Journal: PeerJ

Article Title: CMTM6 significantly relates to PD-L1 and predicts the prognosis of gastric cancer patients

doi: 10.7717/peerj.9536

Figure Lengend Snippet: The clinicopathological characteristics of GC patients.

Article Snippet: The CMTM6 antibody was obtained from Novus Biologicals (NBP1-31183; Littleton, CO, USA) ( ).

Techniques:

Journal: PeerJ

Article Title: CMTM6 significantly relates to PD-L1 and predicts the prognosis of gastric cancer patients

doi: 10.7717/peerj.9536

Figure Lengend Snippet: (A–C) Negative, weak positive and strong positive expression of CMTM6 (7×) (D–F) Negative, weak positive and strong positive expression of PD-L1 (7×).

Article Snippet: The CMTM6 antibody was obtained from Novus Biologicals (NBP1-31183; Littleton, CO, USA) ( ).

Techniques: Expressing

Journal: PeerJ

Article Title: CMTM6 significantly relates to PD-L1 and predicts the prognosis of gastric cancer patients

doi: 10.7717/peerj.9536

Figure Lengend Snippet: (A) X-tile plots Pattern diagram. The vertical axis represents all possible “high” populations, with the size of the high population increasing from top to bottom. The horizontal axis represents all possible “low” populations, with the size of the low population increasing from left to right. Coloration of the plot represents the strength of the association at each division, ranging from low (black) to high (gray or white). 0.5 is the best cutoff value of CMTM6 expression for this set of samples by X-title software. (B) Kapla–Meier analysis of overall survival (OS) with variable CMTM6 expression in GC patients. The black curve represents CMTM6 high expression group, and the gray curve represents CMTM6 low expression group.

Article Snippet: The CMTM6 antibody was obtained from Novus Biologicals (NBP1-31183; Littleton, CO, USA) ( ).

Techniques: Expressing, Software

Journal: PeerJ

Article Title: CMTM6 significantly relates to PD-L1 and predicts the prognosis of gastric cancer patients

doi: 10.7717/peerj.9536

Figure Lengend Snippet: The univariate and multivariate Cox proportional hazard regression analyses between the clinical related factors and survival in GC patients.

Article Snippet: The CMTM6 antibody was obtained from Novus Biologicals (NBP1-31183; Littleton, CO, USA) ( ).

Techniques:

Journal: PeerJ

Article Title: CMTM6 significantly relates to PD-L1 and predicts the prognosis of gastric cancer patients

doi: 10.7717/peerj.9536

Figure Lengend Snippet: (A) Kapla–Meier analysis of overall survival (OS) with variable PD-L1 expression in GC patients. The black curve represents PD-L1 positive expression group, and the gray curve represents PD-L1 negative expression group. (B) The correlation of CMTM6 and PD-L1 expression in the GC tissues.

Article Snippet: The CMTM6 antibody was obtained from Novus Biologicals (NBP1-31183; Littleton, CO, USA) ( ).

Techniques: Expressing

Journal: PeerJ

Article Title: CMTM6 significantly relates to PD-L1 and predicts the prognosis of gastric cancer patients

doi: 10.7717/peerj.9536

Figure Lengend Snippet: The univariate and multivariate logistic regression analysis between the clinical related risk factors and PD-L1 expression in GC patients.

Article Snippet: The CMTM6 antibody was obtained from Novus Biologicals (NBP1-31183; Littleton, CO, USA) ( ).

Techniques: Expressing

Journal: iScience

Article Title: Genetic distinction between functional tissue-resident and conventional natural killer cells

doi: 10.1016/j.isci.2023.107187

Figure Lengend Snippet: IL-21 and IL-18 play accessory roles in facilitating trNK and cNK activation, respectively (A) Flow cytometric analysis of IL-21R expression on trNK and cNK from liver, uterus, and SGs. The same gating strategy as Figure S1 A is used. (B) Statistical calculation of IL-21 expression level on trNK and cNK from liver, uterus, and SGs, based on its mean fluorescence intensity (MFI) in (A) (n = 4 per group). (C) Flow cytometric analysis of IL-18R1 expression on trNK and cNK from liver, uterus, and SGs. The same gating strategy as Figure S1 A is used. (D) Statistical calculation of IL-18R1 expression level on trNK and cNK from liver, uterus, and SGs, based on its mean fluorescence intensity (MFI) in (C) (n = 4 per group). (E) Flow cytometric analysis of IFN-γ and TNF-α expression in liver trNK and cNK kept unstimulated or stimulated by the indicated conditions. (F) Statistical calculation of IFN-γ and TNF-α expression changes in liver trNK and cNK under the indicated conditions, and relative to PMA/ionomycin stimulation alone induced IFN-γ and TNF-α expression changes in trNK and cNK, respectively (n = 4 per group). Data are shown as the mean ± SEM. P. values are calculated by unpaired t-test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are representative of at least three independent experiments.

Article Snippet: Recombinant Mouse IL-18 , Sinobiological , Cat# 50073-MNCE.

Techniques: Activation Assay, Expressing, Fluorescence

Journal: iScience

Article Title: Genetic distinction between functional tissue-resident and conventional natural killer cells

doi: 10.1016/j.isci.2023.107187

Figure Lengend Snippet: Additional IL-21 stimulation enhances the activation features of trNK (A) Workflow of the RNA-seq analysis of ex vivo stimulated liver trNK and cNK. Sorting strategy is the same as Figure S4 A. (B) Pearson correlation between trNK and cNK under the indicated stimulatory conditions, using trNK and cNK genesets. (C) Pearson correlation of trNK and cNK under the indicated stimulatory conditions according to their whole transcriptomes. (D) PCA of trNK (left) and cNK (right) under the indicated stimulatory conditions (n = 2 per group). Dashed arrows in purple and blue respectively indicate additional IL-21 (purple) and IL-18 (blue) stimulation induced changes to PMA/ionomycin-activated trNK (right) and cNK (left). (E) Heatmap profiling of upregulated (left) and downregulated (right) genes by additional IL-21 stimulation in trNK. The genes are screened based on, (1) reaching to the highest or lowest expression in trNK stimulated by additional IL-21, and (2) exhibiting at least 2-fold changes compared to either unstimulated or PMA/ionomycin-stimulated trNK. (F) Go analysis of the upregulated (top) and downregulated (bottom) genes in trNK by additional IL-21 stimulation in (E).

