Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: The molecular structure of SOX11. The SOX11 protein is composed of 441 amino acids. SOX11 contains two functional domains, the N-terminal HMG domain and the conserved TAD. SOX11, sex-determining region Y-related high-mobility-group box transcription factor 11; HMG, high mobility group; TAD, C-terminal transactivation domain.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Functional Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: Overview of SOX11 function reported in diverse types of cancer.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Expressing, Over Expression, Biomarker Discovery, Migration, Methylation, DNA Methylation Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: Overall carcinogenic actions of SOX11 on the hallmarks of tumor biology. SOX11 exerts tumor-stimulative effects through increasing cell proliferation, repressing cell differentiation, inducing angiogenesis, and promoting metastasis. BCL6, B-cell lymphoma 6; BNIP3, B-cell lymphoma 2 (BCL2)/adenovirus E1B 19 kDa protein-interacting protein 3; CIC, cancer-initiating cell; PAX5, Paired box protein 5; PDGFA, platelet-derived growth factor A; SETMAR, SET domain and mariner transposase fusion gene; SOX11, Sex-determining region Y-related high-mobility-group box transcription factor 11; TANK, TRAF family member-associated NF-κB activator.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Cell Differentiation, Derivative Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: Different prognostic significance of SOX11 in tumor cases.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Expressing, Microarray, Over Expression

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 1. The IJM region is necessary for collagen-induced DDR2 activation. (a) Overall topology and alignment of the transmembrane (TM) domain and the intracellular juxtamembrane region (IJM) of DDR1a and DDR2. The IJM was divided into three regions: JM1, JM2 and JM3. (b) Schematic diagram of various DDR2 constructs used in our study. (c) HEK293T cells transiently transfected with plasmids encoding the full-length DDR2 and F-DJM1-JM2 mutant were stimulated by Type I collagen for 60 min. Tyrosine phosphorylation of the F-DJM1-JM2 mutant was inhibited compared to that of the full-length DDR2. (d) HEK293T cells were transfected with plasmids encoding full-length DDR2, F- DJM1 and F-DJM2 and were stimulated by collagen. The F-DJM2 mutant showed a significant decrease in tyrosine phosphorylation. **p < 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Activation Assay, Construct, Transfection, Mutagenesis, Phospho-proteomics

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 2. DDR2 dimerizes via the JM2 of the IJM region. (a and b) HEK293T cells were transiently cotransfected with plasmids encoding TM- JM1-JM2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blot analysis showed that the cytoplasmic domains of DDR2 bind to each other via the intact TM-JM1-JM2 domain and form homodimers. Asterisks indicate the expected size of TM-JM1-JM2. (c) HEK293T cells were transfected with plasmids encoding F-DJM1 and F-DJM2 mutants. A crosslinking assay showed that dimers of F-DJM1 were not changed compared to full-length DDR2, whereas dimers were significantly decreased for F-DJM2.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Immunoprecipitation, Western Blot, Transfection

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 3. JM2 has a dominant-negative effect on DDR2 activation. (a and b) HEK293T cells were cotransfected with plasmids encoding full- length DDR2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blotting showed that the full-length DDR2 and TM-JM1-JM2 bind to each other to form heterodimers. The immunoprecipitates obtained with anti-IgG antibodies were used as a negative control. (c) H1299 cells transfected with plasmids encoding full-length DDR2 and TM-JM1-JM2 and HeLa cells were lysed, and the whole cell lysates (W) were separated into the plasma membrane (P) and cytosol (C) fractions. EGFR and a-tubulin were used as positive controls for the plasma mem- brane and cytosol fractions, respectively. Endogenous full-length DDR2 (HeLa cells), forced-expressed full-length DDR2 and TM-JM1-JM2 pro- teins were appropriately localized in the plasma membrane. (d) HEK293T cells were transfected with plasmids encoding a C-terminally myc- tagged full-length DDR2 and TM-JM1-JM2. Only under the permeabilized condition, full-length DDR2-myc and TM-JM1-JM2-myc were visual- ized, indicating that the C-termini of these proteins were located in the cytosol and not extracellular space. Full-length DDR2 was used as a positive control. Bar, 50 mm. (e) HEK293T cells were cotransfected with plasmids encoding full-length DDR2 (500 ng) and an increasing amount of TM-JM1-JM2 (100, 300 and 500 ng) as indicated and then stimulated with collagen. Tyrosine phosphorylation gradually decreased with an increasing amount of TM-JM1-JM2. (f) HeLa cells were transiently transfected with a plasmid encoding TM-JM1-JM2 and were then stimulated. Tyrosine phosphorylation was significantly decreased in endogenous DDR2. **p < 0.01, Student’s t-test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Dominant Negative Mutation, Activation Assay, Immunoprecipitation, Western Blot, Negative Control, Transfection, Clinical Proteomics, Membrane, Positive Control, Phospho-proteomics, Plasmid Preparation

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 4. JM2 regulates the collagen-binding affinity of DDR2. (a and b) HEK293T cells transiently transfected with various DDR2 constructs were harvested, and protein expression was verified by Western blot- ting (a). Collagen-binding affinities were reduced in F-DJM2, F-DJM1- JM2 and extra mutants but not the F-DJM1 mutant in a dose- dependent manner (b). Nontransfected (NC) and TM-JM1-JM2 samples were used as negative controls. **p< 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Binding Assay, Transfection, Construct, Expressing, Western Blot, Mutagenesis

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 5. Colony formation and proliferation of tumor cells are suppressed by overexpression of JM2. (a and b) Formalin-fixed tissue micro- array slides were used in immunohistochemistry experiments. DDR2 was overexpressed in bladder, testis, lung, kidney, prostate and stom- ach cancers. Bar, 50 mm. (c) Stable TM-JM1-JM2–expressing H1299 cells were stimulated by collagen and then harvested. Immunoprecipitation and Western blot analysis showed that tyrosine phosphorylation of DDR2 was decreased by TM-JM1-JM2 overexpres- sion (labeled JM1/2), but phosphorylation of DDR1 was unaffected. (d) A colony-forming assay of H1299 cells showed that the number and projected area of colonies were decreased by TM-JM1-JM2 overexpression. Bar, 100 mm. (e) Proliferation of H1299 cells was assessed by cell counting (left) and an MTT assay (right). Cell proliferation was inhibited by TM-JM1-JM2 overexpression. **p < 0.01, Student’s t-test; control, nontransfected cells; Mock, empty vector stably transfected cells; JM1/2, TM-JM1-JM2 stably transfected cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Over Expression, Microarray, Immunohistochemistry, Expressing, Immunoprecipitation, Western Blot, Phospho-proteomics, Labeling, Cell Counting, MTT Assay, Control, Plasmid Preparation, Stable Transfection, Transfection

