Journal: Oncotarget

Article Title: TRAF3IP2, a novel therapeutic target in glioblastoma multiforme

doi: 10.18632/oncotarget.25710

Figure Lengend Snippet: ( A ) TRAF3IP2 expression (brown) was localized by IHC. Hematoxylin was used as a counterstain (blue). Images representing glioblastoma tumor tissues from ten independent subjects are shown (5 females and 5 males, age of each subject is indicated on the image). The right panels show the images representing lack of TRAF3IP2 expression in adjacent non-tumor tissues. Scale bar, 100 μm. ( B ) TRAF3IP2 knockdown in U87 and U118 cells. TRAF3IP2 mRNA expression in U118, U118 control shRNA , U87, U87 control shRNA , U118 TRAF3IP2 KD , U87 TRAF3IP2 KD , and SVG p12 cells was analyzed by RT-qPCR. Results were normalized to values obtained in U87 and U118 cells respectively ( n = 9/cell type; P < 0.05). ( C ) Western blot analysis of TRAF3IP2 expression in U87 TRAF3IP2 KD and U87 control shRNA cells. ( D ) Immunofluorescent detection of GFP (green) and TRAF3IP2 (red) in U87 TRAF3IP2 KD (top panels) and U87 control shRNA cells (bottom panels), counterstained with DAPI (blue) to visualize nuclei. Scale bar, 100 µm. ( E ) Effect of silencing TRAF3IP2 on sphere forming ability of U87 TRAF3IP2 KD , U118 TRAF3IP2 KD , U87 control shRNA , U118 control shRNA . Cells were incubated in sphere media for up to 96 hours. 20 spheroids/cell type were randomly selected for measurement at 24 and 96h time points. The spheres were imaged using a Nikon microscope. Spheroid diameters were measured using a microscope, and volumes computed ( * P < 0.05; ** P < 0.01). ( F ) Analysis of U87 TRAF3IP2 KD and U87 control shRNA cell proliferation by XTT assay. Statistically significant differences at every time point; ** P < 0.01; *** P < 0.001. ( G ) Silencing TRAF3IP2 alters cell morphology. Morphology of U87 TRAF3IP2 KD and U87 control shRNA cells analyzed by uranyl acetate staining and viewed under electron microscopy (scale bar represents 500 nm). ( H ) Silencing TRAF3IP2 alters cell cycle profile. Mean and SEM of relative numbers of cells in G0/G1, S-Phase and G2/M phase of U87 TRAF3IP2 KD and U87 control shRNA cells ( * P < 0.05; *** P < 0.001; **** P < 0.0001, n = 18).

Article Snippet: Human malignant glioblastoma cell lines U87 and U118, and non-malignant SVG p12 were purchased from ATCC (Rockville, MD, USA).

Techniques: Expressing, Knockdown, Control, shRNA, Quantitative RT-PCR, Western Blot, Incubation, Microscopy, XTT Assay, Staining, Electron Microscopy

Journal: Oncotarget

Article Title: TRAF3IP2, a novel therapeutic target in glioblastoma multiforme

doi: 10.18632/oncotarget.25710

Figure Lengend Snippet: ( A ) Hierarchical clustering displayed genes differentially expressed in U87 TRAF3IP2 KD and U87 control shRNA cells. The extent of blue (decreased fold change) or red (increased fold change) color is directly proportional to the magnitude of differential expression of these genes. ( B ) Reactome ( http://reactome.org/ ) was used for Gene Ontology Tree, representing functional characterization of genes differentially expressed in U87 TRAF3IP2 KD cells and U87 control shRNA cells in comparition to the number (and significance) of gene ontologies and shows a dendrogram comparison of gene ontology (biological process) specifically differential between U87 TRAF3IP2 KD cells and U87 control shRNA cells. Yellow, ontologies enriched in U87 TRAF3IP2 KD cells; gray, ontologies not affected in U87 TRAF3IP2 KD cells. Inset, the top four biological processes hit. Of particular interest is the specific and significant enrichment of proteins involved in cell cycle, DNA replication, immune system, programmed cell death, cellular responses to external stimuli, extracellular matric organization, DNA repair and metabolism. ( C ) Pathway analysis (using Reactome) of a cluster of 1297 perturbed gene expressions in U87 TRAF3IP2 KD cells revealed a statistically significant preponderance of genes involved in cell cycle, metabolism, apoptosis, angiogenesis, immune system, aging, extracellular matrix organization, and cytokine-cytokine interaction. The chart displays genes representative of each pathway displaying greater than 5-fold change in U87 TRAF3IP2 KD versus U87 control shRNA cells ( P < 0.05). ( D ) Fold change expression of perturbed genes involved in angiogenesis in U87 TRAF3IP2 KD versus U87 control shRNA cells ( P < 0.05).

Article Snippet: Human malignant glioblastoma cell lines U87 and U118, and non-malignant SVG p12 were purchased from ATCC (Rockville, MD, USA).

Techniques: Control, shRNA, Quantitative Proteomics, Functional Assay, Comparison, Expressing

Journal: Oncotarget

Article Title: TRAF3IP2, a novel therapeutic target in glioblastoma multiforme

doi: 10.18632/oncotarget.25710

Figure Lengend Snippet: ( A ) p-p65 levels were analyzed by ELISA. Silencing TRAF3IP2 inhibits TNF-α or TNF-α+IL-17-induced p-p65 levels in U87 TRAF3IP2KD and U87 control shRNA cells. ( B ) Western blot analysis demonstrating significantly reduced p-p65 levels in U87 TRAF3IP2 KD versus U87 control shRNA cells. U87 TRAF3IP2 KD and U87 control shRNA cells showed higher expression of p-p65 after TNF-α treatment. However, the magnitude of increase is less in U87 TRAF3IP2KD cells. ( C and D ) RT 2 -qPCR analysis: fold changes of U87 TRAF3IP2KD versus U87 control shRNA cells for IL-8, IL-1β, IL-6, IL-10, CCND1 and VEGF. ( E ) Dot-blot comparative protein analysis of conditioned media from U87 TRAF3IP2KD cells showing decreased expression of G-CSF, GM-CSF, GRO, IL-6, IL-8, MCP-1 and MIF compared to U87 control shRNA cells. Protein levels of G-CSF, GM-CSF, GRO, IL-6, IL-8 and MCP-1 were below the detection limit in U87 TRAF3IP2KD cells conditioned media. ( F ) RT-qPCR analysis: fold changes expression of upstream (IL-17R) and downstream (NF-κB, IL-1β, IL-6, and IL-8, in addition to VEGF) signaling of TRAF3IP2 in U87 spheroids compared to adherent U87 cultures. ( G ) Gene expression analysis of U87 TRAF3IP2KD versus U87 control shRNA spheroids ( n = 6; * P < 0.05, ** P < 0.001, *** P < 0.0001).