Article Snippet: Recombinant Mouse IL-18 , Sinobiological , Cat# 50073-MNCE.

Techniques: Activation Assay, RNA Sequencing Assay, Ex Vivo, Expressing

Journal: iScience

Article Title: Genetic distinction between functional tissue-resident and conventional natural killer cells

doi: 10.1016/j.isci.2023.107187

Figure Lengend Snippet:

Article Snippet: Recombinant Mouse IL-18 , Sinobiological , Cat# 50073-MNCE.

Techniques: Recombinant, Sequencing, Infection, Software

Journal: bioRxiv

Article Title: Human 5’-tailed Mirtrons are Processed by RNaseP

doi: 10.1101/2021.10.15.464553

Figure Lengend Snippet: A) Accumulation of expression, determined by small RNA sequencing, from three mirtron constructs (WT, PolyG and Insert) transfected into HEK 293T. Visualization of small RNA read alignments from transfection using the “IGV” software. Left shows the modifications made to the different mirtrons. C) Quantification of luciferase activity from a hsa-miR-5010 sensor following cotransfection with WT, Poly G and Insert hsa-miR-5010 expression plasmids. D) Quantification of hsa-miR-5010 by high through sequencing after transfection of control or Rpp30 siRNAs followed by WT hsa-miR-5010 expression construct. E) Effects of Rpp30 knockdown on 5’ arm heterogeneity. Dominant position found are listed as 1,2,3 as show on the hairpin base diagram. Percent of each found in control and Rpp30 libraries are shown below F) Incubation of radiolabeled hsa-miR-5010 primary intron with immunoprecipitated RNaseP (Rpp20 Ab), mock pull down (cont Ab), or whole cell lysate. Processing products were separated on a PAGE-urea gel. G) RT-PCR of RNaseP complex RNA subunit to verify complex isolation.

Article Snippet: The insoluble fraction was removed by centrifugation at 14,400 rpm for 15 minutes at 4° C. After that the supernatant was incubated for 2 hour with 10 uL of Rpp20 Antibody (Novus Biologicals) immobilized DynabeadsTM Protein G Immunoprecipitation Kit (Invitrogen).

Techniques: Expressing, RNA Sequencing, Construct, Transfection, Software, Luciferase, Activity Assay, Cotransfection, Sequencing, Control, Knockdown, Incubation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Nature communications

Article Title: Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

doi: 10.1038/ncomms11601

Figure Lengend Snippet: Figure 2 | SLC39A14 deficiency causes hypermanganesemia and neurodegeneration that responds to chelation treatment with Na2CaEDTA. (a) Liver MRIs of a patient with SLC30A10 deficiency, individual E-II-2 with SLC39A14 mutations and a control subject. The extensive signal hyperintensity on T1-weighted imaging caused by hepatic Mn deposition in SLC30A10 deficiency is absent in individual E-II-2. There is only a subtle degree of T1- hyperintensity when compared with the control subject. Signal intensity of the liver (yellow arrow) was compared with that of the spleen (blue arrow). (b) Brain histology from post-mortem examination of subject D-II-1. Sections of globus pallidus and dentate nucleus stained with hematoxylin and eosin (H&E) show marked neuronal loss with only occasional remaining neurons (arrow) accompanied by reactive astrocytosis (shown within the ribbon of the dentate nucleus (between arrows)). Scale bar, 100 mm. Luxol fast blue/cresyl violet stain of a section of the cerebral white matter demonstrates patchy loss of myelin associated with coarse vacuoles (arrow). Scale bar 200 mm. (c) Graph showing whole-blood Mn levels and urinary Mn excretion over four courses of Na2CaEDTA treatment in individual E-II-2. Arrows indicate timing of Na2CaEDTA courses (day 1, 34, 52 and 84). Administration of Na2CaEDTA causes a significant increase in urinary Mn excretion (red) accompanied by a drop in whole-blood Mn levels (blue).

Article Snippet: Immunohistochemistry on normal human brain tissue from the Oregon Brain Bank was performed by hand using the rabbit polyclonal anti-SLC39A14 antibody (1:1,000; NBP1-81551, Novus) with DAB/HRP development as above.

Techniques: Control, Imaging, Staining

Journal: Nature communications

Article Title: Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

doi: 10.1038/ncomms11601

Figure Lengend Snippet: Figure 3 | SLC39A14 isoforms 1 and 2 show differences in tissue expression, Mn uptake and transcriptional regulation. (a) Immunostaining for SLC39A14 (all isoforms) in healthy control liver shows cell membrane expression (yellow arrow) and punctate cytoplasmic staining (black arrow); scale bar, 50mm; Abcam anti-SLC39A14 antibody (ab106568, 1:100); and in globus pallidus (GP) from a healthy control shows positively stained large neurons (black arrow); scale bar, 100 mm; Novus anti-SLC39A14 antibody (NBP1-81551, 1:1,000). (b,c) Confocal images demonstrating the subcellular localization of fluorescently tagged human SLC39A14 isoform 1 (b) and isoform 2 (c) expressed in zebrafish embryos. Immunostaining for EGFP and mCherry at 6 hpf shows that both isoforms are expressed at the cell membrane (co-localization with membrane mCherry) and in the cytoplasm. 40,6-diamidino-2-phenylindole (DAPI) was used as a nuclear stain. Scale bar, 50mm. (d) RT–PCR of adult and fetal human tissues showing differences in mRNA expression between isoform 1 (ubiquitous expression in the tissues examined) and isoform 2 (* absent expression in brain, heart, skeletal muscle and skin). Amplicons for isoform 1 and 2 span 139 bp and 109 bp, respectively. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as a housekeeping gene. L, 100 bp ladder (Promega). (e) Graph showing Mn uptake in HEK-293 cells transiently transfected with wild-type SLC39A14 isoform 1 and 2, and empty pCS2þ vector following 15 and 30min of MnCl2 (1mM) exposure. Both isoforms facilitate Mn uptake. Cells transfected with isoform 2 have significantly higher Mn levels (P ¼ 0.009). Data are presented as means±s.d. from two independent experiments. Statistical analysis was performed using one-way ANOVA (P ¼ 0.0002 (15 min), P ¼ 0.0002 (30 min)) and Tukey’s multiple comparison test (***Po0.001). (f) Graph showing slc39a14 transcript levels assessed by qRT–PCR in 5 dpf zebrafish larvae after exposure to 500 mM MnCl2 for 24h. Overall transcript levels are increased (P ¼ 0.035). Transcript levels of isoform 1 are unchanged (P ¼ 0.41) while those of isoform 2 show a 4.6-fold increase (P ¼ 0.005). Data are presented as means±s.d. from three independent experiments. Statistical analysis was performed using Student’s two-tailed t-test on individual DCt values (*Po0.05, **Po0.01). ANOVA, analysis of variance.

