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: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Global analyses of IKZF1 and c‐FOS binding to the myeloma genome. (A) Upper panel: Average plot (middle) and heatmap (left) of IKZF1 chromatin immunoprecipitation (ChIP)‐seq reads over all transcription start sites (TSSs) ± 5000 bp. The pie chart shows the gene section breakdown (right). Lower panel: Average plot (middle) and heatmap (left) of c‐FOS ChIP‐seq reads over all TSSs ± 5000 bp. The pie chart shows the gene section breakdown (right). Genes (rows) were ordered in the same way in heatmaps. (B) Left panel: Overlap of IKZF1‐ ( n = 13 932) and c‐FOS‐binding ( n = 10 173) sites in MM.1S cells. Right panel: Status of IKZF1 and c‐FOS binding at promoter/enhancer regions of representative genes based on ChIP‐seq data. (C) Nucleotide sequences of IKZF1‐ and c‐FOS‐binding sites deduced from ChIP‐seq analyses of MM.1S cells. The ChIP‐seq data were analyzed using a Partek Flow genomic analysis software v10.0 (Partek Inc.).

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Binding Assay, Chromatin Immunoprecipitation, ChIP-sequencing, Software

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: c‐FOS‐binding sites are present in nearly one‐half of IKZF1‐target genes in multiple myeloma (MM). (A) Using chromatin immunoprecipitation (ChIP)‐seq data of MM.1S cells, we visualized IKZF1 binding near transcription start sites (TSSs) (red triangles) of the indicated genes in the UCSC genome browser. The genes possessing the activator protein‐1 (AP‐1)‐binding motif based on a TRANSFAC search ( https://genexplain.com/transfac/ ) are marked in red, and those without the conventional AP‐1‐binding motif are marked in blue. (B) The peak values of IKZF1 binding and the presence of AP‐1‐binding sites in selected genes. (C) An example of the co‐occupancy of IKZF1 and c‐FOS in MM.1S cells.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Binding Assay, Chromatin Immunoprecipitation, ChIP-sequencing

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Co‐occupancy of IKZF1 and c‐FOS at promoter/enhancer regions of actively transcribed genes in multiple myeloma (MM). Upper panel: chromatin immunoprecipitation (ChIP)‐seq data of IKZF1 and c‐FOS binding in MM.1S cells were aligned with acetylated histone H3K27 marks in the UCSC genome browser. The transcription start site (TSS) of each gene is shown with red triangles. Lower panel: Gene expression was assayed using Affymetrix U133 plus 2.0 microarrays. The data are unpaired GCRMA‐normalized expression signals for each gene in CD138‐positive cells from patients with (1) monoclonal gammopathy of undetermined significance, (2) newly‐diagnosed MM and (3) plasma cell leukaemia ( n = 8 each). ⁵³ (A) The results of three representative genes that are highly expressed in plasma cell disorders. (B) The results of three representative genes not expressed in plasma cell disorders.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Chromatin Immunoprecipitation, ChIP-sequencing, Binding Assay, Gene Expression, Expressing, Clinical Proteomics

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Biological functions of activator protein‐1 (AP‐1) family proteins in multiple myeloma (MM). (A) We determined the correlation of expression levels between IKZF1‐target genes (y‐axis) and the indicated genes (x‐axis) using the GenomicScape tool ( http://www.genomicscape.com ). ³² The data were extracted from DNA microarray analyses of gene expression in newly‐diagnosed MM patients. ⁵³ (B) Cell lysates were prepared from the indicated MM cell lines and analyzed by immunoblotting using antibodies against c‐FOS, FOSB, c‐JUN and GAPDH (loading control). (C) We examined the expression of FOS , FOSB and JUN transcripts in normal plasma cells ( n = 22), CD138‐positive cells derived from patients with monoclonal gammopathy of undetermined significance (MGUS; n = 44) and MM cells from newly‐diagnosed patients ( n = 12). The data were extracted from DNA microarray analyses deposited in the GenomicScape database by the Arkansas group. ⁵³ (D) Left panel: We transduced the indicated MM cell lines with shRNA against FOS or scrambled sequences (sh‐control) and evaluated the expression of candidate IKZF1‐target genes ( IRF4 , SLAMF7 , BSG and CD48 ) along with GAPDH (Control) and FOS (for the evaluation of knockdown efficiency) using quantitative real‐time reverse transcription‐PCR after 24 h. The data were quantified by the 2 –∆∆Ct method using GAPDH as a reference and are shown as the fold changes against the values of sh‐control‐transfected cells. * p < .05 by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests. Right panel: Correlation between the expression levels of FOS mRNA and those of IRF4 or CD48 mRNA in MM cell lines.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Expressing, Microarray, Gene Expression, Western Blot, Control, Clinical Proteomics, Derivative Assay, shRNA, Knockdown, Reverse Transcription, Transfection, Comparison

