Journal: The Journal of Biological Chemistry

Article Title: TGF-β/SMAD3 Pathway Stimulates Sphingosine-1 Phosphate Receptor 3 Expression

doi: 10.1074/jbc.M116.740084

Figure Lengend Snippet: Up-regulation of S1PR3 in human lung adenocarcinomas. A, qPCR quantitation of S1PR3 mRNA in cDNA arrays of human lung adenocarcinoma specimens (OriGene, HLRT101 and HLRT105). **, p < 0.01, Student's t test. B, qPCR quantitation of S1PR2 mRNA in a cDNA array of human lung cancers (OriGene, HLRT105). **, p < 0.01, Student's t test. C, HEK293 cells were transfected with S1PR3 or pcDNA vector. Transfected cells were immunostained with anti-S1PR3 (Cayman Chemical) (IMF, left panels). Arrows, nonspecific fluorescent precipitates used for image orientation. Scale bar = 33 μm. D, anti-S1PR3 staining of human lung adenocarcinoma tumor microarray (Accumax 306). AdC, adenocarcinoma; N, adjacent normal lung tissue. E, immunostaining intensity was quantitated with the National Institutes of Health ImageJ software. Data, analyzed with GraphPad Prism 5 software, are shown as mean ± S.E. Statistical significance was analyzed by Student's t test. F, representative images of anti-S1PR3 staining of human lung adenocarcinoma and the respective adjacent normal lung epithelial tissue. G, quantitation of anti-S1PR3 staining of human lung squamous carcinoma microarray (Accumax 306). Data are mean ± S.E. Statistical significance was analyzed by Student's t test. H, representative images of anti-S1PR3 staining of human lung squamous carcinoma and the respective adjacent normal lung epithelial tissue.

Article Snippet: B , qPCR quantitation of S1PR2 mRNA in a cDNA array of human lung cancers (OriGene, HLRT105).

Techniques: Quantitation Assay, Transfection, Plasmid Preparation, Staining, Microarray, Immunostaining, Software

Journal: The Journal of Biological Chemistry

Article Title: TGF-β/SMAD3 Pathway Stimulates Sphingosine-1 Phosphate Receptor 3 Expression

doi: 10.1074/jbc.M116.740084

Figure Lengend Snippet: TGF-β/SMAD3 signaling axis up-regulates S1PR3. A, HBEC2-KT cells were treated with TGF-β (1 ng/ml) for various times. mRNA levels of S1P receptors were measured by qPCR analysis. Data are mean ± S.D. of triplicate determinations. *, p < 0.05, Student's t test. B, protein levels of S1PR3 in TGF-β (1 ng/ml)-treated HBEC2-KT cells. Lower panel, Western blot intensity was quantitated by National Institutes of Health ImageJ. Data (normalized to actin) are mean ± S.D. of triplicate determinations. * and **, p < 0.05 and 0.01, respectively, Student's t test. C, CHO cells were transduced with adenoviral particles (multiplicity of infection of 200) carrying S1PR1, S1PR2, or S1PR3 vector for 20 h as we described (8). Extracts were blotted with antibody against S1PR3 (Cayman), S1PR2 (Cayman), or S1PR1 (E49) (8). D, mRNAs of S1PR3 and TGF-β in minced C57BL/6 mouse lungs (∼1–2 mm3) infected with adenoviral active TGF-β (Ad-TGF-β, 1 × 108 pfu/ml) or empty vector (Ad-Ctrl) (37 °C, 24 h). **, p < 0.01, n = 5, Student's t test. E, mRNA levels of SphK1 and SphK2 in TGF-β-treated HBEC2-KT cells. F, HBEC2-KT cells (2 × 106 cells in 100-mm dish, 10 ml of cultural medium) were treated with TGF-β (1 ng/ml) for 24 h. Medium was quantitated for S1P, ceramide (Cer), and sphingomyelin (SPM) by LC-MS/MS (29, 46). G, HBEC2-KT were pretreated for 30 min with inhibitors. S1PR3 levels were measured by qPCR, following TGF-β treatment (4 h). The following inhibitors were used: SB4, TGF-β receptor I (SB-431542, 10 μm); SIS3, SMAD3 (2 μm); SB2, p38 kinase (SB-203580, 50 nm); BAY, NFκB (BAY11-7085, 10 μm); JII, JNK (JNK inhibitor II, 10 μm). *, p < 0.05; dashed line, non-statistical significance; n = 3, ANOVA. Each experiment was repeated 2–3 times with similar results. H, cells were pretreated for 30 min with inhibitors, followed by stimulation with TGF-β (1 ng/ml). Activation of p38, JNK, and NFκB was measured by Western blotting with phospho-p38 (P-p38), phospho-JNK (P-p54JNK and P-p46JNK), and phospho-IκBα (p-IκBα). Inhibitors used are: SB-203580 (50 nm) for p38 kinase, JNK inhibitor II (10 μm) for JNK, and BAY11-7085 (10 μm) for NFκB.