Article Snippet: Immunohistochemistry on normal human brain tissue from the Oregon Brain Bank was performed by hand using the rabbit polyclonal anti-SLC39A14 antibody (1:1,000; NBP1-81551, Novus) with DAB/HRP development as above.

Techniques: Expressing, Immunostaining, Control, Membrane, Staining, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Comparison, Quantitative RT-PCR, Two Tailed Test

Journal: Nature communications

Article Title: Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

doi: 10.1038/ncomms11601

Figure Lengend Snippet: Figure 4 | In vitro expressed mutant SLC39A14 shows compromised Mn uptake despite normal subcellular localization. (a) Immunoblot of whole-cell lysates of stably transfected HEK-293 cells showing expression of the wild-type and three mutant SLC39A14 proteins. Actin was used as a loading control. (b) Quantification of SLC39A14 protein levels relative to actin expression revealed no significant difference between wild-type and mutant SLC39A14. Data are presented as means±s.d. from three repeat experiments. Statistical analysis was performed using one-way ANOVA (P ¼ 0.071). (c,d) Confocal images of stably transfected HEK-293 cells expressing FLAG-tagged SLC39A14 (green) showing that wild-type and mutant transporters co-localize with wheat germ agglutinin (WGA)-labelled plasma membrane (red) in (c) non-permeabilized HEK-293 cells and show additional intracellular localization in (d) permeabilized HEK-293 cells. Nuclei are stained with DAPI. Scale bars, 10 mm. (e) Mn influx studies in HEK-293 cells stably expressing wild-type (WT) and mutant forms of SLC39A14 show a decrease in Mn uptake for all SLC39A14 mutants (P ¼ 0.000 [F98V], P ¼ 0.000 [G383R], P ¼ 0.02 [N469K]. Data are presented as means±s.d. from three technical replicates. Statistical analysis was performed using one-way ANOVA (P ¼ 0.000) and Tukey’s multiple comparison test (*Po0.05, ***Po0.001). ANOVA, analysis of variance.

Article Snippet: Immunohistochemistry on normal human brain tissue from the Oregon Brain Bank was performed by hand using the rabbit polyclonal anti-SLC39A14 antibody (1:1,000; NBP1-81551, Novus) with DAB/HRP development as above.

Techniques: In Vitro, Mutagenesis, Western Blot, Stable Transfection, Transfection, Expressing, Control, Clinical Proteomics, Membrane, Staining, Comparison

Journal: Nature communications

Article Title: Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

doi: 10.1038/ncomms11601

Figure Lengend Snippet: Figure 5 | Zebrafish slc39a14 is expressed during early zebrafish development and significantly reduced in slc39a14U801 mutants. (a) RT–PCR showing slc39a14 expression between 3 and 120 hpf in zebrafish. L, 100 bp ladder (Promega). (b) Whole-mount in situ hybridization using a DIG-labelled antisense RNA probe showing slc39a14 expression in the proximal convoluted (black arrows) and straight (red arrows) pronephric tubules in zebrafish larvae at 4 dpf. Top, lateral view; bottom, dorsal view. Scale bar, 200 mm. (c) DNA sequence of the region within exon 5 of slc39a14 targeted by a CRISPR guide RNA is highlighted in yellow and the 2-bp deletion introduced in the slc39a14U801 mutant indicated by dashes. Pam sequence underlined. (d) qRT–PCR demonstrates a 2.2-fold reduction in slc39a14 expression in homozygous slc39a14U801 mutants (*P ¼ 0.0117). Primers were designed to detect all slc39a14 transcripts (Supplementary Table 4). Ef1a was used as a reference gene. Data are presented as means±s.d. from three independent experiments. Statistical analysis was performed using Student’s two-tailed t-test on individual DCt values (*Po00.5).

Article Snippet: Immunohistochemistry on normal human brain tissue from the Oregon Brain Bank was performed by hand using the rabbit polyclonal anti-SLC39A14 antibody (1:1,000; NBP1-81551, Novus) with DAB/HRP development as above.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, In Situ Hybridization, Sequencing, CRISPR, Mutagenesis, Quantitative RT-PCR, Two Tailed Test