Article Snippet: Human malignant glioblastoma cell lines U87 and U118, and non-malignant SVG p12 were purchased from ATCC (Rockville, MD, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Control, shRNA, Western Blot, Expressing, Dot Blot, Quantitative RT-PCR, Gene Expression

Journal: Oncotarget

Article Title: TRAF3IP2, a novel therapeutic target in glioblastoma multiforme

doi: 10.18632/oncotarget.25710

Figure Lengend Snippet: ( A ) Immunodeficient NIH-III mice were injected with U87 TRAF3IP2KD cells (1 × 10 6 cells) into the flank region. Control animals were injected with U87 control shRNA cells (1 × 10 6 cells). Tumor size was measured weekly using calipers. ( B ) U87 TRAF3IP2KD cells formed smaller tumors. ( C ) Immunohistochemical localization of TRAF3IP2, IL-8, and VEGF in tumors derived from U87 TRAF3IP2KD and U87 control shRNA cells. Scale: 100 µm.

Article Snippet: Human malignant glioblastoma cell lines U87 and U118, and non-malignant SVG p12 were purchased from ATCC (Rockville, MD, USA).

Techniques: Injection, Control, shRNA, Immunohistochemical staining, Derivative Assay

Journal: Oncotarget

Article Title: TRAF3IP2, a novel therapeutic target in glioblastoma multiforme

doi: 10.18632/oncotarget.25710

Figure Lengend Snippet: ( A ) Suppression of glioblastoma tumors by TRAF3IP2 shRNA-LV injected subcutaneously onto tumors compared to scrambled shRNA-LV injected tumors. Frequency of administration is shown in the graph. ( B ) Tumor size was measured biweekly ( * P < 0.05; ** P < 0.001). ( C ) Animals imaged for luciferase weekly. Immunohistochemical localization of TRAF3IP2, caspase 8, Ki67, IL-8, and VEGF in tumors treated with TRAF3IP2 shRNA-LV or scrambled shRNA-LV. Scale: 100 µm.

Article Snippet: Human malignant glioblastoma cell lines U87 and U118, and non-malignant SVG p12 were purchased from ATCC (Rockville, MD, USA).

Techniques: shRNA, Injection, Luciferase, Immunohistochemical staining

Journal: Oncotarget

Article Title: TRAF3IP2, a novel therapeutic target in glioblastoma multiforme

doi: 10.18632/oncotarget.25710

Figure Lengend Snippet: ( A ) Clustergram of genes analyzed by RT 2 -based PCR array. Xenograft glioblastoma tumors treated with TRAF3IP2 shRNA-LV were compared with scrambled shRNA-LV treated tumors ( n = 3/group). ( B ) Clustering performed using data analysis software (Qiagen). Intensity of green (decreased fold change) or red (increased fold change) is directly proportional to the magnitude of differentially expressed genes. Expression of genes displaying a +/− 2-fold change in TRAF3IP2 shRNA-LV-treated xenograft tumors. Values normalized to scrambled shRNA-LV treated tumors ( * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Human malignant glioblastoma cell lines U87 and U118, and non-malignant SVG p12 were purchased from ATCC (Rockville, MD, USA).

Techniques: shRNA, Software, Expressing

Journal: Oncotarget

Article Title: TRAF3IP2, a novel therapeutic target in glioblastoma multiforme

doi: 10.18632/oncotarget.25710

Figure Lengend Snippet: In addition to blocking inflammation (green line), our novel findings show that silencing TRAF3IP2 inhibits cell cycle progression, angiogenesis, cell metabolism, and matrix metalloproteinase expression, while increasing apoptosis of glioblastoma cells (orange lines), resulting ultimately in tumor regression, and possibly elimination.

Article Snippet: Human malignant glioblastoma cell lines U87 and U118, and non-malignant SVG p12 were purchased from ATCC (Rockville, MD, USA).

Techniques: Blocking Assay, Expressing

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Characterization of Pttg2-depleted HCT116 cells. ( a ) Schematic diagram showing the sequences and location of shPttg1 and shPttg2. Multiple sequence alignments were made using the Clustal W software ( www.clustal.org ). ( b ) Relative quantification of Pttg1 and Pttg2 mRNA levels in Pttg1 and Pttg2-silenced HCT116 wild-type and HCT116 pttg1 −/− cells by real-time PCR. Expression of each gene is shown as mean±S.E.M. of three independent experiments. Data are shown as 2 −ΔCT normalized to HPRT1. ( c ) Analysis of PTTG1 and PTTG2 protein levels 72 h after shRNA-Pttg1 or shRNA-Pttg2 treatment in HCT116 wild-type and HCT116 pttg1 −/− cells. β -Actin was used as loading control. Densitometric analysis for western blots of PTTG1 and PTTG2 protein expression (% of control) in control and shRNA-treated (shPttg1 or shPttg2) wild-type and Pttg1 −/− HCT116 cells

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Sequencing, Software, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Expressing, shRNA, Control, Western Blot

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Pttg1 and Pttg2 show different biochemical properties. ( a ) PTTG2 does not interact with Separase. Extracts of HEK293T cells expressing N-terminally Flag-tagged separase, C-terminally Myc-tagged PTTG2, or both were used for immunoprecipitation with anti-Myc antibodies. The immunoprecipitates were analyzed by immunoblotting with anti-FLAG and anti-Myc antibodies. The asterisks indicate the immunoglobulin light chains. As positive control, the interaction between PTTG1 and separase was tested using Flag-separase and Myc-tagged PTTG1. ( b ) PTTG2 lacks transactivation capacity. The ORF of Pttg2 was fused to the GAL4 DNA-binding domain (pGAL4-DBD-Pttg2) and tested for transcription activation in HEK293T cells cotransfected with a GAL4 site-dependent reporter plasmid driving expression of the luciferase gene (pGAL4-4RE-Luc). pGAL4-PTTG1 and pGAL4-VP16 were used as positive controls. Luciferase values from at least three independent transfections are plotted as fold activation over control

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Expressing, Immunoprecipitation, Western Blot, Positive Control, Binding Assay, Activation Assay, Plasmid Preparation, Luciferase, Transfection, Control

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Silencing of Pttg2 impairs cell adhesion and proliferative capacity of HCT116 cells. ( a ) Phase-contrast images ( × 10 objective) of Pttg2-silenced HCT116 cells 72 h (upper panels) and 1 week post-infection (PI) (lower panels). Inserts show close-up views of representative cell culture areas. Pttg2-depleted cells show rounder morphology than control cells, indicating a defect in cell adhesion properties. ( b ) Spheroid formation of Pttg2 and Pttg1-silenced HCT116 (wt) cells in suspension. Seventy-two hours post-infection, cells were transferred to poly-HEMA-coated plates at 6 × 10 4 cells/ml. After 48 h in suspension, cells were photographed at 10 × . Control and shPttg1-treated cells formed dense spheroids whereas shPttg2-silenced cells adhere to each other very weakly. Representative areas of each culture were selected. ( c ) Percentage of BrdU-positive cells after shRNA-Pttg2 treatment. HCT116 cells were infected and assayed for BrdU incorporation 72 h post-infection. Bars represent the mean±S.E.M. ( d ) Analysis of mitotic index in Pttg2-depleted HCT116 cells. Cells were treated with shRNA-Pttg2 lentivirus and incubated with nocodazole 1 μ M 72 h post-infection, for 24 h. Percentage of H3P-positive (mitotic) cells was measured by flow cytometry. Bars represent the mean±S.E.M. ( e ) Percentage of cell death following shRNA Pttg2 or shRNA Pttg1 treatment of HCT116 cells growing under adherent or suspension conditions. Seventy-two hours post-infection, cells were transferred onto control or poly-HEMA-coated (PH) plates in the absence or presence of methylcellulose (MC) and the levels of apoptosis was determined 48 h later by measuring the percentage of cells containing a subG1 DNA content by flow cytometry. Results represent the means of three independent experiments±S.E.M. ( f ) p21 induction in Pttg2-depleted HCT116 cells. HCT116 cells treated with shRNA-Pttg2 lentivirus for 72 h were harvested and the levels of p21 determined by immunoblotting using specific p21 antibodies. β -Actin was used as loading control

