summarizing alternative splicing events (ASEs) detected in RNA-seq of control and RBM39-depleted SNU-449 cells and control and ARG1/AGMAT-expressing SNU-449 cells after analysis with the R package NxtIRFcore. IR, intron retention by algorithm; RI, intron retention curated; SE, skipped exon; A3SS, alternative 3′ splice site; A5SS, alternative 5′ splice site; AFE, alternative first exon; ALE, alternative last exon; MXE, mutually excluded exon (see also Table S4 ). (N) Read counts of TRIM27 (Tripartite motif-containing protein 27), DUSP11 (Dual specificity protein phosphatase 11), THEM4 (Thioesterase superfamily member 4), and RFC4 (Replication factor C subunit 4) from RNA-seq of control and RBM39-depleted SNU-449 cells displayed with integrated genome viewer (IGV). Regions highlighted with arrows indicate skipped exons (SE) or intron retention (IR). Blue line indicates introns, and blue boxes indicate exons. Arrow below blue line indicates gene orientation. (O) Representative endpoint PCR of TRIM27 (exon 3–8) in control and RBM39-depleted cells (as in J). (P) Read counts of ASNS , PSAT1 , GLUT3 , and HK2 from RNA-seq of control and RBM39-depleted SNU-449 cells displayed with IGV (as in N). (Q) Relative luciferase-based promoter activity of ASNS and PSAT1 in SNU-449 shCtrl, sh RBM39_1 , and sh RBM39_2 cells grown under arginine-restricted conditions. N = 4–6. (R) Relative luciferase-based promoter activity of ASNS and PSAT1 in control and ARG1/AGMAT-expressing SNU-449 cells grown under arginine-restricted conditions. N = 5–8. (S) Immunoblots of SNU-449 cells expressing full-length, ΔN, or ΔN-NLS cMYC RBM39(G268V)-FLAG treated with indisulam or DMSO. Calnexin serves as loading control. s.e., short exposure; l.e., long exposure. (T) mRNA levels of ASNS in SNU-449 cells expressing ΔN-NLS cMYC RBM39(G268V)-FLAG treated with indisulam for two days in arginine-restricted conditions or in arginine-repleted conditions (400 μM). N = 6. (U) Representative endpoint PCR of TRIM27 (exon 3–8) in SNU-449 cells expressing full-length, ΔN, or ΔN-NLS cMYC RBM39(G268V)-FLAG treated with indisulam. (V) Relative clonogenic growth of SNU-449 cells expressing full-length, ΔN, or ΔN-NLS cMYC RBM39(G268V)-FLAG treated with indisulam. N = 3. (W) Representative images of livers from L-dKO mice injected with AAV-shCtrl or AAV-sh Rbm39 . (X) Liver-to-body-weight ratio of L-dKO mice injected with indisulam or vehicle. n = 4 (vehicle), n = 5 (indisulam). n.s. = not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by unpaired t test (A–E, H, I, Q, R, T, and X) and one-way ANOVA (V).

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), eIF2α (Cell Signaling, Cat# 2103), eIF2α-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: Binding Assay, Control, Expressing, Knockdown, Growth Assay, Western Blot, RNA Sequencing Assay, Alternative Splicing, Luciferase, Activity 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: 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 log 2 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. (E) Kaplan-Meier survival estimate curve for The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) patients ranked by expression of ARG1 and AGMAT . n = 89 (low), n = 109 (normal). (F) Urea cycle metabolites in tumors (T) relative to paired non-tumor (NT) liver tissues (log 2 ratio). n = 11. (G) Immunoblots of RBM39 in tumor lysate (Input) and elution after purification with leucine (Leu)- or arginine (Arg)-coupled agarose beads from three HCC patients. Calnexin serves as input and negative control. (H) Dose-response curve of 20 HCC patient-derived organoids treated with indisulam. Data are presented as the percentage of control DMSO-treated tumor organoids. (I) Model. In liver cancer cells, loss of ARG1 and AGMAT preserves arginine, which in turn binds RBM39 to promote metabolic reprogramming. Arginine-RBM39-mediated ASNS expression further enhances arginine uptake. Trsx, transcription. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 by unpaired t test (C), log rank test (E), and multiple t test (F).