Journal: Nature communications

Article Title: Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

doi: 10.1038/ncomms11601

Figure Lengend Snippet: Figure 6 | Loss of slc39a14 function in zebrafish leads to increased Mn accumulation and sensitivity as well as impaired locomotor behaviour. (a) Mn levels assessed in homozygous slc39a14U801 and wild-type (WT) larvae show that mutant larvae have significantly raised Mn levels at 5 dpf (P ¼ 0.001) and 14 dpf (P ¼ 0.0002), and Mn accumulation on MnCl2 exposure (50 mM from 2 dpf) is significantly higher in mutant compared with WT larvae (P ¼ 0.000) at 5 dpf. Measurements were taken from pools of 10 larvae. Data are presented as means±s.d. from a minimum of five independent experiments. Statistical analysis was performed using Student’s two-tailed t-test (***Po0.001). (b) Graph showing Fe, Zn and Cd levels in 14 dpf mutant and WT larvae. Levels of all three metals are not significantly different between the two groups (P ¼ 0.906 [Fe], P ¼ 0.257 [Zn], P ¼ 0.834 [Cd]). Measurements were taken from pools of 10 larvae. Data are presented as means±s.d. from five independent experiments. Statistical analysis was performed using Student’s two-tailed t-test (NS, not significant). (c) Graph presenting the lethality in homozygous slc39a14U801 and WT larvae at 5 dpf on MnCl2 exposure between 2 and 5 dpf. Median lethal concentration (LC50) of MnCl2 determined by Probit regression analysis was 661 mM for WT (95% confidence interval (CI) 548–808 mM) and 377 mM (95% CI 313–455 mM) for mutant fish. Data are presented as means±s.e.m. from nine independent experiments. (d) Locomotor behaviour studies of homozygous slc39a14U801 and WT larvae show that in unexposed conditions there is no significant difference in locomotor activity; and on MnCl2 exposure, locomotor activity is markedly reduced in mutant larvae compared with WT. The locomotor behaviour was tracked during 4 and 7 dpf using automated analysis software. s/min, movement in seconds per minute. Data are presented as means±s.e.m. 12 larvae were analysed per condition. Statistical analysis was performed using two way ANOVA (i, P ¼ 0.18; ii, P ¼ 0.000) (***Po0.001; NS, not significant). ANOVA, analysis of variance.

Article Snippet: Immunohistochemistry on normal human brain tissue from the Oregon Brain Bank was performed by hand using the rabbit polyclonal anti-SLC39A14 antibody (1:1,000; NBP1-81551, Novus) with DAB/HRP development as above.

Techniques: Mutagenesis, Two Tailed Test, Concentration Assay, Activity Assay, Software

Journal: Nature communications

Article Title: Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

doi: 10.1038/ncomms11601

Figure Lengend Snippet: Figure 8 | Proposed disease mechanism in patients with SLC39A14 mutations. (a) Under normal conditions nutritional Mn (red) is absorbed in the duodenum and enters the enterohepatic circulation via the portal vein from which it is transported into the liver via SLC39A14, a Mn uptake transporter (orange). Any excess Mn is rapidly removed from the systemic circulation by uptake into the liver via SLC39A14 and excreted into the bile via SLC30A10 (brown), a Mn efflux protein. (b) Dysfunction of SLC39A14 (indicated by a black X) impairs hepatic uptake of Mn for subsequent biliary excretion. Consequently, Mn accumulates in the blood and brain leading to hypermanganesemia and neurotoxicity, respectively. IVC, inferior vena cava.

Article Snippet: Immunohistochemistry on normal human brain tissue from the Oregon Brain Bank was performed by hand using the rabbit polyclonal anti-SLC39A14 antibody (1:1,000; NBP1-81551, Novus) with DAB/HRP development as above.

Techniques:

Journal: iScience

Article Title: Maternal milk-derived lipids dictate neonatal macrophage phenotype and function

doi: 10.1016/j.isci.2025.114058

Figure Lengend Snippet: Serum of Milk-D-fed adult mice exhibits neonatal mouse serum-like macrophage modulatory properties (A) The serum levels of FFA, TAG, and total cholesterol, free cholesterol, HDL, and LDL/VLDL in mice fed for four weeks with ChowD or MilkD were measured by ELISA. Mean ± SD of data from six samples is presented. Statistical analyses were performed using Mann-Whitney tests. ∗∗ p < 0.01. (B) Adult mouse peritoneal macrophages were incubated with ChowD-fed mouse serum or MilkD-fed mouse serum containing media, and LDs were detected after BODIPY staining. Representative images of three independent experiments are shown. Scale bar lengths are 200 μm. MFI of cells was quantified using ImageJ software. Statistical analyses were performed using Mann-Whitney tests. ∗∗∗ p < 0.001. (C) The levels of FABP4 ( N = 5 per group), GDF-15 ( N = 5 per group), and MFG-E8 ( N = 6 per group) in ChowD- and MilkD-fed mouse serum were measured by ELISA. Mean ± SD of data from each group is presented. Statistical analyses were performed using Mann-Whitney tests. ∗ p < 0.05. (D) Adult peritoneal macrophages were exposed to ChowD- or MilkD-fed adult mouse serum for 20 h, and the changes in the percentage of CD11b + pre-gated CD206 + or IL-4Rα + cells were analyzed by FACS ( N = 5 per group). Mean ± SD of data from each group is presented. Statistical analyses were performed using Mann-Whitney tests. ns, not significant, ∗ p < 0.05. (E) mRNA was isolated from ChowD- or MilkD-fed adult mouse serum-treated adult peritoneal macrophages and subjected to qPCR analysis. Heat maps of the hypoxia, M1-phenotype, M2-phenotype, antigen presentation, chemokines, lipid transport, and lipid metabolism-associated genes are shown. (F) Extracted mRNA was also subjected to RNA-seq analysis, and the enrichment of genes associated with the M1-phenotype, M2-phenotype, TAM, antigen processing and presentation, as well as phagocytosis pathways, were assessed using GSEA. (G) Adult mouse peritoneal macrophages were incubated with ChowD- or MilkD-fed adult mouse serum for 16 h. The uptake of opsonized fluorescent-labeled latex beads by macrophages was assessed by FACS. Data in panel G are representative of three independent experiments and shown as the mean ± SD of triplicates. p -values in panel G were calculated using two-way ANOVA with Šídák multiple comparisons test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also .

