Journal: Cell Communication and Signaling : CCS

Article Title: Cardiac function in zebrafish embryos is linked to an androgen receptor-adrenomedullin-proepicardium axis

doi: 10.1186/s12964-026-03107-4

Figure Lengend Snippet: Ar LOF increases adm2a expression and ablation of Adm2a partially rescues heart function and AVC patterning. A , Chromatin immunoprecipitation in HEK293T cells transfected with Flag-Ar with control IgG or anti-Flag antibody reveals enrichment of a DNA fragment in exon 2 of human ADM2 . Shown are results normalized to the input fraction. n =6. ** p =0.0087. Two-tailed Mann-Whitney test. B , Chromatin immunoprecipitation in HEK293T cells transfected with Flag-Ar with control IgG or anti-Flag antibody showing enrichment of a DNA fragment upstream of the transcription start site of human ADM2 . Shown are results normalized to the input fraction. n =4. ** p =0.0286 Two-tailed Mann-Whitney test. C , Luciferase reporter assay in HEK293T cells transiently transfected with zebrafish Ar and either empty control vector (ev), a negative control (neg. ctrl) or luciferase under the control of sequences upstream of the transcription start site of zebrafish adm2a . Shown are basal levels and such stimulated with dihydrotestosterone (DHT). n =4. **** p <0.0001, * p =0.0358. One-way ANOVA with Sidak’s multiple comparison test. D , qPCR showing increased expression of adm2a in PF-998,425-treated embryos. n =7. * p =0.0156. Two-tailed Wilcoxon matched-pairs signed rank test. E , Ar promoter crispants have increased adm2a expression compared to Cas9-injected embryos. n =8. * p =0.0493. Two-tailed paired t-test. F , Adm2a transcripts are elevated upon Ar knockdown. n =12. ** p =0.0071. Two-tailed paired t-test. G , qPCR of isolated hearts demonstrates adm2a upregulation in PF-998,425-treated embryos. Graph shows absolute expression normalized to gapdh . n =4 experiments. * p =0.0410. Two-tailed ratio paired t-test. H , Administration of the ADM2 inhibitor AM 22–52 partially rescues edema formation in embryos treated with an Ar blocker. n =3 experiments with 80–86 embryos in total. ** p =0.0025. One-way ANOVA with Holm-Sidak’s multiple comparison test. I , AM 22–52 significantly increases the heart rate of Ar LOF embryos. n =3 experiments with 80–84 embryos in total. * p =0.0169, **** p <0.0001. Kruskal-Wallis test with Dunn’s multiple comparison test. J , AM 22–52 rescues arrhythmia induced by Ar blockade. n =3 experiments with 80–84 embryos in total. * p =0.0433. Kruskal-Wallis test with Dunn’s multiple comparison posttest. K , Adm2a depletion using transient Crispr/Cas9 reduces the percentage of Ar LOF embryos developing edema. n =4 experiments with 80–84 embryos in total. * p =0.0255. One-way ANOVA with Holm-Sidak’s multiple comparison test. L , Adm2a abrogation by Crispr/Cas9 significantly increases the heart rate of Ar blocker-treated embryos. n =5 experiments with 159–177 embryos in total. **** p <0.0001. Kruskal-Wallis test with Dunn’s multiple comparison test. M , Crispr/Cas9 against adm2a significantly rescues arrhythmia in embryos undergoing Ar blockade. n =5 experiments with 159–173 embryos in total. ** p =0.0031. One-way ANOVA with Holm-Sidak’s multiple comparison test. N , gRNA injection against adm2a results in more Ar LOF embryos expressing tbx2b in the AVC compared to Ar blockade alone. n =4 experiments with 81–107 embryos in total. * p =0.01701. Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 multiple comparison test. O , Administration of AM22-52 increases the percentage of embryos with notch1b enrichment in the AVC. n =4 experiments with 80–97 embryos in total. * p =0.0261. One-way ANOVA test with Holm-Šídák’s multiple comparisons test. All data obtained at 48 hpf. Red line: median. Red dotted lines indicate experimental pairs

Article Snippet: Adrenomedullin 22–52 was purchased from MedChemExpress, ARN-509 [ ] and flutamide [ ] were from Selleckchem, and PF-998,425 [ ], tolterodine [ ] and tricaine [ ] were obtained from Sigma-Aldrich.