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Infection, Cell Culture, Control, Suspension, shRNA, BrdU Incorporation Assay, Incubation, Flow Cytometry, Western Blot

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Defective adhesion in Pttg2-depleted HCT116 cells results in p53- and p21-dependent apoptosis. ( a ) Phase-contrast images of Pttg2-silenced HCT116 cells after z-VAD treatment. A round morphology was still observed in Pttg2-depleted cells, suggesting a defective cell adhesion. ( b ) Apoptosis can be rescued in Pttg2-silenced HCT116 cells after caspase inhibition treatment. The percentage of apoptotic cells (subG1) after PTTG2 depletion was drastically reduced after treatment with the caspase inhibitor z-VAD. Bars represent the mean±S.E.M. ( c ) Levels of apoptotic indicators in Pttg2-silenced HCT116 cells. Cells were harvested after 72 h post-infection and assayed for p53, p21, BIM and cleaved-Capase 3 levels by western blot using specific antibodies. β -Actin was used as loading control. ( d , e ) Role of p53 in Pttg2-dependent anoikis. ( d ) Phase-contrast images of Pttg2-depleted HCT116 p53 −/− cells. The absence of PTTG2 in HCT116 p53 −/− results in cell rounding similar to wild-type HCT116 cells. ( e ) FACS analysis of Pttg2-depleted HCT116 p53 −/− cells. In the absence of PTTG2, the percentage of apoptotic cells (subG1) was partially reduced in HCT116 p53 −/− cells compared with HCT116 wild-type cells. Bars represent the mean±S.E.M. ( f ) The induction of p21 in Pttg2-treated cells occurs in a p53-dependent manner. Western blot analysis of p21 levels in HCT116 p53 −/− cells 72 h post-infection with shPttg2 or control lentivirus. β -Actin was used as loading control. ( g ) Role of p21 in anoikis in Pttg2-depleted cells. The percentage of apoptotic cells in HCT116 p21 −/− following treatment with shPttg2 was significantly reduced compared with HCT116 wild-type cells. Bars represent the mean±S.E.M.

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Inhibition, Infection, Western Blot, Control

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Loss of cell adhesion in shPttg2-treated cells precedes the onset of apoptosis. ( a ) Spheroid formation of Pttg2-depleted cells after z-VAD treatment. Wild-type HCT116 cells were infected for 72 h in the presence or absence of the caspases inhibitor z-VAD and the cell–cell adhesion capacity assayed on poly-HEMA (PH)-coated plates. The experiments were repeated three times, and representative areas of each culture are shown. ( b ) Cell-cycle profile of Pttg2-silenced HCT116 cells growing under poly-HEMA conditions after z-VAD treatment. The percentage of apoptotic cells (subG1) was significantly reduced in the presence of z-VAD, whereas the other cell-cycle phases (G1, S and G2/M) were not modified. Data are representative of two independent experiments. ( c ) Phase-contrast images to assess multicellular aggregates formation in shPttg2-treated HCT116 p53 −/− cells growing on poly-HEMA-coated plates. Images show a representative region of each condition. ( d ) Analysis of the cell-cycle profile of Pttg2-depleted HCT116 p53 −/− cells growing under poly-HEMA conditions. ShPttg2-treated cells lacking p53 showed a reduced percentage of cell death (subG1). The other cell-cycle phases (G1, S and G2/M) remained unaltered. Data are representative of two independent experiments

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Infection, Modification

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Pttg2-depleted cells exhibit cytoskeletal defects. Microtubule cytoskeleton of PTTG2 depleted cells. Confocal images of Pttg2-silenced HCT116 cells stably expressing Histone H2B (H2B)-cherry fusion gene. Seventy-two hours post-infection, cells were fixed with methanol at −20 °C and stained for α -tubulin (green). Scale bars, 25 μ m

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Stable Transfection, Expressing, Infection, Staining

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Pttg2 depletion results in deregulated E-cadherin and vimentin levels and induction of the epithelial-to-mesenchymal transition. ( a ) Summary of gene expression profile according to Gene Ontology. Pie charts show the percentage of genes included in each category (molecular function, biological process and cellular component) and subcategories with a P -value <0.01. The GO analysis was performed using DAVID v.6.7 program. GO analysis of differentially upregulated or downregulated genes are shown in upper and lower panels, respectively. ( b ) Validation of E-Cadherin downregulation in Pttg2-silenced cells by quantitative PCR. HCT116 cells were infected with shPttg2 or control lentivirus and assayed for E-cadherin mRNA levels 72 h post-infection. Values were normalized using the hprt1 gene as an internal control. Columns represent the mean values of 2–3 independent experiments ±S.E.M. ( c ) Western blot analysis of E-Cadherin repression in Pttg2-depleted HCT116 cells. The expression levels of the E-Cadherin protein in shPttg2 cells was compared with control cells expressing an empty lentiviral plasmid. Cells were grown either as a monolayer (attached) or in suspension for 48 h on poly-HEMA-coated plates (+PH) and total lysates analyzed by immunoblotting. ( d ) Vimentin mRNA induction in shPttg2-treated cells. HCT116 cells were infected with shPttg2 or control lentivirus and assayed for vimentin levels 72 h post-infection by quantitative PCR. Values were normalized using the hprt1 gene as an internal control. Columns represent the mean values of 2–3 independent experiments±S.E.M.

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Gene Expression, Biomarker Discovery, Real-time Polymerase Chain Reaction, Infection, Control, Western Blot, Expressing, Plasmid Preparation, Suspension

Journal: Nature Communications

Article Title: PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma

doi: 10.1038/s41467-020-15959-6

Figure Lengend Snippet: a Pbrm1 knockout validation in Renca cells at protein levels by western blot, and b at mRNA levels by real-time PCR. Renca cell were treated with or without 1 ng/ml IFNγ for 8 h. c IFNγ-induced JAK-STAT1 expression and phosphorylation in Renca cells. Control KO or Pbrm1 KO (clone #18) Renca cells were treated with 1 ng/ml IFNγ for 2 or 8 h. Cell lysates were analyzed by immunoblot using antibodies against PBRM1, STAT1, P-STA1 Y701, P-STAT1 S727, JAK2, P-JAK2 Y1007/1008, JAK1, P-JAK1 Y1034/1035, IRF1. β-actin was used an internal control. d IFNγ-induced gene expression in Renca cells. Control KO or Pbrm1 KO (clone #18) Renca cells were treated with 1 ng ml IFNγ for 8 h. mRNA expression of Stat1 , Cxcl9 , Irf1 , and Icam1 were detected by real-time PCR. Gapdh was used as internal control. e IFNγ-induced CXCL9 secretion. Renca cells were cultured in serum-free medium and treated with 1 ng/ml IFNγ for 4 or 10 h. The concentration of CXCL9 was analyzed using Quantikine® ELISA kit. f IFNγ-induced JAK-STAT1 expression and phosphorylation in 786-O cells. Control knockdown (Con KD) or PBRM1 knockdown ( PBRM1 KD) 786-O cells were treated with or without 10 ng/ml IFNγ for 2 h. Cell lysates were analyzed by immunoblot using antibodies against PBRM1, STAT1, P-STA1 Y701, JAK2, P-JAK2 Y1007/1008, and IRF1. β-actin was used an internal control. g IFNγ-induced gene expression in 786-O cells. 786-O cells were cultured in DMEM with 10% FBS, and treated with 10 ng/ml IFNγ for 8 h. mRNA expression of STAT1 , CXCL9 , and IRF1 were detected by real-time PCR. GAPDH was used as internal control. Unpaired t -test was performed with GraphPad Prism 7.03. * P < 0.05 and ** P < 0.001, compared with control knockout or knockdown cells. All data are representative of three independent experiments. Data in the bar graphs represent mean ± S.D., n = 3. Source data are provided as a Source Data file.