Article Snippet: ELISA kits for mouse GDF-15, MFG-E8, S100A8/A9 were purchased from R&D Systems and the ELISA kit for FABP4 was from Novus Biologicals (Centennial, CO).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Incubation, Staining, Software, Isolation, Immunopeptidomics, RNA Sequencing, Labeling

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: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet: Characterization of an AAV6.2FF-hACE2 transduction model for SARS-CoV-2 infection of wild-type mice (A) Diagram of AAV genome expressing hACE2 from the CASI promoter. (B) Western blot of HEK293 cells transduced with AAV6.2FF-hACE and probed with an anti-hACE2 antibody. (C) BALB/c mice were administered 1 x 10 11 vg of AAV-Luc intranasally and imaged 10 days later using an IVIS imager. (D–F) (D) IFA images of lungs harvested from BALB/c mice infected intranasally with 1 x 10 11 vg of AAV-hACE2 or AAV-Luc and euthanized 10 days later. Lungs were stained with a rabbit anit-hACE2 antibody and imaged at 20 X (scale bar, 50mM). Viral RNA (E), and virus TCID50 titers (F) were determined in respiratory tissues on days 2 and 4 post-infection. n = 6 (3M, 3F). Statistical significance determined by Mann-Whitney test. ∗ = p < 0.05, ∗∗ = p < 0.01.

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: Transduction, Infection, Expressing, Western Blot, Staining, MANN-WHITNEY

Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet: SARS-CoV-2 spike DNA antigens protect from viral replication in vivo (A) Mice were immunized once or twice separated by four weeks with 10ug of pS via electroporation. Serum was collected at day 18 post-final immunization. At 35 days post-final immunization mice were infected intranasally with adeno-associated virus expressing human ACE2 (white). 17 days following AAV6-ACE2 transduction, animals were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2 VIDO-01 P2. Four days post infection, animals were sacrificed to quantify viral replication. SARS-CoV-2 specific serum IgG endpoint titers (B) and pseudoviral neutralization titers (C) at day 18 post-final immunization. Replication competent virus (D), and viral RNA (E) in the lungs four-days post-infection. Pearson correlations between virus titer and serum IgG endpoints (F) and neutralization titers (G). Pearson correlations between viral RNA copies and serum IgG endpoints (H) and neutralization titers (I). Each point represents the average of duplicate samples from an individual animal, bars represent the mean, lines represent the median, and error bars represent the SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by student's t-test (A and B), or Kruskall-Wallis ANOVA (D and E). Spearman correlations were used to determine relationships (F-I). Data are representative of one experiment with n = 5 males (squares) and 5 females (circles) per group.

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: In Vivo, Electroporation, Infection, Expressing, Transduction, Neutralization

Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet:

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: Recombinant, Plasmid Preparation, Enzyme-linked Immunospot, Software

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Identification of neutralizing antibodies with a PtY display platform. We first used our preconstructed naïve phage displayed human scFv library to screen binders with biotinylated SARS-CoV-2 RBD protein in the solution phase. After enrichment of phage binders, the scFv DNA from enriched binders was cloned into the yeast display plasmid, resulting in display of scFv on the yeast cell surface. We then performed FACS to isolate potential blocking antibodies that could prevent binding of the SARS-CoV-2 RBD to hACE2. The 0.013% gate contained blocking antibodies with high affinity toward RBD. That is, higher Y axis signal represented higher affinity to labeled RBD, whereas lower X signal represented higher potency in blocking the binding of differently labeled hACE2 to RBD. The potential blocking antibodies were sent for sequencing and transient expression. The purified antibodies were evaluated for affinity, blocking activity, biophysical properties, and virus-neutralizing activity

Article Snippet: The affinity of mAbs for SARS-CoV-2 RBD/S1 and its mutants (SARS-CoV-2 RBD [ACRO, SPD-C52H3], SARS-CoV-2 S1 [ACRO, S1N-C52H4], SARS-CoV-2 RBD [N354D/D364Y] [ACRO, SPD-S52H3], SARS-CoV-2 RBD [V367F] [ACRO, SPD-S52H4], SARS-CoV-2 RBD [N354D] [ACRO, SPD-S52H5], SARS-CoV-2 RBD [W436R] [ACRO, SPD-S52H7], SARS-CoV-2 RBD [R408I] [ACRO, SPD-S52H8], SARS-CoV-2 RBD [G476S] [ACRO, SPD-C52H4], SARS-CoV-2 RBD [V483A] [ACRO, SPD-C52H5], SARS-CoV-2 RBD [A475V] [ACRO, SPD-C52Hd], SARS-CoV-2 RBD [L452R] [ACRO, SPD-C52He], SARS-CoV-2 S1 [D614G] [ACRO, S1N-C5256], SARS-CoV-2 S1 [K417N, E484K, N501Y, D614G] [Sino Biological, 40591-V08H10], SARS-CoV-2 S1 [HV69-70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H] [Sino Biological, 40591-V08H12]) was measured using Octet Red96 (ForteBio, Sartorius).

Techniques: Clone Assay, Plasmid Preparation, Blocking Assay, Binding Assay, Labeling, Sequencing, Expressing, Purification, Activity Assay

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characterization of potential blocking antibodies. (a) Blocking assay was performed by immobilizing 1 µg/ml hACE2 on a plate. Serially diluted antibodies and biotinylated SARS-CoV-2 RBD protein were added for competitive binding to hACE2. IC 50 values were calculated with Prism V8.0 software using a four-parameter logistic curve fitting approach. (b) Epitope binning was carried out by BLI. Biotinylated SARS-CoV-2 RBD was immobilized onto the SA sensor, and a high concentration of the primary antibody was used to saturate its own binding site. Subsequently, a second antibody was applied to compete for the binding site on the SARS-CoV-2 RBD protein. Data were analyzed with Octet Data Analysis HT 11.0 software. (c) Neutralization activities of Ab2001.08 and Ab2001.10 were assessed by live virus assay. Live SARS-CoV-2 and serially diluted (3-fold) antibodies were added to VERO E6 cells. The PRNT 50 values were determined by plotting the plaque number (neutralization percentage) against the log antibody concentration in Prism V8.0 software