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Direct interaction of IKZF1 and c‐FOS in multiple myeloma (MM) cells. (A) Left panel: Nuclear extracts from MM.1S and KMS12‐BM cells were immunoprecipitated with rabbit anti‐IKZF1 antibody or isotype‐matched immunoglobulin (IgG). The immunoprecipitates were analyzed by immunoblotting with specific antibodies against IKZF1, IKZF3, c‐FOS, c‐JUN, IRF4 and rabbit IgG. Input: Immunoblotting of nuclear extracts fractioned before immunoprecipitation. Right panel: The same experiments were carried out with c‐FOS immunoprecipitates. (B) The binding of the IKZF1 complex to oligonucleotides containing an IKZF consensus motif was measured by sandwich immunoassay and is shown as the relative activity against the data obtained in the absence of blocking antibodies. Antibody perturbation was carried out with isotype‐matched immunoglobulin (Control), an anti‐IKZF1 antibody, an anti‐c‐FOS antibody and a combination of the two antibodies. p < .05 by Student's t ‐test against Control ( n = 5). (C) HEK293T cells were transfected with an empty vector (Mock) or expression vectors carrying HA‐tagged full‐length IKZF1 protein, the exon 1‐exon 4 fragment, the exon 5‐exon 6 fragment, or the exon 7 fragment of IKZF1 together with a FLAG‐tagged c‐FOS expression vector. Nuclear extracts were isolated 24 h after transfection and immunoprecipitated with an anti‐HA antibody, followed by immunoblotting with antibodies against HA tag (IKZF1), FLAG tag (c‐FOS) or rabbit immunoglobulin (IgG). Red arrows denote the positions of the transfected IKZF1 fragments. (D) Structure‐based prediction of IKZF1‐c‐FOS interactions using the AlphaFold2 program.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Activity Assay, Blocking Assay, Control, Transfection, Plasmid Preparation, Expressing, Isolation, FLAG-tag

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: c‐FOS mediates lenalidomide resistance in multiple myeloma (MM) cells. (A) Left panel: MM.1S and KMS12‐BM cells were transfected with c‐FOS expression vector or empty vector (Mock) and treated with the vehicle alone (None) or 2.5 μM lenalidomide for 24 h, followed by immunoblot analysis for the expression of the indicated molecules. Right panel: MM.1S and KMS12‐BM cells were transfected with a c‐FOS expression vector or an empty vector (Mock) and treated with various concentrations of lenalidomide for 72 hours. Cell viability was determined by MTT reduction assay with a Cell Counting Kit (Fujifilm Wako Biochemicals). The graphs show the means of triplicate samples; the S.D. was less than 10% and thus omitted. * p < .05 by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests. (B) Upper panel: Schematic representation of the IRF4 promoter region from the chromatin immunoprecipitation (ChIP)‐Atlas data. The relative positions of the putative binding sites of transcription factors are approximated by the symbols shown in the box. TSS: transcription start site. Bidirectional red arrows indicate regions that were PCR amplified in ChIP assays. Lower panel: Chromatin suspensions were prepared from KMS12‐BM cells cultured with vehicle alone (DMSO) or 2.5 μM lenalidomide for 24 h and immunoprecipitated with anti‐IKZF1 (grey bars) and c‐FOS (pink bars) antibodies or IgG (back bars). The resulting precipitates were subjected to PCR to amplify the regions shown in the upper panel. * p < .05 against IgG by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests. (C) The expression of IRF4 protein and mRNA in DMSO‐ or lenalidomide‐treated KMS12‐BM cells. (D) MM.1S and KMS12‐BM cells were transfected with sh‐FOS expression vector or empty vector (Control) and treated with vehicle alone (DMSO) or 10 μM lenalidomide. (E) MM.1S and KMS12‐BM cells were treated with vehicle alone (DMSO), 10 μM lenalidomide, 20 μM T‐5224, or the combination of lenalidomide and T‐5224. Upper panels: The expressions of IRF4 and GAPDH transcripts were examined by quantitative real‐time reverse transcription‐PCR after 24 h. The results were normalized to the values of DMSO‐treated cells. Lower panels: Cell viability was determined by MTT reduction assay after 72 h and is shown as the percentage of untreated cells (%Control). The data are presented as the means of three biological replicates with S.D. (bars). * p < .05 by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, MTT Reduction Assay, Cell Counting, Comparison, Chromatin Immunoprecipitation, Binding Assay, Amplification, Cell Culture, Immunoprecipitation, Control, Reverse Transcription