Article Snippet: B , qPCR quantitation of S1PR2 mRNA in a cDNA array of human lung cancers (OriGene, HLRT105).

Techniques: Western Blot, Transduction, Infection, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: TGF-β/SMAD3 Pathway Stimulates Sphingosine-1 Phosphate Receptor 3 Expression

doi: 10.1074/jbc.M116.740084

Figure Lengend Snippet: S1PR3 regulates growth and lung colonization of lung adenocarcinoma cells. A, H1793 cells were stably transfected with sh-S1PR3 or pRS (sh-Ctrl) vector (11, 16). mRNA levels of S1PR3 were quantitated with qPCR analysis. B, H1793 cells (1 × 106 cells), stably transfected with sh-S1PR3 or sh-Ctrl vector, were subcutaneously inoculated in Scid mice. Tumor volume was measured in two dimensions using calipers, and volume was determined using the formula width2 × length × 0.52 (49). C, 4 weeks after inoculation, tumors were removed and weighed. D, Scid mice were injected with H1793 cells transfected with sh-S1PR3 or sh-Ctrl vector (1 × 106 cells) via tail vein route. 28 days later, tumor nodules on lung surface were scored. E, representative images of lung injected with H1793-sh-Ctrl and H1793-sh-S1PR3 cells. Arrows, tumor nodules. Scale bar = 0.5 cm. F, volume of xenograft tumors in athymic nude mice subcutaneously implanted with H1299 cells stably transfected with S1PR3 or control pcDNA vector (1 × 106 cells) (11, 16). G, qPCR quantitation of S1PR3 levels in H1299/pcDNA and H1299/S1PR3 cells. **, p < 0.01, n = 6, ANOVA.

Article Snippet: B , qPCR quantitation of S1PR2 mRNA in a cDNA array of human lung cancers (OriGene, HLRT105).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Injection, Quantitation Assay

Journal: Cell reports

Article Title: Oxidized mC modulates synthetic lethality to PARP inhibitors for the treatment of leukemia

doi: 10.1016/j.celrep.2023.112027

Figure Lengend Snippet:

Article Snippet: Venetoclax , MedChem Express , HY-15531.

Techniques: Purification, Blocking Assay, Recombinant, Lysis, Staining, Saline, DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Binding Assay, Single Cell Gel Electrophoresis, Software, Protein Array, Membrane

Journal: Cell reports

Article Title: Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

doi: 10.1016/j.celrep.2020.107764

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-FoxO3a (D19A7) , Cell Signaling Technology , Cat# 12829; RRID:AB_2636990.