Article Snippet: The affinity of mAbs for SARS-CoV-2 RBD/S1 and its mutants (SARS-CoV-2 RBD [ACRO, SPD-C52H3], SARS-CoV-2 S1 [ACRO, S1N-C52H4], SARS-CoV-2 RBD [N354D/D364Y] [ACRO, SPD-S52H3], SARS-CoV-2 RBD [V367F] [ACRO, SPD-S52H4], SARS-CoV-2 RBD [N354D] [ACRO, SPD-S52H5], SARS-CoV-2 RBD [W436R] [ACRO, SPD-S52H7], SARS-CoV-2 RBD [R408I] [ACRO, SPD-S52H8], SARS-CoV-2 RBD [G476S] [ACRO, SPD-C52H4], SARS-CoV-2 RBD [V483A] [ACRO, SPD-C52H5], SARS-CoV-2 RBD [A475V] [ACRO, SPD-C52Hd], SARS-CoV-2 RBD [L452R] [ACRO, SPD-C52He], SARS-CoV-2 S1 [D614G] [ACRO, S1N-C5256], SARS-CoV-2 S1 [K417N, E484K, N501Y, D614G] [Sino Biological, 40591-V08H10], SARS-CoV-2 S1 [HV69-70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H] [Sino Biological, 40591-V08H12]) was measured using Octet Red96 (ForteBio, Sartorius).

Techniques: Blocking Assay, Binding Assay, Software, Concentration Assay, Neutralization

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Effects of Fc modification on the ADE activity of JMB2002. (a) Binding of Ab2001.08 and JMB2002 to FcγRs was determined by BLI. His-tagged FcγR was loaded onto the HIS1K sensor, and serially diluted antibodies bound to the receptor on the biosensor. K D values were determined with Octet Data Analysis HT 11.0 software using a 1:1 global fit model. (b-d) ADE activity was measured using a pseudotyped SARS-CoV-2 system containing a luciferase reporter. Pseudotyped viruses were preincubated with serially diluted antibodies for 1 h. The mixture was added to FcγR-expressing cells and incubated at 37°C for 20–28 h. Infection of cells with pseudotyped SARS-CoV-2 was assessed by measuring cell-associated luciferase activity. Trastuzumab was used as the irrelevant IgG control

Article Snippet: The affinity of mAbs for SARS-CoV-2 RBD/S1 and its mutants (SARS-CoV-2 RBD [ACRO, SPD-C52H3], SARS-CoV-2 S1 [ACRO, S1N-C52H4], SARS-CoV-2 RBD [N354D/D364Y] [ACRO, SPD-S52H3], SARS-CoV-2 RBD [V367F] [ACRO, SPD-S52H4], SARS-CoV-2 RBD [N354D] [ACRO, SPD-S52H5], SARS-CoV-2 RBD [W436R] [ACRO, SPD-S52H7], SARS-CoV-2 RBD [R408I] [ACRO, SPD-S52H8], SARS-CoV-2 RBD [G476S] [ACRO, SPD-C52H4], SARS-CoV-2 RBD [V483A] [ACRO, SPD-C52H5], SARS-CoV-2 RBD [A475V] [ACRO, SPD-C52Hd], SARS-CoV-2 RBD [L452R] [ACRO, SPD-C52He], SARS-CoV-2 S1 [D614G] [ACRO, S1N-C5256], SARS-CoV-2 S1 [K417N, E484K, N501Y, D614G] [Sino Biological, 40591-V08H10], SARS-CoV-2 S1 [HV69-70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H] [Sino Biological, 40591-V08H12]) was measured using Octet Red96 (ForteBio, Sartorius).

Techniques: Modification, Activity Assay, Binding Assay, Software, Luciferase, Expressing, Incubation, Infection

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characterization of JMB2002. Binding affinity of JMB2002 for the SARS-CoV-2 RBD (a)/S1 (b) prototype and its variants was determined by BLI. JMB2002 was loaded onto the AHC sensor, and serially diluted antigens were bound to JMB2002 on the biosensor. K D values were determined with Octet Data Analysis HT 11.0 software using a 1:1 global fit model. Blocking activity was assessed using ELISA with hACE2-coated plates. A mixture of biotinylated SARS-CoV-2 RBD (c)/S1 (d) proteins and JMB2002 was added for competitive binding to hACE2. IC 50 values were calculated by Prism V8.0 software using a four-parameter logistic curve fitting approach. Values are displayed as the mean ± standard deviations from three independent experiments. (e) The pseudovirus neutralization activity of JMB2002 was evaluated using a pseudotyped SARS-CoV-2 system, which contained a luciferase reporter. Pseudotyped viruses were preincubated with serially diluted antibodies for 1 h. The mixture was added to hACE2-expressing cells and incubated at 37°C for 20–28 h. Infection of cells with pseudotyped SARS-CoV-2 was assessed by measuring cell-associated luciferase activity. IC 50 values were calculated by plotting the inhibition rate against the log antibody concentration in Prism V8.0 software

Article Snippet: The affinity of mAbs for SARS-CoV-2 RBD/S1 and its mutants (SARS-CoV-2 RBD [ACRO, SPD-C52H3], SARS-CoV-2 S1 [ACRO, S1N-C52H4], SARS-CoV-2 RBD [N354D/D364Y] [ACRO, SPD-S52H3], SARS-CoV-2 RBD [V367F] [ACRO, SPD-S52H4], SARS-CoV-2 RBD [N354D] [ACRO, SPD-S52H5], SARS-CoV-2 RBD [W436R] [ACRO, SPD-S52H7], SARS-CoV-2 RBD [R408I] [ACRO, SPD-S52H8], SARS-CoV-2 RBD [G476S] [ACRO, SPD-C52H4], SARS-CoV-2 RBD [V483A] [ACRO, SPD-C52H5], SARS-CoV-2 RBD [A475V] [ACRO, SPD-C52Hd], SARS-CoV-2 RBD [L452R] [ACRO, SPD-C52He], SARS-CoV-2 S1 [D614G] [ACRO, S1N-C5256], SARS-CoV-2 S1 [K417N, E484K, N501Y, D614G] [Sino Biological, 40591-V08H10], SARS-CoV-2 S1 [HV69-70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H] [Sino Biological, 40591-V08H12]) was measured using Octet Red96 (ForteBio, Sartorius).