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) Immunohistochemistry showing reduced methylated H3K79 levels (H3K79me2) that reflect loss of DOT1L activity in damaged areas from osteoarthritic patients (OA) as compared to their corresponding preserved areas and to cartilage from non-OA patients. Images are representative of images from four different patients. Scale bar, 400 μm. ( b ) Heat maps of differential mRNA expression determined by quantitative PCR in chondrocytes treated with DOT1L inhibitor EPZ-5676 (EPZ) or vehicle (V) from passage 0 (P0) until P2, and from preserved versus damaged areas in OA cartilage. The colour code represents the mean expression level of six and four independent patient samples respectively. ( c ) Immunoblot analysis showing decreased methylated H3K79 levels in mouse articular chondrocytes after intra-articular injection of EPZ into C57Bl/6 wild-type mouse knees. The image is representative of one experiment with protein extracts pooled from two or three mice per condition. Unprocessed original scans of blots are shown in . ( d , e ) C57/Bl6 wild-type mouse knees were injected with EPZ (5 mg kg –1 ) or vehicle and killed after 2 or 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( d ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( e ). One experiment was performed with n =10 and 5. Representative images from the 4 week evaluation are shown. * P <0.05 (two-tailed t -test). Error bars indicate mean±s.e.m.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Immunohistochemistry, Methylation, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) KEGG pathway enrichment analysis of microarray data obtained from human articular chondrocytes treated with EPZ-5676 or vehicle. Nominal P values by EASE modified Fisher Exact test using the DAVID analysis tool (see Methods section) are shown. n =5 independent patient-derived cell cultures. ( b ) Co-immunoprecipitation (Co-IP) using an anti-DOT1L antibody showing interaction between DOT1L and β -catenin in human articular chondrocytes, that is increased upon Wnt activation by LiCl and disrupted upon DOT1L inhibition. The image is representative of three experiments. ( c ) TOP/FOP reporter assay in human articular chondrocytes after Wnt stimulation by LiCl and DOT1L inhibition by EPZ. Activity is compared to untreated cells (dotted line). n =3 biologically independent experiments. *** P <0.001 by one-way ANOVA. ( d , e ) LEF1 , TCF1 and c-MYC expression measured by quantitative PCR in chondrocytes treated with EPZ-5676 and LiCl ( d ) or in LiCl-treated chondrocytes transfected with siRNA directed against DOT1L or scrambled siRNA (siDOT1L or siSCR, respectively) ( e ). Data are from one experiment with three technical replicates. ( f ) Immunohistochemistry demonstrating increased TCF1 levels in the articular cartilage of C57/Bl6 wild-type mice after injection of EPZ-5676. The images are representative of three different animals. Scale bar, 200 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Microarray, Modification, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Activation Assay, Inhibition, Reporter Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunohistochemistry, Injection

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: All experiments were performed in healthy human articular chondrocytes: treated as indicated with DOT1L inhibitor EPZ-5676, Wnt activator LiCl, SIRT1 antagonist EX527 or SIRT1 agonist SRT1720; or transfected with DOT1L or scrambled siRNA. All data are presented as mean±s.e.m. ( a ) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis of DOT1L and methylated H3K79 and ( b ) acetylated H3K9 (H3K9Ac) and methylated H3K4 (H3K4me3) as markers of active transcription on the transcriptional start site (TSS) of Wnt target genes. Data are from two to five experiments. ( c ) Expression levels of TCF1 Wnt target gene measured by quantitative PCR in chondrocytes transfected with indicated specific or scrambled siRNA (siSCR). Data are from one experiment with technical triplicates. ( d ) TCF1 expression measured by quantitative PCR in the presence of SIRT1 agonist and antagonist. Data from two experiments each with technical triplicates. ( e ) Co-IP analysis using the indicated antibodies demonstrating the interaction of DOT1L and SIRT1. The image is a representative image of three biologically independent experiments. ( f ) SIRT1 activity relative to vehicle-treated cells (dotted line). Data are from three biologically independent experiments. * P <0.05, *** P <0.001 by one-way ANOVA. ( g ) ChIP-qPCR analysis of SIRT1, PPARGC1A, GCN5 and EP300 binding on the TCF1 promoter and ( h ) TCF1 expression after siRNA transfection with indicated specific or scrambled siRNA. Data from two biologically independent experiments.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Methylation, Expressing, Co-Immunoprecipitation Assay, Activity Assay, Binding Assay

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a – c ) Inactivation of SIRT1 protects against DOT1L inhibitor-induced osteoarthritis: ( a ) C57/Bl6 wild-type mouse knees were injected with DOT1L inhibitor EPZ-5676 (5 mg kg −1 ) and SIRT1 inhibitor EX527 (1.25 mg kg –1 ), or vehicle (V) and killed after 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( a ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( b ). One experiment was performed with n =3 (vehicle), 8 (EPZ) and 10 (EPZ+EX527). * P <0.05 by one-way ANOVA. Error bars indicate mean±s.e.m. ( c ) Immunohistochemistry of TCF1 in the indicated groups. TCF1 levels are increased after EPZ treatment and normalized by additional EX527 treatment. The images are representative of three different animals. Scale bar, 200 μm. ( d , e ) Loss of DOT1L function causes severe growth retardation as demonstrated by skeletal staining ( d ) and histology of the growth plate ( e ) of 4-week-old Dot1l fl/fl ;Col2-Cre −/− (Cre-neg) and Dot1l fl/fl ;Col2-Cre +/− (Dot1l Cart-KO ) mice. ( f , g ) Increased TCF1 levels in Dot1l Cart-KO mice as shown by immunohistochemistry in the indicated mice strains in the articular cartilage ( f ) and growth plate ( g ). The images are representative of three different animals. Scale bar, 200 and 100 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Injection, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: Upon Wnt signalling activation, DOT1L-containing complexes bind Wnt target gene chromatin. DOT1L interacts with SIRT1 and inhibits its function, preventing Wnt pathway hyper-activation. When Wnt signalling is activated in the absence of DOT1L function, high SIRT1 activity mediates the recruitment of transcriptional activators to LEF1 and TCF1 genes. High Wnt signalling leads to deleterious downstream effects and loss of cartilage homeostasis.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Activation Assay, Activity Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Immunohistochemistry staining with antibody against phosphorylated Smad2 (pSmad2) and TGFβ receptor II (TβRII) showed increased nuclear signal for pSmad2 in the invasive ECdnT organotypic cultures. Scale bar 50 micron. (b) Analysis of immunohistochemistry staining for TβRII and pSmad2 in 83 ESCC cases in a tissue microarray shows no significant correlation. Fisher’s exact test, two tailed p= 0.3182. (c) Five paired normal adjacent and ESCC tissues (GSE17531) were analyzed for INHBA mRNA expression, which identified upregulation of INHBA in four ESCC samples. (d) Waterfall plot of a publically available dataset (GSE23400) represented upregulation of INHBA in the ESCC (grey bars) samples vs. normal (black bars).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Immunohistochemistry, Staining, Microarray, Two Tailed Test, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures with either fetal esophageal fibroblasts (FEF) or cancer-associated fibroblasts (CAF) embedded in the underlying matrix. Immunofluorescence staining with antibody against αSMA (green) and podoplanin (red) showed similar expression pattern in the cultures. Scale bar is 50 micron. (b) Activin A concentration in conditioned media from organotypic cultures is higher in invasive cultures as measured using indirect ELISA. * p=0.003, ** p= 0.005, *** p=0.03 (c) Stimulation of epithelial cells with Act A in monolayer plastic culture demonstrated phosphorylation of Smad. Neutralizing antibody against Activin (nAb) prevented the induction of pSmad2 by Act A. Following stimulation with Act A or with conditioned media from organotypic culture increased expression of vimentin was detected after 48 hours by Western Blot. The increase was reversed in the presence of neutralizing antibody (nAb). (d) Inhibition with the Act A antagonist, Follistatin, or a pan-TGFβ inhibitor A83-01 suppressed MMP-9 secretion in E, EC and ECdnT cells as measured by gelatin zymography. Upper bands reflect pro-MMP, lower bands activated, cleaved MMP (arrow).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Immunofluorescence, Staining, Concentration Assay, Indirect ELISA, Phospho-proteomics, Western Blot, Inhibition, Zymography