Techniques: Expressing, Chromatin Immunoprecipitation, Transfection, Control, Two Tailed Test, MANN-WHITNEY, Luciferase, Reporter Assay, Plasmid Preparation, Negative Control, Comparison, Injection, Knockdown, Isolation, CRISPR

Journal: Cancer cell

Article Title: Coordinated Splicing of Regulatory Detained Introns within Oncogenic Transcripts Creates an Exploitable Vulnerability in Malignant Glioma

doi: 10.1016/j.ccell.2017.08.018

Figure Lengend Snippet: (A) Tissue origin and numbers of cell lines used in screen (pie chart) and schematics of EPZ dosing pipeline. (B) Cell line sensitivity parameters. (C) Linear regressions of indicated cell line drug response (left two graphs). Color indicates tissue origin, as in (A). EPZ dose curves of indicated cell lines (right two graphs). (D) Correlation scheme linking cell line EPZ sensitivity to gene expression. (E) GSEA FDR-q values and normalized enrichment scores for Reactome gene sets (left) and examples of GSEA-derived enrichment plots (right). (F) Log odds ratios of the p value for the single gene of each pair most correlated with activity score over the p value of the paired metagene. (G) Correlation between C/R ratio-based biomarker signature and measured activity scores. (H) Schematic of competition between CLNS1A and RIOK1 for PRMT5. (I) Correlation between predicted and measured activity scores. (J) U-87 MG competition assay with indicated shRNAs after 6 days vehicle/10 µM EPZ treatment. Ratio: % shRNA+ cells in drug over vehicle treated cells. Error bars = +/− SEM. See also Figure S5/Table S3.

Article Snippet: They were then transferred onto PVDF membranes and probed with antibodies against Symmetric Di-Methyl Arginine Motif/SDMA (Cell Signaling, #13222, MultiMab rabbit monoclonal antibody mix, 1:1000), HSP90 (BD, #610418, Clone 68, 1:10,000), PRMT5 (Cell Signaling, #2252S, 1:1000), GAPDH (Santa Cruz, #sc-365062, Clone G-9, 1:1000), SOS1 (Cell Signaling, #5890, 1:1000), STAT5B (Santa Cruz, #sc-1656, 1:200), RAF1 (Cell Signaling, #9422, 1:1000), AURKB/AIM1 (BD, #611082, Clone 6, 1:1000), SNRPB (Sigma, #HPA003482, 1:200), SNRPD3 (Sigma, #HPA001170, 1:200), SART3/TIP110 (Bethyl, #A301-521A, 1:10,000), PRP3 (MBL, #D171-3, 1:1000), SNRNP40 (MBL, #RN096PW, 1:1000), anti-H4R3me2s (Abcam, #5823, 1:500) and EIF4E (Cell Signaling, #9742, 1:1000).

Techniques: Expressing, Derivative Assay, Activity Assay, Biomarker Assay, Competitive Binding Assay, shRNA

Journal: Cancer cell

Article Title: Coordinated Splicing of Regulatory Detained Introns within Oncogenic Transcripts Creates an Exploitable Vulnerability in Malignant Glioma

doi: 10.1016/j.ccell.2017.08.018

Figure Lengend Snippet: Clustered, Z-score normalized coding isoform percentages. Enrichment of DIs upregulated upon Prmt5 KO within each cluster is shown as a circle. Circle size: proportional to number of DIs, Significance: Bonferroni-corrected hypergeometric p value. DI expression trends indicated schematically. Network maps display significantly enriched gene sets within the indicated cluster. Functionally related gene sets labeled. See also Figure S8/Table S6.

Article Snippet: They were then transferred onto PVDF membranes and probed with antibodies against Symmetric Di-Methyl Arginine Motif/SDMA (Cell Signaling, #13222, MultiMab rabbit monoclonal antibody mix, 1:1000), HSP90 (BD, #610418, Clone 68, 1:10,000), PRMT5 (Cell Signaling, #2252S, 1:1000), GAPDH (Santa Cruz, #sc-365062, Clone G-9, 1:1000), SOS1 (Cell Signaling, #5890, 1:1000), STAT5B (Santa Cruz, #sc-1656, 1:200), RAF1 (Cell Signaling, #9422, 1:1000), AURKB/AIM1 (BD, #611082, Clone 6, 1:1000), SNRPB (Sigma, #HPA003482, 1:200), SNRPD3 (Sigma, #HPA001170, 1:200), SART3/TIP110 (Bethyl, #A301-521A, 1:10,000), PRP3 (MBL, #D171-3, 1:1000), SNRNP40 (MBL, #RN096PW, 1:1000), anti-H4R3me2s (Abcam, #5823, 1:500) and EIF4E (Cell Signaling, #9742, 1:1000).