Techniques: Binding Assay, Software, Blocking Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Luciferase, Expressing, Incubation, Infection, Inhibition, Concentration Assay

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Prophylactic and therapeutic efficacies of JMB2002 against SARS-CoV-2 infection in rhesus macaques. (a) Schematic representation of the design of the in vivo animal experiment. Five monkeys were divided into three groups: the control group (one animal, C1), prophylactic group (two animals, PA1 and PA2), and therapeutic group (two animals, AC1 and AC2). In the prophylactic group, a single dose of 20 mg/kg JMB2002 was intravenously injected into the animals before SARS-CoV-2 infection. The next day, all monkeys were infected with virus (1 × 10 5 TCID 50 ) via intratracheal inoculation. In the therapeutic group, 50 mg/kg JMB2002 was injected at 1 and 3 dpi, whereas in the control group, a single dose of 20 mg/kg irrelevant IgG control was administered at 1 dpi. (b) The viral load in oropharyngeal swabs was monitored for 7 days by qRT-PCR. The dotted line indicates the copy number detection limit. (c) Histopathological and immunohistochemical characterization of lung tissues. All animals were euthanized and necropsied at 7 dpi. The tissue samples were collected, fixed in 10% formalin solution, embedded in paraffin, sectioned, and stained with hematoxylin and eosin or Masson’s trichrome before observation by light microscopy. Scale bar = 100 μm

Article Snippet: The affinity of mAbs for SARS-CoV-2 RBD/S1 and its mutants (SARS-CoV-2 RBD [ACRO, SPD-C52H3], SARS-CoV-2 S1 [ACRO, S1N-C52H4], SARS-CoV-2 RBD [N354D/D364Y] [ACRO, SPD-S52H3], SARS-CoV-2 RBD [V367F] [ACRO, SPD-S52H4], SARS-CoV-2 RBD [N354D] [ACRO, SPD-S52H5], SARS-CoV-2 RBD [W436R] [ACRO, SPD-S52H7], SARS-CoV-2 RBD [R408I] [ACRO, SPD-S52H8], SARS-CoV-2 RBD [G476S] [ACRO, SPD-C52H4], SARS-CoV-2 RBD [V483A] [ACRO, SPD-C52H5], SARS-CoV-2 RBD [A475V] [ACRO, SPD-C52Hd], SARS-CoV-2 RBD [L452R] [ACRO, SPD-C52He], SARS-CoV-2 S1 [D614G] [ACRO, S1N-C5256], SARS-CoV-2 S1 [K417N, E484K, N501Y, D614G] [Sino Biological, 40591-V08H10], SARS-CoV-2 S1 [HV69-70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H] [Sino Biological, 40591-V08H12]) was measured using Octet Red96 (ForteBio, Sartorius).

Techniques: Infection, In Vivo, Injection, Quantitative RT-PCR, Immunohistochemical staining, Staining, Light Microscopy

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: SPINK2 is highly expressed in CD34 + bone marrow cells (A) Uniform manifold approximation and projection (UMAP) graph shows the CD34 + cell clusters within human bone marrow as annotated by Setty et al. (B) UMAP graph showing SPINK2 expression in CD34 + cells. (C) SPINK2 and SPINK9 expression values are reported as averaged normalized CPM (top graph), and percentage of SPINK2 or SPINK9 positive cells in different population of CD34 + cells within the bone marrow (bottom graph). A cell is considered positive if normalized CPM value is > 0. Numbers of analyzed cells (n) in each population are the following: HSC, hematopoietic stem cell (n = 4690); HMP, hematopoietic multipotent progenitor (n = 4306); CMP, common myeloid progenitor (n = 2328); GMP, granulocyte-monocyte progenitor (n = 3713); DP, dendritic progenitor (n = 2075); MP, megakaryocyte progenitor (n = 507); EP, erythroid progenitor (n = 3463); CLP, common lymphoid progenitor (n = 3237); (D) Averaged normalized CPM (left graph) and percentage of SPINK positive cells (right graph) in different populations of hematopoietic cells within the bone marrow of mouse C57BL/6. LT-HSC, Long Term-HSC (n = 216); HSCs/HMPs, hematopoietic stem and progenitor cells (n = 852); MP/EP/CMP/GMP, megakaryocyte progenitor/erythroid progenitor/common myeloid progenitor/granulocyte-monocyte progenitor (n = 851).

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Expressing

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: Expression of putative tPRSS in human bone marrow Averages of normalized CPM values (top graph) and percentage of positive cells (bottom graph) in different CD34 + cell subpopulations for (A) PRSS1, PRSS2, and PRSS3; (B) PRSS27, PLAT, and DPP7; and (C) PRSS57, CTSG, ELANE, and PRTN3. (D) qRT-PCR analysis of PRSS1, PRSS2, and PRSS57 transcripts in purified mobilized CD34 + blood cells. CD34 − cells were used as calibrator for fold change calculation. (E) SPINK2/PRSS57 or SPINK2/PRSS2 ratios in different subpopulations of HSPCs. (F) Percentage of PRSS2+ and PRSS57+ cells in SPINK2+ cell population. (G) Percentage of SPINK2+ cells in PRSS2+ cell population and in PRSS57+ cell population.