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures in the presence of recombinant Activin A (Act A), its antagonist Follistatin or a neutralizing antibody against Activin A (nAb); H&E staining. Stimulation with Act A inhibited invasion of E and EC cells, but failed to suppress ECdnT cell invasion. Follistatin increased cell invasion in all cell types, while the neutralizing antibody prevented invasion of E and EC cells, without an effect on ECdnT cells. (b) Immunohistochemistry staining with ki67-antibody showed no differences in cell proliferation. Scale bars are 50 micron. (c) Indirect ELISA with antibody against Act A measured increased levels after addition of recombinant Act A in fibroblasts (FEF) and ECdnT. Untreated ECdnT cells (Control) secreted higher baseline levels of Act A than FEF, which were reduced by Follistatin. (d) TGFβ1 concentration was increased in response to stimulation with Act A and overall baseline secretion was higher in control ECdnT cells than fibroblasts as measured by indirect ELISA. Follistatin inhibited TGFβ1 secretion.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Recombinant, Staining, Immunohistochemistry, Indirect ELISA, Control, Concentration Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Separating the cellular matrix and epithelium of the organotypic cultures growing ECdnT cells through a collagen I layer, dashed lines, prevented cell invasion in the absence (control) and presence of Act A (+Act A). When the cellular matrix of the organotypic culture was treated with puromycin to kill the embedded fibroblasts before the ECdnT cells were seeded, epithelial formation occurred but invasion was inhibited with and without Act A stimulation. (b) Treatment of ECdnT organotypic cultures with a pan-MMP inhibitor, GM6001, suppressed cell invasion, which was not restored in the presence of Act A. Untreated (no tx) control ECdnT cells in organotypic culture invaded into the underlying matrix. TGFβ1 treatment inhibited epithelial cell invasion. Scale bars are 50 micron. (c) Immunohistochemistry showed nuclear localization of phosphorylated Smad (pSmad2, red) in control and Act A stimulated conditions. Collagen IV, red, was disrupted in invasive cultures after Act A treatment. Loss of the fibroblasts (FEF), labeled green with antibody against vimentin (no staining in the lower panels), had no effect on the nuclear localization of pSmad2. The collagen IV layer was not disrupted in non-invasive cultures in the absence of FEFs.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Control, Immunohistochemistry, Labeling, Staining

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 1. Loss of NF1 reduces RAC1-driven melanoblast migration. A. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 9 h and 12 h using either WT or NF1+/−melanoblasts (MB) in the presence of a RAC1 activator (CN04). B. RAC1 activity was measured by G-lisa in WT and NF1+/−melanoblasts (MB). C. Scratch-like migration assay after 3 h, 6 h, 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1). D. Expression status of NF1 and expression of phosphorylated and non-phosphorylated ERK and AKT in NF1+/−melanoblasts by western blot. α-actinin was used as a loading control. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody. E. Scratch-like migration assay representing the percentage of cell coverage after 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1) and in the presence or absence of a RAC1 activator (CN04). *: SCR vs. siNF1, #: -CN04 vs. +CN04. F. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody in the presence or absence of a RAC1 activator (CN04). **P < 0.01, *P < 0.05, ns: not significant (unpaired Student's t-test). All error bars represent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Activity Assay, Transfection, Expressing, Western Blot, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 2. Loss of NF1 increases melanoma migration and is associated with increased PREX1 expression. A. NF1 mRNA expression under NF1 silencing with two siRNAs (NF1.6 and NF1.11) in SK-mel-23, Mel501, and SK-mel-103 melanoma cell lines. B. PREX1 mRNA expression under NF1 silencing with two siRNAs in SK-mel-23, Mel501, and SK- mel-103 cell lines. C. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 12 h and 24 h under NF1 silencing in SK-mel-23, Mel501, and SK-mel- 103 cell lines. D. Scratch-like migration assay as in C, after additional transfection with siRNA control (scramble) or with PREX1 siRNA (siPREX1). E. Scratch-like migration assay as in C. in the absence (control) or presence (RAC1 inhibitor) of a RAC1 inhibitor. ***P < 0.001, **P < 0.01, *P < 0.05 (unpaired Student's t-test). All error bars rep- resent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Expressing, Transfection, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 4. PREX is upregulated in low NF1 expressing melanoma metastases. A. Representative microphotographs of Tissue Microarray (TMA) containing primary and metastatic melanoma samples analysed by immunohistochemistry using a specific antibody against NF1, RAC1 and PREX1. Bar, 100 μm. B. Scoring of the immunohistochemistry staining was performed according to our previously described protocol [24]. Duplicates of valid punch samples are represented for each condition. Significance was tested using two-tailed t-test with *P < 0.05 and ns: not significant.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Two Tailed Test