Article Snippet: PBRM1 antibody (A301-591A) was from Bethyl Laboratories.

Techniques: Knock-Out, Biomarker Discovery, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Phospho-proteomics, Control, Gene Expression, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: Nature Communications

Article Title: PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma

doi: 10.1038/s41467-020-15959-6

Figure Lengend Snippet: a BRG1 and STAT1 binding to Cxcl9 and Cxcl10 promoter in Renca cells. b BRG1 and SP1 binding to Ifngr2 promoter. Chromatin immunoprecipitation (ChIP) with BRG1, STAT1 or SP1 antibody as indicated in figures was performed using SimpleChIP® Plus Enzymatic Chromatin IP Kit. Isotype IgG was used as negative control. Immunoprecipitated DNA was amplified and quantified by real-time PCR. Protein relative occupancy on promoter was expressed as a percent of the total input chromatin. Con KO or Pbrm1 KO Renca cells were treated with 1 ng/ml IFNγ for 2 h for IFNγ-induced STAT1 binding to Cxcl9 or Cxcl10 promoter. c IFNγ receptor subunits, Ifngr1 and Ifngr2 , mRNA expression detected by real-time PCR. Gapdh was used as internal control. d IFNγ receptor subunits, IFNGR1 and IFNGR2, protein expression was detected by western blot. β-actin was used an internal control. e IFNGR2 membrane expression was detected by flow cytometry. Unpaired t -test was performed with GraphPad Prism 7.03. * P < 0.05 and ** P < 0.001, compared with control knockout cells. Data in the bar graphs represent mean ± S.D. Source data are provided as a Source Data file.

Article Snippet: PBRM1 antibody (A301-591A) was from Bethyl Laboratories.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Negative Control, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, Membrane, Flow Cytometry, Knock-Out

Journal: Nature Communications

Article Title: PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma

doi: 10.1038/s41467-020-15959-6

Figure Lengend Snippet: a The confidence interval plots represented the differences in these genes between the two groups ( Pbrm1 KO minus Pbrm1 WT). The x -axis is the difference in group means of Pbrm1 knockout group minus Pbrm1 wild-type group, so negative values correspond to genes downregulated in the Pbrm1 knockout group. The lines depicted 95% confidence intervals, and genes with a significant difference between the groups (adjusted P -value <0.05) were marked in orange. The P -values were obtained from a two-sample t-test on the log2-transformed values, and the resulting P -values were adjusted for multiple comparisons using the Benjamini–Hochberg method across the gene set. b mRNA expressions of Ifng , Cxcl9 , Cxcl10 , and Pdcd1 and c the mRNA expression of Pbrm1 , Ifngr1 , Ifngr2 , and Cd274 were detected by real-time PCR. Each dot represents the mean value of triplicated tumor samples. Unpaired t -test was performed with GraphPad Prism 7.03. d The coordinated differences of the genes as listed in ( a ) across the two groups assessed by gene set enrichment analysis (GSEA). e T cell infiltration and quantification. Murine Renca tumor microarrays, with triplicate formalin-fixed tissue cores for each case, were immunohistochemically stained with antibodies against CD3, CD8, CD4, PD-1, and P-STAT1 Y701. The percentages of positively stained cells were analyzed using inForm software. Unpaired t-test was performed with GraphPad Prism 7.03. Scale bar, 100 µm. f Multiplex Opal Immunofluorescence staining. The slides were stained with primary antibodies against CD8 and PD-1, corresponding HRP conjugated secondary antibodies, and subsequently TSA dyes to generate Opal signal (CD8, 520 nm; PD-1, 570 nm). PD-1 Opal signals are artificially colored as red. Scale bar, 50 µm. g Immunomodulatory gene expression signature score in pre-malignant murine kidneys following loss of Vhl alone ( Vhl −/− ), or Vhl in combination with Pbrm1 ( Vhl −/− Pbrm1 −/− ). Student t -test. h Gene expression-based inference of total T cell and CD8 T-cell infiltrates in pre-malignant murine kidneys following loss of Vhl alone, or Vhl in combination with Pbrm1 . Student t -test. Data in the graphs represent mean ± S.D. Source data are provided as a Source Data file.

Article Snippet: PBRM1 antibody (A301-591A) was from Bethyl Laboratories.

Techniques: Knock-Out, Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Staining, Software, Multiplex Assay, Immunofluorescence, Gene Expression

Journal: Nature Communications

Article Title: PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma

doi: 10.1038/s41467-020-15959-6

Figure Lengend Snippet: a The confidence interval plots represented the differences in immunomodulatory genes between the two groups (mutant PBRM1 minus wild-type PBRM1 ) in TCGA KIRC dataset. MUT, mutant PBRM1 ; WT, wild-type PBRM1 . The lines depicted 95% confidence intervals, and genes with a significant difference between the groups (adjusted P -value <0.05) were marked in orange. The P -values were obtained from a two-sample t -test on the log2-transformed values, and the resulting P -values were adjusted for multiple comparisons using the Benjamini–Hochberg method across the gene set. b Comparison of gene expression in tumors with mutant PBRM1 (MUT) versus wild-type PBRM1 (WT) by GSEA using the immunomodulatory genes as listed in ( a ) and other predefined immune-related gene sets in the TCGA, IMmotion150 and ICGC patient cohorts. c Comparison of CD8 infiltrates as assessed by immunohistochemistry for CD8 in the IMmotion150 patient cohort. Rank-sum test. d CD8 immunostaining levels positively correlated with gene expression-based inference. Spearman correlation and associated P -value inset. e Gene expression-based inference of CD8 T cell infiltrates in patients stratified by PBRM1 mutation status. Rank-sum test. f Comparison of immune cell PD-L1 expression in patients from the IMmotion150 cohort stratified by PBRM1 mutation status. Cochran–Armitage test for trend. g Treatment naive RCC tumor microarray from 20 untreated RCC patients, including 15 samples with wild-type PBRM1 and 5 with PBRM1 mutations, were immunohistochemically stained with antibodies against CD3, CD45RO, CD8, and CD4. Each tumor was triplicated, and the n values indicate the number of the intact cores. The percentages of positively stained cells were analyzed using inForm software. Non-parametric Mann–Whitney test was performed with GraphPad Prism 7.03. Scale bar, 100 µm. h Multiplex Opal immunofluorescence staining. The slides were stained with primary antibodies against CD8 and PD-1, corresponding HRP conjugated secondary antibodies, and subsequently TSA dyes to generate Opal signal (CD8, 520 nm; PD-1, 620 nm). Scale bar, 50 µm. Data in the graphs represent mean ± S.D. Source data are provided as a Source Data file.