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Expressing, Quantitative RT-PCR, Purification

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: SPINK2 transcript and protein are highly expressed in CD34 + bone marrow cells (A) qRT-PCR by 2 −ΔΔCT method confirms high levels of SPINK2 in purified CD34 + cells. ELANE, typically expressed in differentiated neutrophil granulocytes and granulocyte-monocyte progenitors, has been evaluated as negative control. (B) SPINK2 and PRSS57 are highly expressed at protein level in CD34 + HSPCs. Protein intensity of SPINK2 and PRSS57 is expressed as normalized label-free (LF) score in CD34 + HSPC (Hennrich et al. ). Negative control results in bone marrow mesenchymal stromal cells (MSC) are also shown. (C) Western blot showing SPINK2 enrichment in CD34 + cells. GAPDH and ACTB were the loading control. (D) SPINK2 (P20155) and SPINK1 (P00995) structures have been downloaded from AlphaFold Protein Structure Database developed by DeepMind and EMBL-EBI ( https://alphafold.ebi.ac.uk/entry/P20155 ; https://alphafold.ebi.ac.uk/entry/P00995 ). , Kazal domain is highlighted in green square. (E) SPINK2 (NP_066937, 84 amino acids) and SPINK1 (NP_001366539) protein alignment was performed by Constrain-based Multiple Alignment tool (Cobalt, https://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi ), and as showed in Figure 2 E the method highlights in red color highly conserved aminoacidic positions based on the relative entropy threshold of the residue, while blue color indicates lower conservation. Kazal domain is underlined (green) and is located among amino acids in position 36 and 84 and includes 6 typical cysteines forming three pairs of disulfide bonds that stabilize its conformation.

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Quantitative RT-PCR, Purification, Negative Control, Western Blot, Control, Residue

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: SPINK2 expression in human tissues and cancer cell lines (A) SPINK2 expression in 1,293 cancer cell lines assembled for tissue of origin in 26 groups. Averages of TPM±SD and percentage of positive cell lines (>10 TPM) are shown in top and bottom histograms, respectively. (B) SPINK2 expression levels in AML cell lines (n = 42); (C) in ALL cell lines (n = 45); and (D) in Lymphoma cell lines (n = 86). (E) qRT-PCR confirmed SPINK2 expression in Jurkat cell line, a model of ALL, while other leukemia cell lines (HL-60, K562, KASUMI-1) did not express SPINK2. HCT116, a colon cancer cell line, was used as calibrator for fold change calculation.

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: Kinetic properties of trypsin inhibition by SPINK2 (A) Graph showing experimental initial reaction rates at different concentrations of SPINK2 (range 3.75–120 nM) and fixed concentration of total enzyme (0.0032 μM) and initial substrate (125 μM); K m was constrained at 2,450 μM. Ki was determined by fitting experimental data to Morrison’s equation by GraphPad Prism 8.0.2. Results are expressed as averages ±SEM of two independent experiments. (B) Initial reaction rates expressed as % of uninhibited reaction rate versus Log 10 concentration of SPINK2. Blue lines and points are calculated values with a Ki = 0.011 μM; red points are experimental values. (C) Experimental “product vs. time” curves; enzymatic reactions were followed for 17 h by monitoring spectrophotometrically the conversion of the substrate FVR-NA in p -nitroaniline at 410 nm. Trypsin (3.2 nM) was incubated with two different SPINK2 concentrations (3.75 and 60 nM) corresponding to an inhibitor/enzyme ratio of 1.2 and 19, respectively. (D) Calculated data obtained with TI-Model, showing the different “product vs. time” curves obtained with temporary and persistent inhibition; (E) Graph showing the “% of initial SPINK2 concentration” vs. Time obtained by TI-Model with Ki = 3 nM and increasing concentration of SPINK2. A SPINK2 k cat of 1.2 x 10 −3 sec −1 and a fixed concentration of 3.2 nM trypsin were used in the simulation. (F) In vitro degradation of recombinant SPINK2 by trypsin at different times. Each incubation was performed in duplicate. Averages of densitometric areas of western blotting bands (shown in the inset) are expressed as percentages of value at time zero (t0). (G) Kinetic reaction scheme for enzyme activity, competitive enzyme inhibition, and inhibitor degradation. Differential rate equations and abbreviations for kinetic constants (k0-k5) and chemical species are also reported in the figure.

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Inhibition, Concentration Assay, Incubation, In Vitro, Recombinant, Western Blot, Activity Assay, Enzyme Inhibition Assay

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: Schematic drawing illustrating the TI-D theory for SPINK2 role in hematopoietic stem cell niche

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques:

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: The temporary inhibition-diffusion model for SPINK2 in hematopoietic stem cell niche (A) The concentration of the complex tPRSS SPINK2, indicated as [EI] and shown in top graphs, and the velocity of conversion of protein substrate ( S v), shown in bottom graphs, were calculated at different distances from the source cell using different values of D (μ 2 /sec), as indicated above each plot. Results obtained for temporary or persistent inhibitor are shown in each plot. A SPINK2/tPRSS ratio = 2 is used in these simulations. (B) The velocity of conversion of protein substrate ( S v) per unit volume was calculated at different distances from the source cell using different ratios [I]/[E] at the source boundary, as indicated above each plot. Results obtained for a temporary inhibitor or a persistent one are compared in each plot. A D value = 5 μ 2 /sec is used in these simulations. (C) The concentration of the complex [EI] is shown in top panels, and the velocity of conversion of protein substrate (Sv) per unit volume in bottom panels. A fixed total concentration of [E] is used in left panels, and an inverse concentration gradient in the right panels. Results obtained for a temporary inhibitor or a persistent one are compared in each plot. A SPINK2/tPRSS ratio = 2 at the boundary of inhibitor source and a D value = 5 μ 2 /sec are used in these simulations.

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Inhibition, Diffusion-based Assay, Concentration Assay

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet:

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Recombinant, Sequencing, Modification, Isolation, SYBR Green Assay, Mass Spectrometry, Extraction, Synthesized, Software

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: Primers details used for qRT-PCR

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques:

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-53BP1 , Novus Biologicals , Cat# NB100-904.

Techniques: Transduction, Recombinant, Protease Inhibitor, SYBR Green Assay, Mutagenesis, Purification, Gel Extraction, Imaging, Sequencing, Negative Control, Real-time Polymerase Chain Reaction, Software, Expressing