Journal: Scientific Reports

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

doi: 10.1038/s41598-025-00465-w

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

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

Techniques: Immunohistochemistry, Ubiquitin Proteomics

Journal: Scientific Reports

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

doi: 10.1038/s41598-025-00465-w

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

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

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

Journal: Scientific Reports

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

doi: 10.1038/s41598-025-00465-w

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

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

Techniques: Ubiquitin Proteomics, Immunohistochemical staining

Journal: Scientific Reports

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

doi: 10.1038/s41598-025-00465-w

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

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

Techniques: Ubiquitin Proteomics

Journal: Oncotarget

Article Title: Secretory leukoprotease inhibitor is required for efficient quercetin-mediated suppression of TNFα secretion

doi: 10.18632/oncotarget.12415

Figure Lengend Snippet: A. Slpi expression from the microarray data of BMDCs exposed to vehicle or ROBs-QP at day 5 and 7 and treated with 1 μg/mL of LPS for 6 hours (n=4, **P<0.01). B. Time course mRNA expression of Slpi mRNA measured by qPCR of BMDCs exposed to vehicle (black bars) or ROBs-QP (white bars) at day 5 and 7 and treated with 1 μg/mL of LPS. Fold change are expressed relative to vehicle at time 0. mRNA was extracted at indicated time points and bars represent the mean ± SEM of 3 independent experiments. (*P<0.05, **P<0.01).

Article Snippet: Gene expression of Slpi and GAPDH was performed with TaqMan Gene Expression Assays (Thermo Fisher Scientific, MA, USA) - murine probes: Mm00441530_g1 and Mm00484668_m1, respectively; human probes: Hs00268204_m1 and Hs02758991_g1, respectively.

Techniques: Expressing, Microarray

Journal: Oncotarget

Article Title: Secretory leukoprotease inhibitor is required for efficient quercetin-mediated suppression of TNFα secretion

doi: 10.18632/oncotarget.12415

Figure Lengend Snippet: A. BMDCs were cultured from WT and Slpi-KO mice and treated with quercetin at day 5 and 7. BMDCs cultures were exposed to 1 μg/mL of LPS and the secretion of TNFα was determined by ELISA after 24 hours. Bars represent mean cytokine concentration ± SEM (n=4) for BMDCs from WT or Slpi-KO mice treated with and without quercetin (black and white bars, respectively, ***P<0.001). B. BMDCs were transfected with siRNA for Slpi and Lipofectamine® 3000 reagent (mock) at day 4, before the administration of quercetin. qPCR for Slpi demonstrated a good efficiency for siRNA transfection (left). Bars represent a mean fold change ± SEM (n=3) between LPS stimulated BMDCs +/− quercetin relative to LPS unstimulated cells +/− quercetin, respectively. **P<0.01. The secretion of TNFα was determined 24 hours after LPS stimulation by ELISA (right). Bars represent mean cytokine concentration ± SEM (n=3) for DCs treated with and without quercetin (black and white bars, respectively). ***P<0.001. C. BMDCs were treated with quercetin on day 5 and transfected with siRNA for Slpi at day 6 followed by quercetin administration at day 7 and a subsequent exposure to LPS on day 8. A good reduction in Slpi expression was observed by qPCR analysis (left). Bars represent a mean fold change ± SEM (n=3) between LPS stimulated BMDCs +/− quercetin relative to LPS unstimulated cells +/− quercetin, respectively. **P<0.01. The secretion of TNFα was determined 24 hours after LPS stimulation by ELISA (right). Bars represent mean cytokine concentration ± SEM (n=3) for DCs treated with and without quercetin (black and white bars, respectively). *P<0.05.

Article Snippet: Gene expression of Slpi and GAPDH was performed with TaqMan Gene Expression Assays (Thermo Fisher Scientific, MA, USA) - murine probes: Mm00441530_g1 and Mm00484668_m1, respectively; human probes: Hs00268204_m1 and Hs02758991_g1, respectively.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection, Expressing

Journal: Oncotarget

Article Title: Secretory leukoprotease inhibitor is required for efficient quercetin-mediated suppression of TNFα secretion

doi: 10.18632/oncotarget.12415

Figure Lengend Snippet: A. Supernatant collected from WT BMDCs culture stimulated at day 5 and day 7 with quercetin was added 1:1 to an unrelated BMDCs culture (left). After 1 hour the recipient BMDCs were stimulated with 1μg/ml of LPS for 24 hours and the amount of TNFα was tested by ELISA relative to SN untreated BMDCs (panel B, white bar and black bar, respectively). *P<0.05. B. To discriminate the contribution of the endogenous and the extracellular form of Slpi, the SN collected from WT BMDCs plus quercetin was treated with an anti-Slpi antibody (BAF1735, R&D, panel A, right). Supernatant incubated with the antibody was treated with LPS as before and tested by ELISA for TNFα secretion (panel B, stripped bar). Bars represent mean cytokine concentration ± SEM (n=3) for BMDCs from WT mice.

Article Snippet: Gene expression of Slpi and GAPDH was performed with TaqMan Gene Expression Assays (Thermo Fisher Scientific, MA, USA) - murine probes: Mm00441530_g1 and Mm00484668_m1, respectively; human probes: Hs00268204_m1 and Hs02758991_g1, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay

Journal: Oncotarget

Article Title: Secretory leukoprotease inhibitor is required for efficient quercetin-mediated suppression of TNFα secretion

doi: 10.18632/oncotarget.12415

Figure Lengend Snippet: Gavage of quercetin or vehicle was administered at day 0, 3, 5 and 7. Starting from day 4, 2%DSS was administered for 3 days in drinking water. At day 7 mice were sacrificed, the colon collected and the mRNA extracted. Slpi expression was measured by qPCR in the colon of vehicle (black bars) and quercetin (white bars) treated mice exposed or not to DSS. Bars represent mean expression ± SEM (n=3) for each treatment. ***P<0.001.

Article Snippet: Gene expression of Slpi and GAPDH was performed with TaqMan Gene Expression Assays (Thermo Fisher Scientific, MA, USA) - murine probes: Mm00441530_g1 and Mm00484668_m1, respectively; human probes: Hs00268204_m1 and Hs02758991_g1, respectively.