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Graphical abstract. c‐FOS, a subunit of the activator protein‐1 (AP‐1) transactivator, is an integral component of the IKZF1 complex and is primarily responsible for the activator function of the complex in multiple myeloma (MM) cells. Left panel: The IKZF complex binds to the enhancer/promoter regions of the genes involved in the growth and survival of MM cells such as IRF4 and SLAMF7 through the canonical IKZF‐binding motif CTTCC with c‐FOS/c‐JUN. Middle panel: Lenalidomide induces ubiquitin‐dependent degradation of IKZF1/IKZF3; however, residual c‐FOS, the level of which is often increased by lenalidomide treatment, binds to the AP‐1 consensus sequences, which present in the vicinity of IKZF‐binding sites of certain genes including IRF4 and SLAMF7 , leading to sustained expression of these genes and lenalidomide resistance. Right panel: A selective AP‐1 inhibitor, T‐5224, binds to the DNA‐binding domain of c‐FOS and mitigates the residual activity of the MM‐specific activator complex, resulting in complete IRF4 down‐regulation and augmentation of the anti‐MM effects of lenalidomide.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Binding Assay, Ubiquitin Proteomics, Expressing, Activity Assay

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 1 | Identification of CD177+ and CD275+ ADE subpopulations. a, Schematic representation of hESC differentiation toward DE. b,c, Representative FACS plots of apparently homogeneous FOXA2+/SOX17+ DE (b) showing a heterogenous population marked by CXCR4+/CD117+ cells (c) (n = 3 (b), n = 6 (c) biologically independent experiments). d–g, Gene expression profiles of CXCR4+/CD117−, CXCR4high/CD117high, CXCR4mid/CD117mid and CXCR4low/CD117low cells for FOXA2 (d), SOX17 (e), CER1 (f) and HHEX (g) (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h, Summary of the antibody screen identifying and isolating CD177 and CD275 as markers of ADE subpopulations. CXCR4 and FOXA2 are used as controls to identify the whole DE. i, hPSCs and hPSC-derived DE stained for CXCR4, CD177 and CD275 as shown by live-cell FACS (n = 10 biologically independent experiments). AA, activin A; D, day.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Gene Expression, Derivative Assay, Staining

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 2 | Molecular profiling of CD177+, CD275+ and CXCR4+ DE subpopulations reveals distinct signatures. a, Summary of differentiation protocol toward DE/ADE followed by MACS to enrich for CD177, CD275 and CXCR4 populations. b, Principal component analysis showing that mRNA-derived transcriptome profiles are characteristic of different DE/ADE subpopulations (n = 3 biologically independent experiments). c–e, Bar graphs of selected and significantly enriched gene ontology terms in CD275+ versus CXCR4+ (c), CD177+ versus CD275+ (d) and CD177+ versus CXCR4+ (e) DE populations (n = 3 biologically independent experiments). Enrichment P values are calculated by HOMER findGO.pl based on the cumulative hypergeometric distribution. f,g, Validation of the microarray analysis by qPCR for noncanonical WNT/PCP components and ligands (f) and canonical WNT components and ligands (g). Data were normalized to 18S (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h,i, Western blot analysis (h) and quantification (i) of WNT/PCP components such as p-JNK and DVL2 in ADE subpopulations (n = 3 biologically independent experiments). GAPDH is used as a loading control. Data are represented as mean ± s.e.m. j, Immunofluorescence analysis validated the exclusive localization of β-catenin in the membrane in CD177+ ADE cells and in the cytoplasm and nucleus in CD275+ ADE and CXCR4+ DE cells (n = 3 biologically independent experiments). FOXA2 is used as a nuclear marker. Scale bars, 20 µm and 10 µm in inset. PC1/2, principal component 1/2.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Derivative Assay, Biomarker Discovery, Microarray, Western Blot, Control, Immunofluorescence, Membrane, Marker