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), eIF2α (Cell Signaling, Cat# 2103), eIF2α-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, Expressing, Purification, Negative Control, Derivative Assay

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: ARG1 and AGMAT are decreased and arginine, RBM39, and ASNS are increased in HCC patient tumors that are sensitive to RBM39 depletion by indisulam, related to Figure 7 (A) RBM39 mRNA levels in liver tumor tissue (T) from HCC patients compared to adjacent non-tumor tissue (NT), displayed as log 2 ratio. n = 73 (Edm. low), n = 49 (Edm. high). (B) RBM39 protein levels in liver tumor tissue (T) from HCC patients compared to adjacent non-tumor tissue (NT), displayed as log 2 ratio. n = 30 (Edm. low), n = 21 (Edm. high). (C) ASNS mRNA levels in liver tumor tissue (T) from HCC patients compared to adjacent non-tumor tissue (NT), displayed as log 2 ratio. n = 73 (Edm. low), n = 49 (Edm. high). (D) ASNS protein levels in liver tumor tissue (T) from HCC patients compared to adjacent non-tumor tissue (NT), displayed as log 2 ratio, if applicable. BW, black-and-white, i.e., only detected in tumor tissues. n = 3 (Edm. low), n = 8 (Edm. high). (E) Staging of ARG1 and AGMAT IHC staining in tissue micro array. (F) mRNA expression of ARG1 , AGMAT , RBM39 , and ASNS in early-stage HCC (data from Jiang et al. ). log 2 fold-change tumor (T) relative to non-tumor (NT) tissues. n = 35. (G) Kaplan-Meier survival estimate curve for TCGA-LIHC patients ranked by expression of ARG1 . n = 135 (low), n =155 (normal). (H) Kaplan-Meier survival estimate curve for TCGA-LIHC patients ranked by expression of AGMAT . n = 136 (low), n = 158 (normal). (I) Polyamine species in tumors (T) relative to paired non-tumor (NT) liver tissues (log 2 ratio). n = 11. (J) Arginine content in paired non-tumor (NT) and tumor (T) tissues of HCC patients. n = 10. (K) Total polyamine content in paired non-tumor (NT) and tumor (T) tissues of HCC patients. n = 10. (L) Volcano plot of the −log 10 (adjusted p value) against the log 2 fold-change of 600 proteins identified by MS (in minimum 2 out of 3 samples) after purification from HCC tissues by arginine (Arg)- compared to leucine (Leu)-coupled agarose beads. Red dot highlights RBM39. (M) Dose-response curve of 20 HCC patient-derived organoids treated with sorafenib. Data are presented as the percentage of control DMSO-treated tumor organoids. (N) IC 50 of indisulam- and sorafenib-treated HCC patient-derived organoids. n = 20. (O and P) Rbm39 and Asns mRNA levels in embryonic day 14 (E14), E18, and adult mouse liver as reads per kilobase of exon per million reads mapped (RPKM). Data from NBCI Gene. n.s. = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by paired t test (A–C, J, K, and N), multiple t test (F and I), and log rank test (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), eIF2α (Cell Signaling, Cat# 2103), eIF2α-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: Immunohistochemistry, Microarray, Expressing, Purification, Derivative Assay, Control

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: 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), eIF2α (Cell Signaling, Cat# 2103), eIF2α-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: Recombinant, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, RNA Sequencing Assay, Control, Mutagenesis, CRISPR, Plasmid Preparation, shRNA, Software

Journal: bioRxiv

Article Title: Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

doi: 10.1101/2020.03.24.006544

Figure Lengend Snippet: Coronavirus antigens on microarray.

Article Snippet: Coronavirus , HKU1 , HKU1 , S1 , Q0ZME7 , HEK293 , Sino Biological , N-(AA13-756)-His-C , 40602-V08H.

Techniques: Microarray, Expressing, Construct

Journal: bioRxiv

Article Title: Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

doi: 10.1101/2020.03.24.006544

Figure Lengend Snippet: Content of coronavirus antigen microarray.

Article Snippet: Influenza , H3N2 , A/Texas/50/2012 , HA1 , AGL07159.1 , HEK293 , Sino Biological , N-(AA1-345)-His-C , 40354-V08H1.

Techniques: Microarray

Journal: bioRxiv

Article Title: Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

doi: 10.1101/2020.03.24.006544

Figure Lengend Snippet: Non-coronavirus respiratory virus antigens on microarray.

Article Snippet: Influenza , H3N2 , A/Texas/50/2012 , HA1 , AGL07159.1 , HEK293 , Sino Biological , N-(AA1-345)-His-C , 40354-V08H1.

Techniques: Microarray, Expressing, Construct

Journal: STAR Protocols

Article Title: An antigen microarray protocol for COVID-19 serological analysis

doi: 10.1016/j.xpro.2021.100815

Figure Lengend Snippet: An overview of the observed response for 48 patient sera to the S1 fragment of spike protein for each of the four HCoV viruses as well as SARS-CoV-2

Article Snippet: Human coronavirus (HCoV-NL63) Spike/S1 Protein (S1 Subunit, His Tag) , Sino Biological , Cat#40600-V08H.