Techniques: Expressing

Journal: Oncotarget

Article Title: Secretory leukoprotease inhibitor is required for efficient quercetin-mediated suppression of TNFα secretion

doi: 10.18632/oncotarget.12415

Figure Lengend Snippet: A-D. Representative immunohistochemistry images for SLPI expression in normal (panels A and B) and inflamed (panels C and D) human intestinal biopsies. FFPE sections were prepared at 20 μm thickness, stained for Slpi and visualized using BioCare polymer IHC kit. E. SLPI expression was measured on human normal and inflamed areas by qPCR relative to untreated non-inflamed biopsies. Bars represent mean ± SEM (n=7). **P<0.01; ***P<0.001. F. qPCR on human inflamed tissues treated with quercetin relative to inflamed untreated ones. Bars represent mean ± SEM (n=7). *P<0.05.

Article Snippet: Gene expression of Slpi and GAPDH was performed with TaqMan Gene Expression Assays (Thermo Fisher Scientific, MA, USA) - murine probes: Mm00441530_g1 and Mm00484668_m1, respectively; human probes: Hs00268204_m1 and Hs02758991_g1, respectively.

Techniques: Immunohistochemistry, Expressing, Staining, Polymer

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: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: RT-PCR primer list

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques:

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Absence of TRKB receptors result in reduced expression of Jagged1 and NOTCH target genes in the mouse ovary. Panel A, Decrease in Jagged1 mRNA content in the ovary of 7-d-old TrkB−/− mice detected using cDNA microarrays. Changes in mRNA content are expressed as fold-decrease with respect to mRNA values in TrkB+/+ mice of the same age. Filled squares represent the values detected in independent microarray determinations. Panel B, Jagged1 mRNA content was reduced in the ovary of 7-d-old TrkB−/− mice, as assessed by real-time PCR. Inset, Jagged1 mRNA content increased in 7-d-old WT ovaries treated in vitro with BDNF (100 ng/ml, 8 h) compared to control (C) ovaries incubated with vehicle. Relative mRNA values are expressed as arbitrary units (AU), normalized using 18s RNA or Ppia mRNA values as the normalizing unit. Panel C, Hes1 and Hey2, but not Notch, mRNA abundance, was also reduced in TrkB-null ovaries. Panel D, JAGGED1 immunoreactive material (green color) mostly localizes to oocytes in the ovary from 7-d-old TrkB+/+ mice. Panel E, JAGGED1 immunoreactivity was noticeably decreased in oocytes of TrkB−/− mice. Panel F, Section incubated without JAGGED1 antibodies. Cell nuclei stained with the DNA-binding dye Hoechst are shown in blue. White arrows point to examples of oocytes showing JAGGED1 staining. Columns in B and C represent means from four to five animals per group, and vertical lines are sem. *, P < 0.05; **, P < 0.01 vs. WT controls. Scale bars, 50 μm.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction, In Vitro, Incubation, Staining, Binding Assay

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Changes in ovarian content of Jagged1, Notch2, Hes1, and Hey2 mRNA during the first postnatal week of life of the mouse, as assessed by real-time PCR. A, Jagged1 mRNA. B, Notch2 mRNA. C, Hes1 mRNA. D, Hey2 mRNA. Relative mRNA values are expressed as arbitrary units (AU), normalized using 18s RNA values as the normalizing unit. E–J, In situ hybridization, using a mouse-specific 35S-uridine triphosphate-labeled Jagged1 cRNA probe, shows that Jagged1 mRNA is exclusively expressed in oocytes and that the abundance of Jagged1 mRNA increases during the first 12 d of postnatal life. Bright field images are shown in E–G and dark field images in H–J. Black and white arrows point to examples of Jagged1 mRNA-containing oocytes. Bars represent the mean of four to five mice per group, and vertical lines are sem. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. 0-d-old group. Scale bar, 50 μm.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Real-time Polymerase Chain Reaction, In Situ Hybridization, Labeling

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Lentiviral-mediated delivery of Jagged1, using the Gdf9 promoter to target expression of a JAGGED1-HA fusion protein to oocytes, correctly targets JAGGED1 to the cell membrane of oocytes. A, Map of the lentiviral delivery construct (LV-Jagged1) used in this study. The lentiviral vector employed has been previously described (79). The 3′LTR of this vector contains a 400-bp deletion that results in the self-inactivation (SIN) of the vector. The other components include the packaging signal (ψ), the Rev response element binding site (RRE), the central polypurine tract (cPPT), and the woodchuck-hepatitis-virus posttranslational regulatory element (wPRE). The LV-Jagged1 construct contains a bicistronic transgene cassette in which expression of a Jagged1-HA cDNA is driven by the rat Gdf9 promoter (Gdf9p). The Jagged1-HA cDNA is linked to an enhanced green fluorescent protein (eGFP) cDNA via an internal ribosome entry site (IRES). A construct lacking Jagged1-HA (LV-no Jagged1) was used as a negative control. B–D, Immunohistofluorescent images of sections from 3-d-old mouse ovaries cultured for 4 d in the presence of LV-Jagged1 and stained with monoclonal antibodies against the HA epitope. E, Section from an ovary not infected with LV. F, Section from an ovary infected with LV-Jagged1 and incubated without HA antibodies. G, Section from an ovary infected with LV-no Jagged1. JAGGED1 immunoreactive cells are seen in red, and cell nuclei stained with the DNA-binding dye Hoechst are shown in blue. Scale bar, 50 μm.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Construct, Plasmid Preparation, Binding Assay, Negative Control, Cell Culture, Staining, Infection, Incubation

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Oocyte-specific restoration of JAGGED1 expression, via lentiviral-mediated gene transfer, rescues the deficit in follicle growth of TrkB−/− ovaries. A, Increased number of secondary follicles in TrkB−/− ovaries incubated for 4 d with LV-Jagged1 in comparison with TrkB−/− ovaries infected with LV-no Jagged1. B, Section from a TrkB−/− ovary infected with LV-no Jagged1. C, Section from a TrkB−/− ovary infected with LV-Jagged1. Arrows point to secondary follicles, which contain an oocyte surrounded by two layers of GC. Scale bar, 50 μm. Columns represent the mean of four mice per group, and vertical lines are sem. One ovary from each animal was infected with LV-Jagged1 and the contralateral ovary from the same animal with LV-no Jagged1. *, P < 0.05.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Incubation, Infection

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Oocyte-specific restoration of JAGGED1 expression, via lentiviral-mediated gene transfer, rescues the deficit in GC proliferation of TrkB−/− ovaries. A, Percent of follicles showing at least one PCNA-positive GC. B, Number of PCNA-positive GC per follicle (primary and secondary). C, Image of a section from a TrkB−/− ovary incubated for 4 d with LV-no Jagged1. D, A section from a TrkB−/− ovary incubated for 4 d with a LV-Jagged1. E, Ovarian section immunostained in absence of primary antibodies. Columns represent the mean of four mice per group, and vertical lines are sem. In each group, four sections per ovary were used for quantification. One ovary from each animal was infected with LV-Jagged1 and the contralateral ovary from the same animal with LV-no Jagged1. Scale bar, 50 μm. ***, P < 0.001 vs. LV-no Jagged1.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Incubation, Infection