Article Snippet: PBRM1 antibody (A301-591A) was from Bethyl Laboratories.

Techniques: Mutagenesis, Transformation Assay, Comparison, Gene Expression, Immunohistochemistry, Immunostaining, Expressing, Microarray, Staining, Software, MANN-WHITNEY, Multiplex Assay, Immunofluorescence

Journal: Nature Communications

Article Title: PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma

doi: 10.1038/s41467-020-15959-6

Figure Lengend Snippet: a Correlogram between the expression of immunomodulatory and angiogenic genes IMmotion 150 cohort (top line of each square), TCGA cohort (middle line of each square) and ICGC cohort (bottom line of each square). b Patients with mutant PBRM1 demonstrated increased CD31 immunostaining levels in the IMmotion150 cohort. Rank-sum test. c CD31 immunostaining levels positively correlated with angiogenesis expression score. Spearman correlation and associated P -value inset ( N = 119). d PBRM1 mutated tumors were associated with increased angiogenesis score in 3 indicated patient cohorts. Rank-sum test. e Pbrm1 knockout was associated with increased angiogenesis score in pre-malignant murine kidneys. Student t -test. f Pbrm1 knockout Renca tumors demonstrated increased CD31 immunostaining levels. Student t -test. Data in the bar graphs represent mean ± S.D. Source data are provided as a Source Data file.

Article Snippet: PBRM1 antibody (A301-591A) was from Bethyl Laboratories.

Techniques: Expressing, Mutagenesis, Immunostaining, Knock-Out

Journal: Nature Communications

Article Title: PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma

doi: 10.1038/s41467-020-15959-6

Figure Lengend Snippet: a Early treatment with anti-PD-1 blockade. PD-1 antibodies were administrated at day 3, day 6, and day 9 after tumor inoculation. First dose was 400 µg/mouse, and the following two doses were 200 µg/mouse. b Delayed treatment with anti-PD-1 blockade. Anti-PD-1 antibody (200 µg/mouse) was administrated every third day once the tumors reached 100-200 mm 3 . Treatment schemas, In vivo tumor growth rates and survival rates of mice are shown. Two-way ANOVA and Log-rank (Mantel-Cox) analyses were performed with GraphPad Prism 7.03. Data in the graphs are means ± SEM. UnTX, untreated control. ns, P > 0.05, * P < 0.05, and ** P < 0.001. c Patient response in the IMmotion150 cohort following treatment with either atezolizumab (Atezo) or atezolizumab in combination with bevacizumab (Atezo + Bev). Mono, PBRM1 mutation only, Dual, PBRM1 mutation in combination with a BAP1 or SETD2 mutation; WT, wild type; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. Cochran–Mantel–Haenszel test. d Comparison of patient response rate, defined as either a complete or partial response, in the IMmotion150 cohort. Cochran–Mantel–Haenszel test. e Overall survival of RCC patients treated with ICB from the MSKCC IMPACT cohort stratified by PBRM1 status. Log-rank test. f Overall survival of ccRCC patients from TCGA stratified by PBRM1 mutation status. Log-rank test. g Model for PBRM1 mediated IFNγ-STAT1 signaling and tumor immune microenvironment modulating. PBRM1 ensures IFNγ-induced STAT1 activity and autonomous expression of downstream genes involved in T-cell recruitment (e.g., CXCL9 ). Infiltrating, activated T cells in turn produce more IFNγ which stimulates tumor cells to secrete immunostimulatory chemokines and cytokines, and in parallel upregulate checkpoint pathways on T cells and tumors cells. Thus, the immunogenic TME of PBRM1 proficient tumors is primed to respond to ICB. On the other hand, PBRM1 loss reduces IFNγ-STAT1 signaling and downstream T cell attracting factors, which prevents T cell infiltration and IFNγ secretion. Such a non-immunogenic TME of PBRM1 mutated tumors blunts ICB response. Source data are provided as a Source Data file.

Article Snippet: PBRM1 antibody (A301-591A) was from Bethyl Laboratories.

Techniques: In Vivo, Control, Mutagenesis, Comparison, Activity Assay, Expressing

Journal: Cell Stem Cell

Article Title: Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis

doi: 10.1016/j.stem.2020.11.004

Figure Lengend Snippet: MSCs are fibrosis-driving cells in patients characterized by upregulation of S100A8/A9 (A) Diagnostic BM images of the patients. Representative H&E and reticulin stainings. For additional images (all controls) and detailed patient characteristics, see Figure S5 . (B) UMAP of cells in 1 PMF patient (MF2, n = 243 cells) and two control patients (MF0, n = 255 cells). In the left panel, cells are color coded by their annotated cellular identity, and in the right panel, by their patient source. (C) Top marker genes. Wilcoxon rank-sum test, p < 0.01. (D) Ridgeline plot comparing PMF (blue) versus control (red) condition. Competitive gene set enrichment analysis was used. (E) PROGENy analysis. Sampling-based permutation (10,000 permutations). Pathway activity scores are given as Z scores. (F) Ridgeline plot of S100A8/A9 expression in PMF (blue) or control (red). Significance estimated by modeling the dropout rate as a binomial process with the observed dropout rate per condition as estimator of p for both conditions, respectively. (G) Network plot of ligand-receptor activity in PMF compared to control. (H) Bar plot of top 10 most abundant ligands in all inferred ligand-receptor interactions. (I) Sankey plot of top 20 deregulated TGFB1 -mediated ligand-receptor interactions. The absolute difference in mean LR expression was used as a metric for the extent of deregulation. (J) Sankey plot of top 20 deregulated ligand-receptor interactions mediated by PF4, PF4V1, or PPBP. The absolute difference in mean LR expression was used as a metric for the extent of deregulation. See also Figure S6 and , , , , and .

Article Snippet: Samples were diluted 1:50 - 1:150 and S100A8 concentration was quantified using the Human S100A8 DuoSet ELISA (R&D Systems, DY4570-05) according to the manufacturer’s instructions.

Techniques: Diagnostic Assay, Control, Marker, Sampling, Activity Assay, Expressing

Journal: Cell Stem Cell

Article Title: Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis

doi: 10.1016/j.stem.2020.11.004

Figure Lengend Snippet: Spatial kinetics of S100A8/S100A9 detects disease progression in MPN and their pharmacological targeting ameliorates the disease (A) ELISA of S100A8 (and S100A9) in MPN (blue) and controls (red) plasma. Two-tailed, two-sample Welch test was used. (B) ELISA of S100A8 (and S100A9) in MPN with different MF grades (blue) and controls (red) plasma. Mean ± SEM. One-way-ANOVA with post hoc Tukey’s was used. (C) Frequency of S100A8 + cells BM biopsies; n = 64 patients. One-way-ANOVA with post hoc Tukey’s HSD was used. (D and E) Grading of S100A8 in the non-hematopoietic compartment in BM biopsies. Scale bar, 100 μm. n = 64 patients. Kruskal-Wallis H test with post hoc Wilcoxon rank-sum test was used. p values were adjusted for multiple hypothesis testing by the Holm-Bonferroni method. (F) White blood cell counts of WT mice transplanted with either JAK2 V617F (blue) or JAK2 WT overexpressing HSPCs (red) each either treated with Tasquinimod 30 mg/kg/day or vehicle control. Two-way repeated ANOVA pairwise comparisons were analyzed by estimated marginal means. (G) Spleens at sacrifice as indicated. (H) Relative spleen weights. Mean ± SEM. One-way-ANOVA with post hoc Tukey’s HSD. (I) Reticulin (MF) grade. Kruskal-Wallis H test with post hoc Wilcoxon rank-sum test. See also Figure S6 .