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 1. Verification of the interaction between rat PPAR and CITED2 in vitro. CITED2 was radiolabeled with [35S]methionine and incubated with bacterially expressed PPAR-MBP fusion for 1 h at 4 °C in the presence of amylose resin. Resin was collected and washed three times with cold radioimmune precipitation assay buffer. Bound MBP was eluted from the resin using 10 mM maltose in radioimmune pre- cipitation assay buffer for 1 min at 4 °C. Eluate was resolved on a 12% Tris-glycine gel, dried, and subjected to autoradiography. The image is representative of two independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: In Vitro, Incubation, Autoradiography

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 2. CITED2 interacts with the D domain of rPPAR. COS-1 cells were transiently transfected with the GAL4-DBD fused to rPPAR and VP16 activation domain with or without fused CITED2. Cells were treated with 50 M Wy-14,643 (Wy), 200 M CLA mixture, or Me2SO (DMSO) for 6 h. Domains containing no ligand activation were treated with Me2SO. Luciferase activity was determined and corrected for transfection efficiency and extraction yield. Each domain and treatment group was corrected to their corresponding VP16 value (100%). *, p 0.01 comparing CITED2 bar to corresponding VP16 bar. The graph is representative of three independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transfection, Activation Assay, Luciferase, Activity Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 3. CITED2 is a coregulator for PPAR. A, HepG2 cells were transiently transfected with expression vectors for rPPAR with CITED2 or empty vector control (pcDNA3) and a PPRE-driven lu- ciferase reporter. B, COS-1 cells were transiently transfected with rPPAR fused to the GAL4-DBD and CITED2 or empty vector control with GAL4-respon- sive reporter. Cells were treated with 50 M Wy-14,643 (Wy), 200 M CLA mixture, 100 M ciprofibrate, or Me2SO (DMSO) for 6 h. Luciferase activity was determined and corrected for transfection efficiency and extraction yield. All bars are corrected to untreated pcDNA3 level. *, p 0.05 compar- ing CITED2 bar to corresponding pcDNA3 bar. The graph is representative of three independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Activity Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 4. CITED2 acts as a dose-dependent coactivator of PPAR. A, COS-1 cells were transiently transfected with rPPAR fused to the GAL4-DBD and increasing amounts of CITED2. Cells were treated with 50 M Wy-14,643 for 6 h. Luciferase activity was deter- mined and corrected for transfection efficiency and extraction yield. Each treatment is corrected to luciferase activity with no CITED2 added (100%). Results show that CITED2 can act as a dose-dependent coactivator of PPAR in the presence or absence of ligand. *, p 0.05 comparing within a chemical treatment. Values in parentheses are relative luciferase units (rlu) for the accompanying data point. B, COS-1 cells were transiently transfected with rPPAR fused to the GAL4-DBD with or without CITED2. Cells were treated with 100 nM, 500 nM, 1 M, 5 M, 10 M, or 50 M Wy-14,643 for 6 h. Luciferase activity was determined and corrected for transfection efficiency and extraction yield. Each treatment is corrected to luciferase activity in the absence of Wy-14,643 (Me2SO (DMSO) at 100%). Graphs are representative of three independent experiments. CI, confidence interval.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transfection, Luciferase, Activity Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 5. CITED2 acts as a coactivator for PPAR but not PPAR. A, HepG2 cells were transiently transfected with expression vectors for each PPAR subtype with CITED2 or empty vector control and a PPRE-driven luciferase reporter. B, COS-1 cells were transfected with GAL4-DBD-PPAR fusions for all three subtypes with and without exogenous CITED2. Transfected cells were treated for 6 h with 50 M Wy-14,643 (Wy), 50 M tetradecylthioacetic acid (TTA), 10 M prostag- landin J2 (PGJ2), or Me2SO (DMSO). Luciferase activity was deter- mined and corrected for transfection efficiency and extraction yield. Each treatment is corrected to the luciferase activity for Me2SO for each subtype. *, p 0.05 comparing CITED2 bar with corresponding pcDNA3 bar. The graph is representative of three independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Activity Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 6. CITED2 is ubiquitously expressed in mouse tissues. Total RNA from 10 different mouse tissues and the SV40-transformed mouse hepatocytes was examined for CITED2 mRNA using reverse transcription-PCR. Equivalent amounts of total RNA were tested using primers designed for the 5 -end of mouse CITED2 mRNA. The graph is representative of three independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transformation Assay, Reverse Transcription