Techniques: Expressing, Labeling

Journal: Cancer cell

Article Title: Coordinated Splicing of Regulatory Detained Introns within Oncogenic Transcripts Creates an Exploitable Vulnerability in Malignant Glioma

doi: 10.1016/j.ccell.2017.08.018

Figure Lengend Snippet: (A) In vitro competition assay in U-87 MG cells (n=3) normalized to pre-shRNA induction levels (t=0). (B,C) Competition assay using the U138 (B) or the A172 (C) cell line, n=3/line, assayed 12 days post shRNA induction with Dox. Significance estimated by paired t-tests. (D) Changes in endogenous and ectopic PRMT5 mRNA levels 2 days post shRNA induction. (E) U-87 MG cells with constitutive expression of wild-type, shRNA resistant PRMT5 cDNA and Dox induced expression of shRNAs as indicated, assayed by immunoblot (I. exp. = increased exposure) and competition assay. (F) U-87 MG cells with constitutive expression of PRMT5 mutant cDNA and Dox induced expression of shRNAs as indicated, assayed by immunoblot (left) and competition assay (right). Error bars = +/− SEM. See also Figure S2.

Article Snippet: They were then transferred onto PVDF membranes and probed with antibodies against Symmetric Di-Methyl Arginine Motif/SDMA (Cell Signaling, #13222, MultiMab rabbit monoclonal antibody mix, 1:1000), HSP90 (BD, #610418, Clone 68, 1:10,000), PRMT5 (Cell Signaling, #2252S, 1:1000), GAPDH (Santa Cruz, #sc-365062, Clone G-9, 1:1000), SOS1 (Cell Signaling, #5890, 1:1000), STAT5B (Santa Cruz, #sc-1656, 1:200), RAF1 (Cell Signaling, #9422, 1:1000), AURKB/AIM1 (BD, #611082, Clone 6, 1:1000), SNRPB (Sigma, #HPA003482, 1:200), SNRPD3 (Sigma, #HPA001170, 1:200), SART3/TIP110 (Bethyl, #A301-521A, 1:10,000), PRP3 (MBL, #D171-3, 1:1000), SNRNP40 (MBL, #RN096PW, 1:1000), anti-H4R3me2s (Abcam, #5823, 1:500) and EIF4E (Cell Signaling, #9742, 1:1000).

Techniques: In Vitro, Competitive Binding Assay, shRNA, Expressing, Western Blot, Mutagenesis

Journal: Cancer cell

Article Title: Coordinated Splicing of Regulatory Detained Introns within Oncogenic Transcripts Creates an Exploitable Vulnerability in Malignant Glioma

doi: 10.1016/j.ccell.2017.08.018

Figure Lengend Snippet: (A) Survival plot of C57BL/6J mice following intracranial implantation of Gl261 cells constitutively expressing the indicated shRNA constructs (n=6 per condition). Significance estimated with Log-Rank (Mantel-Cox) test. (B) Intracranial competition assay of U-87 MG cells in nude mice, shRNA induction 5 days post injection. n=5 for REN.713, n=8 for PRMT5.1949. Significance estimated with KS-test. Horizontal lines represent mean. (C, D) Weight (C), and blood (D) profiles of non-tumor bearing C57BL/6J mice dosed with oral EPZ or vehicle. ALT = alanine aminotransferase, WBC = white blood cells, NE = neutrophils, Ly = lymphocytes. (E) Tumor volume over time in mice with subcutaneous (s.c.) U-87 MG tumors treated with vehicle (n=24 tumors/12 mice) or EPZ (n=22 tumors/11 mice). Significance estimated with Mann-Whitney test at the final time point. (F) Survival of mice with intracranial U-87 MG tumors, treated with EPZ (n=9) or vehicle (n=9) for indicated times. Significance: Log-Rank (Mantel-Cox test). (G, H) MALDI-MSI workflow (G, left) and MALDI signal intensity/H&E brain sections of intracranial U-87 MG tumors in nude mice (G, right), and quantification of EPZ concentration (H). (I) Evan’s blue dye extravasation, macroscopic example of dye extravasation with red arrow depicting tumor injection site (left) and Evan’s blue dye intensity in brain samples of nude mice at indicated days post U-87 MG cell injection (right). (J) Volumes of s.c. MGG8 PDX tumors treated with vehicle/EPZ at indicated times, n=10. Significance estimated with Mann-Whitney test at final time point. Error bars: +/− SEM. See also Figure S4.