Techniques: Virus, Mutagenesis, Recombinant, Cell Viability Assay, Viability Assay, CellTox Assay, Cytotoxicity Assay, In Situ, CRISPR, Protein Array, RNA Sequencing, Mass Spectrometry, shRNA, Sequencing, Software

Journal: Cell reports

Article Title: Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

doi: 10.1016/j.celrep.2020.107764

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-phospho-FoxO1 (Ser256) , Cell Signaling Technology , Cat# 9461; RRID:AB_329831.

Techniques: Virus, Mutagenesis, Recombinant, Cell Viability Assay, Viability Assay, CellTox Assay, Cytotoxicity Assay, In Situ, CRISPR, Protein Array, RNA Sequencing, Mass Spectrometry, shRNA, Sequencing, Software

Journal: Cell reports

Article Title: Architecture of androgen receptor pathways amplifying glucagon-like peptide-1 insulinotropic action in male pancreatic β cells

doi: 10.1016/j.celrep.2023.112529

Figure Lengend Snippet: (A–C) GSIS was measured via static incubation in male human islets (n = 3–7 donors, 10 IEQ/condition measured in triplicate) cultured for 24 h with vehicle or DHT (10 nM) and then for 40 min in the presence or absence of (A) a carbonic anhydrase inhibitor AZZ (50 μM), (B) CFTR inhibitor CFTRi (500 μM), or (C) NA + /HCO 3 − co-transporter inhibitor S0859 (2 μM). (D) Schematic representation of bioenergetics, metabolomics, proteomics, and transcriptomics workflow. (E–K) Oxygen consumption rate (OCR) was measured in male C57/BL6 mouse islets treated with vehicle or DHT (10 nM) in a Seahorse analyzer during a mitochondrial stress test using oligomycin, FCCP, and antimycin A/rotenone at the indicated times. Islets were pooled from 10 mice with 150 islets per well in 10 wells for each condition and n = 3 experiments. (E) Line graph of OCR showing the key parameters of mitochondrial function measured in (F) to (K). (L–P) Extracellular acidification rate (ECAR) was measured in a Seahorse analyzer during (L–N) a glycolytic stress test using 2-DG and (O and P) a mitochondrial stress test using antimycin A/rotenone at the indicated times. Islets were pooled from 5 mice with 150 islets per well in 5 wells for each condition and n = 2 experiments. (Q) Human islets (n = 3 donors, 1,200 IEQ/condition) were treated with DHT for 5 min, 30 min, and 18 h, compared with control untreated islets, and studied by reverse-phase protein array (RPPA). Normalized values from the three individual donors were averaged to generate an average Z score and map proteins in the heatmap. *p < 0.05 (t test) and # p < 0.05 (Fisher’s LSD test) compared with control. (R) Human islets (n = 3 donors, 500 IEQ/condition) were treated with DHT at 16.7 mM glucose for 30 min in the presence of [ 13 C]glucose, and metabolites were measured by LC-MS. Results represent the mean ± SE of the ratio 13 C/ 12 C metabolite abundance. (S) Schematic representation of the pathway, enzymes, and metabolites described in (A) to (R). Values represent the mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Islets were then treated with vehicle (95% ethanol), DHT, GLP-1 (10nM; Indiana University Bloomington), GIP (100nM; Tocris), glucagon (20nM; Sigma), forskolin (10μM; Sigma), H89 (10μM; CST), ESI-09 (10μM; Sigma), Exendin9–39 (100nM; Sigma), KH7 (1μM; Sigma Aldrich), LRE1 (10μM; Sigma Aldrich), TDI-10229 (2μM, 5μM, 10μM; Cornell), Acetazolamide/AZZ (50μM; MedChemExpress), CFTRinh-172 (500μM; Selleckchem), S0859 (2μM; MedChemExpress), 2′,5′-Dideoxyadenosine/ddADO (1μM), PF431396 (15nM; Selleckchem), PF562271 (15nM; Selleckchem) PF573228 (10nM; MedChemExpress), PF4618433 (1μM; MedChemExpress), PP2 (1μM; Sigma Aldrich), BKM120 (1μM; Selleckchem), GDC-0941 (1μM; Sigma Aldrich), Rapamycin (27.4nM; Sigma Aldrich), JR-AB2–011 (5μM; TargetMol), Linagliptin (83 μg/ kg; Boehringer Ingelheim), Finasteride (100nM; Sigma Aldrich), Dutasteride (100nM; Sigma Aldrich), Exendin4 (100nM), ExendinPhe1 (100nM; Imperial College London) at 2.8mM and then 16.7mM glucose for 40 min sequentially.