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 7. Ameliorated CITED2 expression leads to decreased PPAR activity. Undifferentiated 3T3-L1 preadipocytes were tran- siently transfected with an RNAi for CITED2 or vehicle control (pSu- per), pM-PPAR, and pFR-luciferase reporter and treated with 50 M Wy-14,643 (Wy), 100 M CLA mixture, 100 M ciprofibrate, or Me2SO (DMSO). Luciferase activity was measured and corrected for transfec- tion efficiency and extraction yield. Luciferase values were standard- ized to uninhibited (CITED2/) untreated (Me2SO) cells (100%). Graphs are representative of two independent experiments. *, p 0.05 comparing CITED2- to pSuper-transfected cells.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Expressing, Activity Assay, Transfection, Control, Luciferase, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 8. CITED2 and CITED2 sta- bly expressing hepatocytes. A, CITED2 inhibition was achieved using double- stranded RNAi molecules (pSUPER and CITED2). The inhibition was verified using Western blot. Lanes labeled pcDNA3 and CITED2 are MuSH wild type cells stably expressing exogenous CITED2. B, MuSH wild type cells that stably overexpress CITED2 or inhibited CITED2 were assayed for growth in re- sponse to Wy-14,643 (Wy). Cells were plated and treated with 50 M Wy-14,643 for 72 h and assayed for relative cell num- ber. Values were corrected for Me2SO (DMSO) control (100%) for the corre- sponding control cell line. The stable cell lines are pooled populations of trans- fected cells. Data represent two independ- ent experiments. neo, RNAi empty vector control. *, p 0.05 comparing untreated to treated cells within the same cell type; , p 0.05 comparing cell types within the same treatment group.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Expressing, Inhibition, Western Blot, Labeling, Stable Transfection, Control, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 9. Regulation of gene expres- sion by peroxisome proliferators is affected by alterations in CITED2 ex- pression. A–E, MuSH wild type cells that were stably transfected with human CITED2 or CITED2 RNAi were treated with 50 M Wy-14,643, 100 M CLA, 100 M ciprofibrate (Cipro), or Me2SO (DMSO) for 6 h. Total RNA was isolated and used in real time reverse transcrip- tion-PCR for known PPAR-regulated genes: Angpl4 (A), HIF1 (B), FOXc2 (C), MKP-1 (D), and VEGF-D (E). The Me2SO level for each corresponding empty vector control was set to 100%. *, different than Me2SO-treated cells within the same cell line (p 0.05). F–J, data are identical to that presented in A–E with grouping based on treatment. *, different than con- trol stably transfected cells within the same treatment (p 0.05). The stable cell lines are pooled populations of trans- fected cells. Graphs are representative of two independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Stable Transfection, Transfection, Isolation, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 10. Analysis of altered gene expression in the CITED2 cells. Genes that were significantly regulated in gene expression microarrays (CITED2/pcDNA3, Table II) were examined using Pathway Assist (Version 2.01). The pathway was built by looking for common regulators of the genes shown in Table II, and the predominant cluster is depicted. The lines and arrows depict observations on regulation of gene expression (, increased expression; , decreased expression) from the literature. The effects of CITED2 overexpression on the amount of mRNA in the microarray experiments are shown (black, significantly increased; gray, significantly decreased; white, no significant effect observed). Following creation of this cluster, the proteins in the gradient filled ovals were included (PPAR, PPAR, Angptl4, and HIF1), and the connections were determined by Pathway Assist or by manually adding (PPAR and CITED2 interaction). EGF, epidermal growth factor; TGF, transforming growth factor; DCN, decorin; AQP5, aquaporin 5; TNF, tumor necrosis factor; IFG1R, insulin-like growth factor I receptor; IL2, interleukin 2; IGFBP2, insulin-like growth factor-binding protein 2; IFN, interferon; LTBP1, latent transforming growth factor--binding protein 1; EPS15, epidermal growth factor receptor pathway substrate 15; TGM2, transglutaminase 2; UCHL1, ubiquitin carboxyl-terminal hydrolase L1; FIGF, c-fos-induced growth factor; GHRL, growth hormone receptor, long form; IL13R, interleukin 13 receptor; GH1, growth hormone 1; ADCY8, adenylate cyclase 8; TSA, trichostatin A; MGP, matrix -carboxyglutamate protein.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Gene Expression, Expressing, Over Expression, Microarray, Binding Assay, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Primary cilia and SHH signaling impairments in human and mouse models of Parkinson’s disease