Article Snippet: They were then transferred onto PVDF membranes and probed with antibodies against Symmetric Di-Methyl Arginine Motif/SDMA (Cell Signaling, #13222, MultiMab rabbit monoclonal antibody mix, 1:1000), HSP90 (BD, #610418, Clone 68, 1:10,000), PRMT5 (Cell Signaling, #2252S, 1:1000), GAPDH (Santa Cruz, #sc-365062, Clone G-9, 1:1000), SOS1 (Cell Signaling, #5890, 1:1000), STAT5B (Santa Cruz, #sc-1656, 1:200), RAF1 (Cell Signaling, #9422, 1:1000), AURKB/AIM1 (BD, #611082, Clone 6, 1:1000), SNRPB (Sigma, #HPA003482, 1:200), SNRPD3 (Sigma, #HPA001170, 1:200), SART3/TIP110 (Bethyl, #A301-521A, 1:10,000), PRP3 (MBL, #D171-3, 1:1000), SNRNP40 (MBL, #RN096PW, 1:1000), anti-H4R3me2s (Abcam, #5823, 1:500) and EIF4E (Cell Signaling, #9742, 1:1000).

Techniques: Expressing, shRNA, Construct, Competitive Binding Assay, Injection, MANN-WHITNEY, Concentration Assay

Journal: Journal of biomedical materials research. Part A

Article Title: PHENOTYPIC EXPRESSION IN HUMAN MONOCYTE-DERIVED INTERLEUKIN-4-INDUCED FOREIGN BODY GIANT CELLS AND MACROPHAGES IN VITRO : DEPENDENCE ON MATERIAL SURFACE PROPERTIES

doi: 10.1002/jbm.a.35280

Figure Lengend Snippet: Antibodies and control cell lysates or proteins for immunoblotting

Article Snippet: Positive control cell lysates or recombinant proteins were employed in all cases to confirm detection, and actin and GAPDH were employed as lysate loading controls ( ) as for our previous studies ( 27 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Function Antibody Source Control (+) CD206 mannose receptor, macrophage fusion Santa Cruz sc-367108 mouse spleen CD98 fusion regulatory protein Santa Cruz sc-7095 HL-60 CD147 cell-cell interactions Santa Cruz sc-374101 HeLa CD13 aminopeptidase, cell-cell interactions Santa Cruz sc-166105 CD13:293T Calnexin ER protein present at fusion interfaces Santa Cruz sc-46669 HeLa TGase-2 protein crosslinking Santa Cruz sc-73612 HeLa Galectin-3 macrophage ‘phagosome’ component Santa Cruz sc-225279 HeLa HLA-DR antigen-presenting molecule Santa Cruz sc-69673 BJAB B7-1 lymphocyte co-stimulator Santa Cruz sc-1633 B7-1:293T B7-2 lymphocyte co-stimulator Santa Cruz sc-19617 B7-2:293T B7-H1 lymphocyte co-stimulator, down-regulatory RnD AF-156 Daudi B7-H2 lymphocyte co-stimulator, up-regulatory RnD AF-165 BJAB CD14 LPS receptor, macrophage marker Santa Cruz sc-58951 CD14:293T CD68 macrophage marker, lysosomal protein Santa Cruz sc-17832 THP-1 CD44 cell-cell interactions Santa Cruz sc-7297 HL-60 Ferritin HC ferritin heavy chain, iron metabolism Santa Cruz sc-376594 HeLa α 2 Mac associated with phagocytic activity Santa Cruz sc-390544 α 2 M:293T TLR-2 PAMP recognition, Gram+, yeast RnD AF-2616 TLR-2:293T TLR-4 PAMP recognition, Gram−, LPS RnD MAB-1478 rhTLR-4 TLR-5 PAMP recognition, bacterial flagellin Santa Cruz sc-16482 rhTLR-5 CD32B IgGRII, B isoform, immune down-regulatory Santa Cruz sc-12815 CD32:293T DC-SIGN PAMP recognition, mannose carbohydrates Santa Cruz sc-74589 DC-SIGN:293T Fascin-1 actin bundling Santa Cruz sc-71100 fascin-1:293T Actin cytoskeletal protein, lysate loading control Santa Cruz sc-1616 GADPH ‘housekeeping’ protein, lysate loading control Santa Cruz sc-166545 Open in a separate window α 2 Mac, α 2 -macroglobulin; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; ER, endoplasmic reticulum; GADPH, glyceraldehyde 3-phosphate dehydrogenase; HLA, human leukocyte antigen; LPS, lipopolysaccharide; PAMP, pathogen-associated molecular pattern; rh, recombinant human, TGase, transglutaminase; TLR, toll-like receptor; 293T, transfected fibroblasts Antibodies and control cell lysates or proteins for immunoblotting