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: TrkB signaling sustains c-Myc and Odc1 expression but not the expression of core regulatory components of the cell cycle. Panel A, c-Myc mRNA content was reduced in 7-d-old TrkB−/− ovaries as compared with WT animals. Inset, In vitro exposure of WT ovaries to NT4/5 (100 ng/ml, 8 h) increased c-Myc mRNA abundance as compared to control (C) ovaries incubated with vehicle. Panel B, Odc1 mRNA abundance was also decreased in TrkB−/− ovaries. Inset, NT4/5 increased Odc1 mRNA abundance in WT ovaries. Panels C and D, The content of mRNA encoding cyclins (CycD2 and CycE1), CDK (Cdk2 and Cdk4), and the CKI of the INK4 family (p15INK4b, p16INK4a, p18INK4c, and p19INK4d) and CIP/KIP family (p21Cip1, p27Kip1, and p57Kip2) remain unaltered in TrkB−/− ovaries as compared with TrkB+/+ mice. Panel E, c-Myc mRNA levels were increased in TrkB−/− ovaries after oocyte-specific restoration of JAGGED1 synthesis. The ovaries from 3-d-old mice were incubated for 4 d with a lentiviral construct carrying the Jagged1-coding region under the control of the Gdf9 promoter. Panel F, Neither p19INK4D nor p27Kip1 mRNA levels changed after lentiviral-mediated restoration of JAGGED1 synthesis. Control ovaries were infected with a LV lacking Jagged1 cDNA (LV-no Jagged1). Each column represents the mean of four to five mice per group, and vertical lines are sem. One ovary from each animal was infected with LV-Jagged1 and the contralateral ovary with LV-no Jagged1. *, P < 0.05; **, P < 0.01 vs. their respective controls. AU, Arbitrary units.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, In Vitro, Incubation, Construct, Infection

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 2 DACH1 deletion PCa enhances AR signaling. A Interrogation of human PCa gene expression data [26], showing candidate genetic drivers ERG, ETV1/ETV4/FLI1, SPOP, FOXA1, and unknown. Samples with DACH1 homozygous (deep) genetic deletions (29/333) are shown as an additional subtype. The AR score (the average of the AR target gene expression) refers to a group of AR-responsive genes [26], and together with the expression Z-score of the AR target genes, are shown as colorimetric scales. The AR score-based gene names are shown. The androgen receptor (AR) activity, inferred by the induction of AR target genes, was increased in DACH1 homozygous (‘deep’) deletion PCa compared with normal (P = 2 × 10−5 by t-test) and ERG mutation groups (P = 0.003 by t-test). B AR mRNA and AR protein levels, shown for each DACH1 deletion sample, were not significantly different. C The iCluster [29], mRNA cluster, and SCNA (somatic copy-number alteration), and DNA methylation status are shown for the PCa classified by the corresponding gene deletion subtypes. D DACH1 homozygous deletions were enriched for iCluster 2 and 3 [29], mRNA cluster 2 (P = 0.0003 by Fisher exact test, SCNA (“more” somatic copy-number alteration, P = 0.0004 by Fisher exact test), but not for DNA methylation.

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Gene Expression, Targeted Gene Expression, Expressing, Activity Assay, Mutagenesis, DNA Methylation Assay

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 3 Prostate-specific Dach1 gene deletion promotes prostate hyperplasia and dysplasia in OncoMice (15 weeks). A Schematic representation of transgenes integrated into mice. B Representative immunohistochemistry for Dach1, with data quantitated as mean ± standard error of the mean (SEM) for N = 20 (4 separate mice, with 5 views per mouse, in each group). C Blinded quantitative histology grading of prostate of multigenic mice at 15 weeks. Data are shown as mean ± SEM for N = 15 (5 separate mice, with 3 prostate areas [anterior, ventral, lateral] per mouse) in each group). H&E staining demonstrates the presence of a focal atypical intraductal proliferation in Dach1−/−prostate, compatible with prostatic intraepithelial neoplasia (PIN). Representative immunohistochemistry with results shown as mean ± SEM for Ki-67 (n = 20, 4 separate mice for each genotype, 5 views per mouse) (D), Beclin 1 (n = 9, 3 separate mice for each genotype, 3 views per mouse) (E); and AR (n = 15 for Dach1wt/wt mice, 3 separate mice, 5 views per mouse) (n = 12 for Dach1fl/flmice, 3 separate mice, 2 views for one mouse and 5 views for other two mice) (F). Scale bars, 50 μm. A Student’s t test was performed for all comparisons.

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Immunohistochemistry, Staining

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 4 Prostate-specific Dach1 gene deletion in TRAMP mice induces PIN lesions with increased TGFβ activity. Genome-wide expression analysis of TRAMP Dach1+/+ vs. Dach1−/−PIN lesions was analyzed for enrichment of known targets of upstream regulators using Ingenuity Pathway Analysis (IPA) and represented as (A) barplot was calculated by IPA activation Z-score labeled and as (B) bubble plot with size of the bubbles proportional to –log10 p values. C IHC was conducted for SMAD activation using SMAD2P, quantitated and shown as (D) mean ± SEM (n = 15 for Dach1wt/wt mice, 3 separate mice, 5 views per mouse) (n = 10 for Dach1fl/flmice, 2 separate mice, 5 views per mouse). E–G Western blot of either PCa cell lines for the presence of DACH1 (E, F) or (G) TGFβ-treated (10 ng/ml for 24 h) PC3 cells illustrating induction of nuclear vimentin and cytoplasmic cyclin D1. Protein loading controls are β-tubulin (a marker of cytoplasmic proteins) and Lamin B1 (a marker for nuclear protein enrichment). H Microarray-based gene expression analysis of PC3 cells stably expressing DACH1, showing restraint of genes mediating TGFβ signaling (shown with blue arrows), including reduction of TGFB2 and TGFBR2 [33].