doi: 10.1038/s41467-022-32229-9

Figure Lengend Snippet: a Immunostainings exemplarily shown for O3H-R1-003. hiPSC pluripotency staining for markers OCT4, NANOG, SOX2. Scale bar=200 µm. hNPC staining for markers SOX1, SOX2, NESTIN, PAX6. Scale bar=100 µm. Neuron staining for markers TUBB3 and DAn marker TH. Scale bar=100 µm. Astrocyte staining for markers GFAP and SLC1A3. Scale bar=100 µm. b Summary of somatic CNVs identified in hNPC clones by chromosomal microarray analysis shown as total number of somatic CNVs detected per analyzed clone and average length of CNVs (in kb; green=copy number gain; orange=copy number loss) per analyzed clone. n = 5 Ctrl and 7 sPD patients. c Circos plot showing the genomic distribution of somatic CNVs in Ctrl (blue) and sPD (red) clones. d Quantification of RBFOX3 (synonym: NeuN) positive as well as TH / RBFOX3 double-positive cells in DAn populations. n = 5 Ctrl and 7 sPD clones, in triplicates. e Characterization of neurite morphologies of DAns. Boxplots show the average number of neurites emerging from TH positive cell bodies, their average number of branch points and their average length. n = 5 Ctrl and 7 sPD clones, in triplicates. Boxplots display the median and range from the 25th to 75th percentile. Whiskers extend from the min to max value. Each dot represents one patient. P -values were determined by two-sided t -test d (right), e ; two-sided Mann–Whitney-U test b, d (left). * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were diluted as follows: AC-TUB (T6793, Sigma-Aldrich; 1:1000), ARL13B (17711-1-AP, Proteintech; 1:500), GFAP (MAB360, Millipore; 1:250), GLI3 (AF3690, R&D; 1:100), NANOG (AF1997, R&D Systems; 1:200), NES (Ma1110, Thermo Fisher Scientific; 1:250), PAX6 (Ab78545, Abcam; 1:200), PITX3 (38-2850, Invitrogen; 1:300), POU5F1 (2840 S, Cell Signaling; 1:500), RBFOX3 (ab104224, Abcam; 1:800), SLC1A3 (NB100-1869, Novus Biologicals; 1:250), SMO (sc166685, Santa Cruz; 1:500), SOX1 (Ab87775, Abcam; 1:500), SOX2 (sc17320, Santa Cruz; 1:500), TH (P40101, PelFreez; 1:600), TUBB3 (T5076, Sigma-Aldrich; 1:1000).

Techniques: Staining, Marker, Clone Assay, Microarray, Full Display Name, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cardiac fibroblasts regulate cardiomyocyte hypertrophy through dynamic regulation of type I collagen

doi: 10.1101/2022.05.25.493406

Figure Lengend Snippet: (A) Whole ventricle mRNA microarray analysis of Col1a2 -/- mouse hearts compared to Col1a2 +/- at 2 months of age, n=3 per genotype. (B) Mass spectrometry analysis of ECM protein changes in Col1a2 -/- mouse hearts compared to Col1a2 +/- hearts at 3 months of age, n=4 per genotype. (C) Representative immunofluorescence images and (D) Western blot analysis of periostin from hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Scale bar: 25 µm. (E) Flow cytometric gate strategy and (F) analysis of cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from dissociated hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. (G) Representative immunofluorescence images of platelet-derived growth factor receptor (PDGFR)-α (purple) in Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Wheat germ agglutinin (WGA) staining is green and shows outlines of cardiomyocytes. Scale bar: 100 µm. Relative mRNA expression of Col1a2 (H), Postn (I), Col3a1 (J) and Col5a1 (K) in sorted cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from Col1a2 +/- and Col1a2 -/- mice at 9 months of age. Student t -test for panels (F), (H), (I), (J) and (K).

Article Snippet: Antibodies against the following proteins were used: periostin (Novus Biologicals NBP1-30042; 1:300 dilution for IF, 1:1000 for Western blot); collagen I (Abcam ab21286; 1:100 for IF); PDGFRα from (R&D Systems AF1062; 1:1000 for IF); collagen 1a2 (Santa Cruz sc-393573; 1:500 for Western blot) Anti-CD31 was from BioLegend (102423; 1:100 for flow cytometry); anti-CD45 was from BD Biosciences (563890; 1:100 for flow cytometry); anti-MEFSK4 was from Miltenyi Biotec (130-120-802; used 1:30 for flow cytometry).

Techniques: Microarray, Mass Spectrometry, Immunofluorescence, Western Blot, Derivative Assay, Staining, 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