Article Snippet: Samples were diluted 1:50 - 1:150 and S100A8 concentration was quantified using the Human S100A8 DuoSet ELISA (R&D Systems, DY4570-05) according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Two Tailed Test, Control

Journal: Cell Stem Cell

Article Title: Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis

doi: 10.1016/j.stem.2020.11.004

Figure Lengend Snippet:

Article Snippet: Samples were diluted 1:50 - 1:150 and S100A8 concentration was quantified using the Human S100A8 DuoSet ELISA (R&D Systems, DY4570-05) according to the manufacturer’s instructions.

Techniques: Recombinant, Staining, Plasmid Preparation, Virus, Multiplex Assay, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Avidin-Biotin Assay, Blocking Assay, Retroviral, Software

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: A small population of ACC cells H295R overexpresses Ptch1 at the plasma membrane . ( A ) H295R were labeled with an anti-Ptch1 antibody directed against the extracellular loop and cells presenting Ptch1 at their plasma membrane (H295R-PM-Ptc+ AF594+ cells) were sorted. AF594+ in blue represents the percentage of cells with Ptch1 at the cell surface (H295R-PM-Ptc+ cells). ( B ) Surface labeling of Ptch1 using anti-Ptch1 antibody directed against the extracellular loop of Ptch1 (Alexa 594 in red) on nonpermeabilized parental H295R and H295R-PM-Ptc+ cells. Nuclei were stained with DAPI (in blue). The histogram represents the mean ± SEM of Alexa 594 fluorescence intensity per cell (****: p -value < 0.00005 ( p -value = 2 × 10 −36 )).

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Clinical Proteomics, Membrane, Labeling, Staining, Fluorescence

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: H295R-PM-Ptc+ cells are more resistant to chemotherapy than parental cells . ( A ) Doxorubicin (dxr) cytotoxicity. H295R and H295R-PM-Ptc+ cells were treated for 48 h with increasing concentrations of dxr before cell viability measure. ( B ) Doxorubicin IC50 of H295R-PM-Ptc+ and H295R parental cells in the absence or the presence of 10 μM of the Ptch1 efflux inhibitor methiothepin. ( C ) H295R-PM-Ptc+ cells accumulate less doxorubicin than parental H295R cells. Cells on coverslips were incubated with 2 μM dxr for 15, 30, 60, 180 and 240 min and immediately fixed with PFA. Dxr fluorescence was acquired using a filter for Alexa 594 and quantified using ImageJ software. About 100 cells (from three wells) were scored per condition per experiment. All data presented are the mean ± SEM of at least 3 independent experiments. Significance is attained at p -value < 0.05 (*), (**** p < 0.00005).

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Incubation, Fluorescence, Software

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: Differentially expressed genes (DEG) between H295R-PM-Ptc+ and parental H295R cells selected for their role in cancer. Genes overexpressed are indicated in red and genes underexpressed are in blue.

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Activation Assay, Expressing, Inhibition, Gene Expression, Migration, Marker, Biomarker Discovery

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: Composition of active modules containing one or more of the identified genes of interest listed in Table 1 (in bold) with genes upregulated in red and genes downregulated in blue, representative enrichment and role of differentially expressed genes (DEGs) in cancers.

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Migration, Cell Differentiation, Activation Assay, Membrane, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Berberine Sensitizes Human Hepatoma Cells to Regorafenib via Modulating Expression of Circular RNAs

doi: 10.3389/fphar.2021.632201

Figure Lengend Snippet: BBR promoted the anti-proliferative effect of regorafenib on HCC cells in vitro . (A,B) After HCC cells were treated with different concentrations of regorafenib (0, 0.01, 0.1, 1, 2, 4, 8, 16, 32 and 64μM) and BBR (0, 5, 10, 20, 40, 80, 160, 320, 640 and 1280μM) for 48h, MTS assay was used to detect cell viability. (C,D) HCC cells were treated with regorafenib (5, 10μM), BBR (100μM) or the combination of regorafenib and BBR for 24h or 48h. MTS assay was used to determine cell viability. (E,F) The combination index of each combined treatment was calculated using CompuSyn software. Points below the dotted line indicated synergy (CI values <1). * p < 0.05.

Article Snippet: Regorafenib and BBR were obtained from Selleck Chemicals (Houston, TX, United States; cat. no. S1178 and S2271).

Techniques: In Vitro, MTS Assay, Software

Journal: Frontiers in Pharmacology

Article Title: Berberine Sensitizes Human Hepatoma Cells to Regorafenib via Modulating Expression of Circular RNAs

doi: 10.3389/fphar.2021.632201

Figure Lengend Snippet: The combination of BBR and regorafenib reduced the proliferative capacity of HCC cells. (A,B) The proliferative capacity of SMMC-7721 cells detected by Edu assay after treatment with regorafenib (5, 10µM), BBR (100µM) alone or in combination for 24 or 48h. (C,D) The combination of BBR and regorafenib reduced the proliferative capacity of Hep3B cells, as determined by Edu assay (magnification, ×200). * p < 0.05.

Article Snippet: Regorafenib and BBR were obtained from Selleck Chemicals (Houston, TX, United States; cat. no. S1178 and S2271).

Techniques: EdU Assay

Journal: Frontiers in Pharmacology

Article Title: Berberine Sensitizes Human Hepatoma Cells to Regorafenib via Modulating Expression of Circular RNAs

doi: 10.3389/fphar.2021.632201

Figure Lengend Snippet: Apoptosis was induced by the combination treatment of BBR and regorafenib in HCC cells. (A) HCC cells were treated with regorafenib (5, 10µM), BBR (100µM) alone or in combination for 24 or 48h. The apoptotic cells were examined by TUNEL assay (200 x). (B) Western blotting assay measured the expression of apoptosis related proteins. GAPDH was used as a loading control.

Article Snippet: Regorafenib and BBR were obtained from Selleck Chemicals (Houston, TX, United States; cat. no. S1178 and S2271).

Techniques: TUNEL Assay, Western Blot, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Berberine Sensitizes Human Hepatoma Cells to Regorafenib via Modulating Expression of Circular RNAs

doi: 10.3389/fphar.2021.632201

Figure Lengend Snippet: BBR enhanced regorafenib induced apoptosis in HCC cells. (A) Flow cytometric assay analyzed the apoptotic rate of SMMC-7721 and Hep3B cells induced by BBR and regorafenib. After HCC cells were treated with BBR (100µM), regorafenib (5, 10µM) or their combination for 48h, they were stained with Annexin V/PI. (B) Histogram displayed the apoptotic rates in different groups from three independent experiments. * p < 0.05.