Techniques: Incubation, Cell Culture, Control, Protein Array, Liquid Chromatography with Mass Spectroscopy

Journal: Cell reports

Article Title: Architecture of androgen receptor pathways amplifying glucagon-like peptide-1 insulinotropic action in male pancreatic β cells

doi: 10.1016/j.celrep.2023.112529

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Islets were then treated with vehicle (95% ethanol), DHT, GLP-1 (10nM; Indiana University Bloomington), GIP (100nM; Tocris), glucagon (20nM; Sigma), forskolin (10μM; Sigma), H89 (10μM; CST), ESI-09 (10μM; Sigma), Exendin9–39 (100nM; Sigma), KH7 (1μM; Sigma Aldrich), LRE1 (10μM; Sigma Aldrich), TDI-10229 (2μM, 5μM, 10μM; Cornell), Acetazolamide/AZZ (50μM; MedChemExpress), CFTRinh-172 (500μM; Selleckchem), S0859 (2μM; MedChemExpress), 2′,5′-Dideoxyadenosine/ddADO (1μM), PF431396 (15nM; Selleckchem), PF562271 (15nM; Selleckchem) PF573228 (10nM; MedChemExpress), PF4618433 (1μM; MedChemExpress), PP2 (1μM; Sigma Aldrich), BKM120 (1μM; Selleckchem), GDC-0941 (1μM; Sigma Aldrich), Rapamycin (27.4nM; Sigma Aldrich), JR-AB2–011 (5μM; TargetMol), Linagliptin (83 μg/ kg; Boehringer Ingelheim), Finasteride (100nM; Sigma Aldrich), Dutasteride (100nM; Sigma Aldrich), Exendin4 (100nM), ExendinPhe1 (100nM; Imperial College London) at 2.8mM and then 16.7mM glucose for 40 min sequentially.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Gene Expression, Software, Single-cell Analysis

Journal: Cell Reports

Article Title: USP15 Deubiquitinase Safeguards Hematopoiesis and Genome Integrity in Hematopoietic Stem Cells and Leukemia Cells

doi: 10.1016/j.celrep.2020.108533

Figure Lengend Snippet:

Article Snippet: sgRNA sequences targeting human USP15 were selected from the Human Brunello CRISPR knockout pooled library ( ) (Addgene #73178) and further selected on the basis of high quality score in two additional online tools: CRISPR Design ( https://zlab.bio/guide-design-resources ) and CRISPRscan ( https://www.crisprscan.org/ ).

Techniques: Recombinant, Gene Knockout, Transfection, Cell Isolation, RNA Sequencing, Knockdown, Mass Spectrometry, Expressing, Knock-Out, Illumina Sequencing, Multiplexing, CRISPR, Control, shRNA, Sequencing, Quantitative RT-PCR, Plasmid Preparation, Software, Quantitative Proteomics, Irradiation, Imaging

Journal: Cell reports

Article Title: Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

doi: 10.1016/j.celrep.2020.107764

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-Raf-1 (E-10) , Santa Cruz Biotechnology , Cat# sc-7267; Lot# I0117; RRID:AB_628196.

Techniques: Virus, Mutagenesis, Recombinant, Cell Viability Assay, Viability Assay, CellTox Assay, Cytotoxicity Assay, In Situ, CRISPR, Protein Array, RNA Sequencing, Mass Spectrometry, shRNA, Sequencing, Software