Techniques:

Journal: STAR Protocols

Article Title: An antigen microarray protocol for COVID-19 serological analysis

doi: 10.1016/j.xpro.2021.100815

Figure Lengend Snippet:

Article Snippet: Human coronavirus (HCoV-NL63) Spike/S1 Protein (S1 Subunit, His Tag) , Sino Biological , Cat#40600-V08H.

Techniques: Concentration Assay, Recombinant, Software, Microarray

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Antibody panels used for immunohistochemistry on HD tissue microarrays.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Immunohistochemistry, Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Immunohistochemical profiling of HTT inclusion body ubiquitination and associated triage protein binding in the HD human middle temporal gyrus. Multiplexed immunohistochemical approaches were used to identify HTT inclusion bodies, ubiquitin species, and triage proteins in neurologically normal and HD human middle temporal gyrus tissue microarray cores. Example images from HD case, HC150, are shown. HTT inclusion body antibodies, EM48 ( A ), EPR ( B ), and MW1 ( C ), were used for labelling together with antibodies for pan-ubiquitin ( D ), K48- and K63-linked polyubiquitination ( E and F ), p62 ( G ), and ubiquilin 2 ( H ), with a Hoechst nuclear counterstain ( I ); scale bars = 20 μm.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Immunohistochemical staining, Ubiquitin Proteomics, Protein Binding, Microarray

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: HTT inclusion bodies are not frequently ubiquitinated, but when ubiquitinated, are predominantly ubiquitinated by K63-linked ubiquitin. Immunohistochemical labelling revealed that EM48, EPR, and/or MW1 HTT inclusion bodies were ubiquitinated by K48- and/or K63-linked ubiquitin ( A ); a representative image of K48- and K63-ubiquitinated HTT inclusion bodies from HD case, HC145, is shown; scale bars = 10 μm. The ubiquitination status of each HTT inclusion body was determined by labelling for pan-, K48-, and K63-linked ubiquitin, where positive labelling was identified if the maximum intensity was above manually determined thresholds. The percentage of EM48 + versus EM48- ( B ), EPR + versus EPR- ( C ), and MW1 + versus MW1- ( D ) HTT inclusion bodies that were ubiquitinated (either pan, K48-, and/or K63-linked) were compared using a Wilcoxon matched-pairs signed rank test. The percentage of ubiquitinated HTT inclusion bodies was determined for each EM48, EPR, and MW1 +/- phenotype per HD case ( E ), and compared between phenotypes using a mixed-effects analysis, with Geisser-Greenhouse correction and Tukey’s multiple comparisons test. The percentage of ubiquitinated HTT inclusion bodies ubiquitinated by K48- versus K63-linked ubiquitin was compared using a Wilcoxon matched-pairs signed rank test ( F ). The percentage of ubiquitinated EM48 + versus EM48- ( G ), EPR + versus EPR- ( H ), and MW1 + versus MW1- ( I ) HTT inclusion bodies ubiquitinated by K48- versus K63-linked ubiquitin were compared using an ordinary two-way ANOVA with Tukey’s multiple comparisons test. The percentage of EM48, EPR, and MW1 +/- immunophenotypes HTT inclusion bodies identified as being ubiquitinated by K48- or K63-linked chains were compared using an ordinary two-way ANOVA with Sidak’s multiple comparisons test ( J ). Data are presented as truncated violin plots ( n = 20). Statistical significance of differences shown for B-D and F-J: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Statistical significance for E is shown in Supplementary Table 3.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Ubiquitin Proteomics, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Summary of HTT inclusion body characteristics. Heatmap organised by HTT inclusion body phenotype, with each column representing a single case and each bar coloured according to that case’s value for the characteristic outlined by the row title ( A ). Schematic illustrating the general characteristics of each HTT inclusion body phenotype: (1) EPR + MW1 + inclusion bodies are more frequently located in the nucleus compared to other phenotypes, (2) HTT inclusion bodies that label for more than one epitope-specific antibody are more frequently ubiquitinated, and that ubiquitination occurs more frequently by K63- compared to K48-linked ubiquitin chains, (3) Ubiquitinated HTT inclusion bodies are more frequently tagged by ubiquilin 2 than p62 ( B ). Schematic summarising our hypothesis of HTT inclusion body immunophenotype, ubiquitination, and triage protein tagging with increasing HD severity ( C ); created in BioRender.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Ubiquitin Proteomics