Article Snippet: Regorafenib and BBR were obtained from Selleck Chemicals (Houston, TX, United States; cat. no. S1178 and S2271).

Techniques: Flow Cytometry, Staining

Journal: Frontiers in Pharmacology

Article Title: Berberine Sensitizes Human Hepatoma Cells to Regorafenib via Modulating Expression of Circular RNAs

doi: 10.3389/fphar.2021.632201

Figure Lengend Snippet: Combined treatment with BBR and regorafenib inhibited the growth of xenograft tumors and induced cell apoptosis in mice. (A) Representative images of the subcutaneous xenografts tumors in mice treated with DMSO, BBR, regorafenib and their combination. (B,C) The weight and volume of xenograft tumors displayed as mean ± SD in DMSO, BBR, regorafenib and the combined treatment groups. (D) Western blotting assay measured the expression of apoptosis related proteins. GAPDH was used as a loading control. (E) TUNEL assay was used to evaluate the apoptosis of xenograft tumor tissue. n = 5, * p < 0.05.

Article Snippet: Regorafenib and BBR were obtained from Selleck Chemicals (Houston, TX, United States; cat. no. S1178 and S2271).

Techniques: Western Blot, Expressing, Control, TUNEL Assay

Journal: Frontiers in Pharmacology

Article Title: Berberine Sensitizes Human Hepatoma Cells to Regorafenib via Modulating Expression of Circular RNAs

doi: 10.3389/fphar.2021.632201

Figure Lengend Snippet: Differential expression of circRNAs in SMMC-7721 cells treated with BBR and regorafenib. (A) Different circRNA expression profiles among samples from the RNA sequencing data shown by heat map. Three DMSO samples and three combined samples of BBR and regorafenib were examined. (B,C) In the volcano and scatter plots, 58 circRNAs were increased and 19 were decreased in the combination treatment group. (D) qRT-PCR performed on the validation of two differentially expressed circRNAs in control, BBR, regorafenib and the combination groups. (E) Interaction network of circRNAs and their potential target miRNAs * p < 0.05.

Article Snippet: Regorafenib and BBR were obtained from Selleck Chemicals (Houston, TX, United States; cat. no. S1178 and S2271).

Techniques: Quantitative Proteomics, Expressing, RNA Sequencing, Quantitative RT-PCR, Biomarker Discovery, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Generation of humanized Zt/g4 antibody and characterization of RON-targeted antibody-drug conjugates: ( a ) Modeling of CDRs from mouse Zt/g4 in the variable regions of human IgG heavy chain and light chain. The framework of human IgG1 molecule was used for Zt/g4 humanization. The models of Zt/g4 CDRs grafted in the variable regions of human IgG1 heavy chain and light chain were generated by using the software PIGS from Automatic Predictions of Immunoglobulin Structures (Tramontano at University of Rome, Italy). ( b ) Binding of subclone H-Zt/g4 molecules to human RON. Different amounts of individual H-Zt/g4 s were incubated with NIH-3 T3 cells expressing human RON followed by addition of goat anti-human IgG1 antibody coupled with FITC. ( c ) Kinetic characterization of H-Zt/g4 interaction with human RON proteins by Octet RED96 system. Pure RON proteins from lysates of NIH3T3 cells expressing RON were immobilized onto the amine reactive sensor and assayed against individual H-Zt/g4 molecules in duplicate. The data set is analyzed with global fitting to produce the antibody-receptor binding affinity ( K D ). Blue curves represent experimental data and red curves represent the statistical fitting of curves. ( d ) Interaction of H-Zt/g4 H1L3 with RONs from different species. NIH3T3 cells expressing human, monkey, or mouse RON were incubated with H-Zt/g4 H1L3 followed by goat anti-human IgG coupled with FITC. Immunofluorescent intensities from individual samples were determined by flow cytometric analysis. ( e ) Schematic representation of H-Zt/g4-MMAE structure. MMAE was conjugated to H-Zt/g4 by the valine-citruline dipeptide linker according to the manufacturer’s instruction ( www.concortis.com ). ( f ) HIC analysis of MMAE conjugated to H-Zt/g4: Individual Zt/g4-MMAEs with different numbers of MMAE (0 to 8) are marked as P0 to P8. A DAR combining P2, P4, and P6 at 3.77:1 was achieved. ( g ) Free MMAE dissociated from H-Zt/g4-MMAE in human plasma. H-Zt/g4-MMAE at 10 μg per ml was incubated with fresh human plasma at 37 °C for 20 days. The amount of free MMAE in plasma was determined using the LC-MS/MS method with slight modifications. ( h ) Samples from ( g ) were used also for measuring MMAE conjugated H-Zt/g4 as detailed in Materials and Methods. A ratio from free MMAE to the total MMAE in H-Zt/g4-MMAE was calculated to determine the percentages of MMAE dissociated from H-Zt/g4-MMAE

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Generated, Software, Binding Assay, Incubation, Expressing, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Effect of H-Zt/g4-MMAE on RON internalization, cell viability, and death: ( a ) H-Zt/g4-induced cell surface RON internalization. PDAC cell lines BxPC-3, FG and L3.6pl (1 × 10 6 cells per dish) were treated at 37 °C with 5 μg/ml of H-Zt/g4-MMAE, collected at different time points, washed with acidic buffer to eliminate cell surface bound IgG , and then incubated with 2 μg/mL of anti-RON mAb Zt/c1 . Immunofluorescence was analyzed by flow cytometer using FITC-coupled anti-mouse IgG. Immunofluorescence from cells treated with H-Zt/g4 at 4 °C was set as 100%. Internalization efficiency (IC 50 ) was calculated as the time required achieving 50% reduction of cell surface RON. ( b ) Intracellular localization of internalized RON. FG cells in a 6-well plate were treated with 5 μg/ml of H-Zt/g4 at 4 °C or 37 °C for 12 h followed by mouse anti-human IgG1-coupled with FITC. Nuclear DNAs were stained with DAPI. LAMP-1 was used as a marker for protein cytoplasmic localization. Similar results also observed in additional three PDAC cell lines (data not shown). ( c ) Effect of H-Zt/g4-MMAE on viability of PDAC cells. Three PDAC cell lines (8000 cells per well in a 96-well plate in triplicate) were treated with different amounts of H-Zt/g4-MMAE for 96 h. Panc-1 cells served as the negative control. Cell viability was determined by the MTS assay. ( d ) Death of PDAC cells after H-Zt/g4-MMAE treatment. PDAC cells were treated with different amounts of H-Zt/g4-MMAE for 96 h. The percentages of cell death were determined by the trypan blue exclusion method. Data shown in ( c ) and ( d ) are derived from one of three experiments with similar results