Techniques: Marker, Activity Assay

Journal: Stem Cells International

Article Title: Generation of Human-Induced Pluripotent Stem Cells by a Nonintegrating RNA Sendai Virus Vector in Feeder-Free or Xeno-Free Conditions

doi: 10.1155/2012/564612

Figure Lengend Snippet: (a) TaqMan Gene Expression Assays of OCT4/POU5F1, SOX2, LIN28, and NANOG were compared among the WA09 hESC line, an episomally derived iPSC line (Epi-line 1), and three Sendai-derived iPSC lines (Sendai 1, 3, and 6). Samples were normalized against the endogenous reference B-actin. (b) TaqMan Protein Assays of OCT4/POU5F1, SOX2, LIN28, and NANOG are compared among the WA09 hESC line, an episomally derived iPSC line (Epi-line 1), and three Sendai-derived iPSC lines (Sendai 1, 3, and 6). Samples were normalized against the endogenous reference 18S ribosomal genomic DNA and plotted relative fold change to the expression level of untransduced human dermal fibroblasts.

Article Snippet: Human neonatal fibroblasts (ATCC Cat. no. CRL-2252) were maintained in fibroblast growth media consisting of DMEM + GlutaMAX (Cat. no. 10569-010), supplemented with 10% ES cell-qualified fetal bovine serum (Cat. no. 16141-079), and 1% nonessential amino acids (Cat. no. 11140-050).

Techniques: Gene Expression, Derivative Assay, Expressing

Journal: Stem Cells International

Article Title: Generation of Human-Induced Pluripotent Stem Cells by a Nonintegrating RNA Sendai Virus Vector in Feeder-Free or Xeno-Free Conditions

doi: 10.1155/2012/564612

Figure Lengend Snippet: ((a) and (b)) Scatter plots comparing microRNA expression profiles using the TaqMan Human MicroRNA Array of BJ fibroblasts, iPSCs generated with the Sendai virus vector (Sen-iPSC), and the WA09 human embryonic stem cell (hESC) line. ((c) and (d)) Scatter plots comparing “pluripotent” microRNA expression profiles.

Article Snippet: Human neonatal fibroblasts (ATCC Cat. no. CRL-2252) were maintained in fibroblast growth media consisting of DMEM + GlutaMAX (Cat. no. 10569-010), supplemented with 10% ES cell-qualified fetal bovine serum (Cat. no. 16141-079), and 1% nonessential amino acids (Cat. no. 11140-050).

Techniques: Expressing, Generated, Virus, Plasmid Preparation

Journal: Experimental & Molecular Medicine

Article Title: Z-FL-COCHO, a cathepsin S inhibitor, enhances oxaliplatin-mediated apoptosis through the induction of endoplasmic reticulum stress

doi: 10.1038/s12276-018-0138-6

Figure Lengend Snippet: a Caki cells were treated with 10 ng/mL TNF-α plus 2.5 μg/mL CHX, 500 ng/mL Fas ligand (Fas L), 1 μM doxorubicin, 3 μg/mL etoposide, 250 μΜ 5-FU, 30 μΜ cisplatin, 0.1 μΜ gefitinib, and 5 μΜ sorafenib in the presence or absence of 2 μM ZFL for 24 h. b – e Caki cells were treated with 25 μM oxaliplatin in the presence or absence of 2 μM ZFL for 24 h. We analyzed cell morphology using interference light microscopy and nuclei condensation and fragmentation using DAPI staining ( c ). Cytoplasmic DNA fragments ( d ) and caspase activity ( e ) were detected using a kit, as described in the Materials and methods section. f , g Caki cells were treated with 2 μM ZFL plus 25 μM oxaliplatin in the presence or absence of 20 μM z-VAD-fmk (z-VAD) for 24 h. Flow cytometry was used to detect the sub-G1 fraction, and western blotting was used to detect the protein levels of PARP, pro-caspase-3, cleaved-caspase-3, and/or actin. The values in ( a , b , d , e , f ) represent the mean ± SD of three independent samples; * p < 0.01 compared to the control; # p < 0.01 compared to ZFL plus oxaliplatin

Article Snippet: The doxorubicin was purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Light Microscopy, Staining, Activity Assay, Flow Cytometry, Western Blot, Control