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Activity Assay, Genome Wide, Expressing, Activation Assay, Labeling, Western Blot, Marker, Protein Enrichment, Microarray, Gene Expression, Stable Transfection

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 6 DACH1 facilitates the recruitment of, and co-accumulates with, Ku70/Ku80 proteins at sites of DNA damage. A Co-accumulation of Ku-70/Ku-80 at laser micro irradiation-induced DSBs sites in Dach1+/+ 3T3 cells. B, C 24 h after transfection, the accumulation of DACH1 and Ku70/Ku80 in Dach1−/−3T3 cells transfected with EGFP or EGFP-tagged DACH1 and red fluorescent protein (RFP)-tagged Ku70 or RFP-tagged Ku80 expression vectors were treated with laser micro-irradiation (403 nm) to induce DSBs. Time is shown after micro-irradiation. Accumulation of the transfected proteins was indicated by EGFP (green) or RFP (red) fluorescence at laser-irradiated sites. Co-accumulation was visualized in yellow merged images. Time is shown in minutes and -fold increase in foci intensity is shown as mean ± SEM for N = 5 separate cells.

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Irradiation, Transfection, Expressing

Journal: Molecular Oncology

Article Title: PLK1 and FoxM1 expressions positively correlate in papillary thyroid carcinoma and their combined inhibition results in synergistic anti‐tumor effects

doi: 10.1002/1878-0261.13610

Figure Lengend Snippet: Tissue microarray (TMA) based immunohistochemistry analysis of PLK1 and FoxM1 in papillary thyroid cancer (PTC) patients. PTC TMA spots showing overexpression of PLK1 (A) and FoxM1 (C). In contrast, another set of TMA spots showing reduced expression of PLK1 (B) and FoxM1 (D). 20×/0.70 objective on an Olympus BX 51 microscope (Olympus America Inc, Center Valley, PA, USA) with the inset showing a 40× 0.85 aperture magnified view of the same TMA spot (scale bar = 500 μm). (E) Recurrence‐free survival (RFS). Kaplan–Meier survival plot showing statistically significant poor RFS in PLK1 high expression cases compared to PLK1 low expression ( P = 0.0009).

Article Snippet: Plasmid DNA encoding human PLK1 and shRNA targeting human PLK1 were purchased from Origene (Rockville, MD, USA).

Techniques: Microarray, Immunohistochemistry, Over Expression, Expressing, Microscopy

Journal: Molecular Oncology

Article Title: PLK1 and FoxM1 expressions positively correlate in papillary thyroid carcinoma and their combined inhibition results in synergistic anti‐tumor effects

doi: 10.1002/1878-0261.13610

Figure Lengend Snippet: Inhibition of PLK1 impedes cell proliferation and induces cell cycle arrest and apoptosis. (A) Basal expression of PLK1 in Nthy‐ori 3–1 and PTC cell lines. Proteins were extracted from Nthy‐ori 3–1 cell line and three PTC cell lines and subjected to immunoblotting using indicated antibodies ( n = 3). (B, C) Volasertib inhibits cell viability. PTC cells (10 4 ) were exposed to increasing doses of volasertib for 24 and 48 h and cell viability was assessed using MTT. Data were presented as mean ± SD ( n = 8). (D, E) Cell cycle analysis of PTC cells treated with volasertib. BCPAP and TPC‐1 cells were exposed to 250 and 500 n m volasertib for 48 h. Following incubation, either cells were analyzed for cell cycle fractions by flow cytometry (D) or proteins were extracted from cells and were subjected to immuno‐blotting to analyze cell cycle markers (E). Data were presented as mean ± SD ( n = 3). (F, G) Volasertib induces apoptosis in PTC cell lines. PTC cells were exposed to indicated doses of volasertib for 48 h. Following incubation, either cells were stained with annexin‐V/PI and analyzed by flow cytometry (F) or proteins were extracted from cells and were subjected immuno‐blotting using antibodies as indicated (G). Data were presented as mean ± SD ( n = 3). (H) Effect of volasertib on Bax activation in PTC cells. PTC cells were treated with volasertib for indicated time course. Following treatment, cells were lysed in 1% Chaps lysis buffer and subjected to immuno‐precipitation with anti‐Bax antibody (sc‐493; Santa Cruz) and immunoblotted with anti‐Bax antibody (B8554; SIGMA‐ALDRICH), ( n = 3). Statistical analyses were conducted employing two‐tailed Student's t ‐tests. * P < 0.05.

Article Snippet: Plasmid DNA encoding human PLK1 and shRNA targeting human PLK1 were purchased from Origene (Rockville, MD, USA).

Techniques: Inhibition, Expressing, Western Blot, Cell Cycle Assay, Incubation, Flow Cytometry, Staining, Activation Assay, Lysis, Immunoprecipitation, Two Tailed Test

Journal: Molecular Oncology

Article Title: PLK1 and FoxM1 expressions positively correlate in papillary thyroid carcinoma and their combined inhibition results in synergistic anti‐tumor effects

doi: 10.1002/1878-0261.13610

Figure Lengend Snippet: PLK1 interacts with FoxM1 in vitro . (A) Basal expression of PLK1 and FoxM1 in PTC cell lines. Proteins were isolated from Nthy‐ori 3–1 cell line and three PTC cell lines and immunoblotted with antibodies against PLK1, FoxM1, and GAPDH ( n = 3). (B) PLK1 interact with FoxM1. Cell lysates extracted from PTC cells were immunoprecipitated with PLK1 or IgG antibody. Interaction of endogenous PLK1 and FoxM1 was detected by immunoblotting ( n = 3). (C) FoxM1 interact with PLK1. Cell lysates extracted from PTC cells were immunoprecipitated with FoxM1 or IgG antibody. Interaction of endogenous FoxM1 and PLK1 was detected by immunoblotting ( n = 3). (D) Forced expression of PLK1 triggers the activation of FoxM1 expression. Nthy‐ori 3–1 cells were transfected with either an empty vector or PLK1 cDNA for 48 h. Proteins were extracted from overexpression clones and were subjected immunoblotting using antibodies as indicated ( n = 3). (E) Knockdown of PLK1 inhibits FoxM1. PTC cells underwent transfection with two distinct PLK1 shRNA sequences, and proteins from the selected clones were later analyzed using immunoblotting ( n = 3). (F) Volasertib treatment reduce the expression of PLK1 and FoxM1 in PTC cells. PTC cells were treated with specified doses of volasertib for 48 h. Proteins were extracted after cell lysis and were subjected to immunoblotting, as indicated ( n = 3). (G, H) Inhibition of FoxM1 has no effect on PLK1 expression. PTC cells were either transfected with FoxM1 siRNA (20 n m ) or treated with thiostrepton (2.5 and 5 μ m ). Following 48 h, the cells were lysed, and the extracted proteins were subsequently subjected to immunoblotting using antibodies as indicated ( n = 3).

Article Snippet: Plasmid DNA encoding human PLK1 and shRNA targeting human PLK1 were purchased from Origene (Rockville, MD, USA).

Techniques: In Vitro, Expressing, Isolation, Immunoprecipitation, Western Blot, Activation Assay, Transfection, Plasmid Preparation, Over Expression, Clone Assay, Knockdown, shRNA, Lysis, Inhibition

Journal: Molecular Oncology

Article Title: PLK1 and FoxM1 expressions positively correlate in papillary thyroid carcinoma and their combined inhibition results in synergistic anti‐tumor effects

doi: 10.1002/1878-0261.13610

Figure Lengend Snippet: Combined inhibition of PLK1 and FoxM1 markedly attenuates PTC cell growth in vitro . (A–B) Volasertib and thiostrepton synergistically inhibits clonogenicity. PTC cells (5 × 10 2 ) after volasertib and thiostrepton treatments were seeded into each of three dishes (60 mm diameter), and grown for an additional 10 days, then stained with crystal violet and colonies were counted. Data were presented as mean ± SD ( n = 3). Statistical analyses were performed using two‐tailed Student's t ‐tests. * P < 0.05. (C) Volasertib and thiostrepton synergistically induce apoptosis. PTC cells were exposed to specified doses either of volasertib and thiostrepton, single or in combination, for 48 h. Subsequently, these cells were stained with annexin‐V/PI and analyzed by flow cytometry. Data were presented as mean ± SD ( n = 3). Statistical analyses were performed using two‐tailed Student's t ‐tests. * P < 0.05. (D) Volasertib and thiostrepton synergistically induce the cleavage of caspase‐3 and PARP. PTC cells after 48 h of indicated treatment, lysed and proteins were subjected to immunoblot analysis using antibodies as indicated ( n = 3).

Article Snippet: Plasmid DNA encoding human PLK1 and shRNA targeting human PLK1 were purchased from Origene (Rockville, MD, USA).

Techniques: Inhibition, In Vitro, Staining, Two Tailed Test, Flow Cytometry, Western Blot

Journal: Molecular Oncology

Article Title: PLK1 and FoxM1 expressions positively correlate in papillary thyroid carcinoma and their combined inhibition results in synergistic anti‐tumor effects

doi: 10.1002/1878-0261.13610

Figure Lengend Snippet: Inhibition of PLK1 decreases the self‐renewal ability of spheroids generated from PTC cells. (A, B) Knockdown of PLK1 impairs the self‐renewal capacity of spheroids. PTC cells were transfected with PLK1 shRNA and subsequently subjected to a sphere‐forming assay (scale bar = 1 mm). The total number of spheroids in the entire well was quantified. Data were presented as mean ± SD ( n = 3). (C) Knockdown of PLK1 reduces the stem cell characteristics of spheroids. PTC cells were transfected with PLK1 shRNA and cultured in a sphere‐forming medium. Subsequently, proteins were extracted from the spheroids and analyzed via immunoblotting using antibodies as indicated ( n = 3). (D) Forced PLK1 expression enhances the growth of spheroids. Nthy‐ori 3–1 cells were transfected with either an empty vector or PLK1 cDNA, and subsequently, these cells were subjected to a sphere‐forming assay. The total number of spheroids in the entire dish was quantified. Data were presented as mean ± SD ( n = 3). (E) Forced expression of PLK1 enhances the stem cell properties of spheroids, as validated through immunoblotting employing stem cell markers. Nthy‐ori 3–1 cells were transfected with either an empty vector or PLK1 cDNA and subsequently cultured in a sphere‐forming medium. Proteins were isolated from spheroids and immunoblotted with antibodies against PLK1, FoxM1, CD44, CD133, NANOG, and GAPDH ( n = 3). (F) Volasertib and thiostrepton synergistically reduces spheroid growth. PTC cells were exposed to specified doses of volasertib and thiostrepton, either single or in combination, for 48 h. Following this treatment, the cells were subjected to a sphere‐forming assay. Spheroids in the entire dish were counted. Data were presented as mean ± SD ( n = 3). (G) PTC cells were exposed to specified doses either of volasertib and thiostrepton, single or in combination, for 48 h. Subsequently, these treated cells were cultured in a sphere‐forming medium and proteins isolated from spheroids were subjected to immunoblotting using antibodies as indicated ( n = 3). Statistical analyses were performed using two‐tailed Student's t ‐tests. * P < 0.05.

Article Snippet: Plasmid DNA encoding human PLK1 and shRNA targeting human PLK1 were purchased from Origene (Rockville, MD, USA).

Techniques: Inhibition, Generated, Knockdown, Transfection, shRNA, Cell Culture, Western Blot, Expressing, Plasmid Preparation, Isolation, Two Tailed Test

Journal: Molecular Oncology

Article Title: PLK1 and FoxM1 expressions positively correlate in papillary thyroid carcinoma and their combined inhibition results in synergistic anti‐tumor effects

doi: 10.1002/1878-0261.13610

Figure Lengend Snippet: Volasertib and thiostrepton synergistically inhibit PTC tumor growth in vivo . TPC‐1 cells were subcutaneously injected into the flanks of 6‐week‐old NU/J mice (4 × 10 6 cells per mouse). Once the tumors reached approximately 100 mm 3 in size, the mice were subjected to intraperitoneal treatment with the specified doses of volasertib (20 mg·kg −1 ) and thiostrepton (20 mg·kg −1 ), either administered individually or in combination. This treatment regimen was repeated twice a week and continued for a duration of 30 days. (A) Representative images of tumors from each group of mice. (B) The tumor volume measurements were taken every week. The average ( n = 5) tumor volume (±SD) in each group of mice was calculated, * P < 0.05. (C) Following 4 weeks of treatment, the mice were euthanized, and the mean tumor weight (±SD) was determined for each group, * P < 0.05. (D) Tissue lysates from tumors were immuno‐blotted with antibodies against PLK1, FoxM1, PARP, Caspase‐3, Cleaved‐caspase‐3, Bcl‐2, Bcl‐xL, and GAPDH. (E) Western blots were quantified and data are the mean ± SD ( n = 3). Statistical analyses were performed using two‐tailed Student's t ‐tests. *Statistically significant difference compared to vehicle control with P < 0.05. # Statistically significant difference compared to volasertib treatment with P < 0.05. @ Statistically significant difference compared to thiostrepton treatment with P < 0.05.

Article Snippet: Plasmid DNA encoding human PLK1 and shRNA targeting human PLK1 were purchased from Origene (Rockville, MD, USA).

Techniques: In Vivo, Injection, Western Blot, Two Tailed Test, Control