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Incubation, Immunofluorescence, Flow Cytometry, Staining, Marker, Negative Control, MTS Assay, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Pharmacokinetic profiles of H-Zt/g4-MMAE in both mouse and cynomolgus monkey: ( a ) PK profiles of H-Zt/g4-MMAE in mouse. Tumor-bearing and -nonbearing mice (athymic nude, 5 mice per group) were injected with a single dose of H-Zt/g4-MMAE at 3, 10, and 20 mg/kg, respectively. Collected blood samples were analyzed using the MMAE ADC ELISA kit (Eagle Biosciences, Inc., Nashua, NH). Various PK parameters were calculated using the software provided by Eagle Biosciences. ( b ) Free MMAE dissociated from H-Zt/g4-MMAE in monkey plasma. A single dose of H-Zt/g4-MMAE at 10 or 30 mg/kg was injected into cynomolgus monkey (3 animals per group). Free MMAE from individual blood samples collected at different time intervals were subjected to the LC-MS/MS analysis. ( c ) PK profiles of H-Zt/g4-MMAE in cynomolgus monkey. Blood samples from ( b ) were analyzed for MMAE coupled H-Zt/g4 using the MMAE ADC ELISA kit as described in ( a ) to obtain various PK parameters

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Software, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Therapeutic efficacy of H-Zt/g4-MMAE in PDAC xenograft tumor models: ( a ) Dose-dependent effect of H-Zt/g4-MMAE: Athymic nude mice (5 mice per group) were subcutaneously inoculated with 5 × 10 6 FG cells. H-Zt/g4-MMAE at 1, 3, 7, 10, and 15 mg/kg was injected through tail vein in the Q6 × 5 regimen after tumors volumes reached to ~ 150 mm 3 . Mice injected with CmIgG-MMAE at 10 mg/kg were used as the control. Xenografts initiated by HT-29 cells served for comparison. ( b ) Effect of H-Zt/g4-MMAE in PDAC xenograft growth and eradication. Individual tumors from different groups described in (A) were collected from euthanized mice. Control mice bearing FG xenografts were sacrificed at day 24 due to rapid growth of tumors. Mice from other groups were killed at day 28 or day 44 dependent on the size of tumors. All tumors were weighted to reach the average tumor weight per group. The number of tumors from individual groups also was counted to determine the eradicating effect of H-Zt/g4-MMAE. NA, no tumors were found in the injected site. ( c ) Effect of H-Zt/g4-MMAE in three PDAC xenograft models: Xenograft tumors in mice (five animals per group) initiated by four PDAC cell lines were used for study. H-Zt/g4-MMAE was used at 20 mg/kg in the Q12 × 2 schedules. To establish the dose-effect relationship, the estimated reduction of H-Zt/g4-MMAE in vivo according to the t½ was marked as red circles. ( d ) Effect of H-Zt/g4-MMAE in tumor growth and eradication: Tumors were collected from mice described in ( b ). Tumor weight, count, and calculation were performed as described in ( b ). NA, no tumors were observed in the injected site

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Drug discovery, Injection, Control, Comparison, In Vivo

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Therapeutic Effect of H-Zt/g4-MMAE on xenograft tumors mediated by PDAC stem-like cells and primary PDX cells: ( a ) Effect of H-Zt/g4-MMAE on PDAC stem-like cell derived xenografts: Athymic nude mice (five mice per group) were subcutaneously inoculated with 5 × 10 5 PSC + 24/44/ESA prepared from BxPc-3, FG, and L3.6pl cells. H-Zt/g4-MMAE at 20 mg/kg was injected through tail vein in the Q12 × 2 regimen after tumors volumes reached to ~ 150 mm 3 . Mice injected with CmIgG-MMAE at 20 mg/kg were used as the control. ( b ) The eradicating effect of H-Zt/g4-MMAE on PDAC stem-like cell derived xenografts. Tumors were collected from mice as described in Fig. b. Tumor weight, count, and calculation were performed as described in Fig. b. ( c ) Mice were injected with individual primary PDX cell lines at 5 × 10 6 cells in 0.1 ml in PBS. H-Zt/g4-MMAE at 10 mg/kg was injected through tail vein in the Q12 × 2 regimen after tumors volumes reached to 150 to 200 mm 3 . Mice injected with CmIgG-MMAE at 10 mg/kg were used as the control. ( d ) Individual tumors were collected from each group of mice as described in Fig. b. Average tumor weight and number per group were measured to determine levels of inhibition and eradication

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Derivative Assay, Injection, Control, Inhibition

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Therapeutic effect of H-Zt/g4-MMAE in comparison with H-Zt/g4-DM1 in inhibition of xenograft tumors derived from human pancreatic and colorectal cancer cells a

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Comparison, Inhibition, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Toxicological activities of H-Zt/g4-MMAE in mouse and cynomolgus monkey. ( a ) and ( b ) Adverse activities of H-Zt/g4-MMAE in blood leukocytes in cynomolgus monkey. H-Zt/g4-MMAE at 10 or 30 mg/kg in a single dose was injected once into cynomolgus monkey. Monkeys without ADC injection served as the control. Peripheral blood samples were collected at different time intervals. Total numbers of leukocytes ( a ) including neutrophil, lymphocytes, and monocytes from each group were countered accordingly. The percentages of blood leukocytes ( b ) were also determined. ( c ) Adverse effect of H-Zt/g4-MMAE on blood erythrocytes and reticulocytes in cynomolgus monkey. Total numbers of erythrocytes and reticulocytes from blood samples collected from each group as described in ( b ) were counted accordingly. ( d ) Adverse effects of H-Zt/g4-MMAE on various enzymes in plasma of cynomolgus monkey. Six enzymatic activities from each group were quantitatively measured using the blood samples collected from individual monkeys as described in ( b )

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Injection, Control, Clinical Proteomics

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Indirect immunofluorescence assay (IFA) of HeLa cells infected with C. muridarum TC0668 wt or TC0668 mut strains. With 2.5 × 10 5 IFU/well inoculum (MOI = 1), Chlamydia -infected HeLa cells were photographed using optical microscopy at 6, 12, 18, and 24 h p.i. Chlamydial inclusion bodies (green) are visible in both TC0668 wt - and TC0668 mut -infected cells, whereas the TC0668 protein (red) is only visible in TC0668 wt -infected HeLa cells. Magnification, ×200. TC0668 wt -infected HeLa cells were photographed by using phase contrast microscopy (A) and IFA (B) at 6, 12, 18, and 24 h p.i. TC0668 mut -infected HeLa cells were photographed by using phase contrast microscopy (C) and IFA (D) at 6, 12, 18, and 24 h p.i.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Immunofluorescence, Infection, Microscopy

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: qRT-PCR analysis of tc0668 gene copy number in C. muridarum TC0668 wt - and TC0668 mut -infected HeLa cells. With 1 × 10 6 IFU/well inoculum (MOI = 1), the copy number of the C. muridarum gene tc0668 was determined using qRT-PCR, and the C. muridarum plasmid gene pgp8 was used as the control. 16S rRNA was used to normalize tc0668 and pgp8 signals. Three biological replicates of each time point were performed, and points represent mean and standard errors. Copy number differences of tc0668 between TC0668 wt - and TC0668 mut -infected cells were statistically significant (one-way ANOVA, P < 0.05).

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Quantitative RT-PCR, Infection, Plasmid Preparation, Control

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Quantitative proteomic analysis of HeLa cells infected with C. muridarum TC0668 wt or TC0668 mut strains. (A) Basic statistics of proteome results from iTRAQ. Spectra, secondary mass spectra after quality control; Unique peptides, identified peptides that belong to only a group of proteins; and protein, identified proteins using Mascot 2.3.02 software. (B) Trends of differentially expressed proteins in TC0668 wt -infected cells at 6, 12, 18, and 24 h p.i. (C) Trends of differentially expressed proteins in TC0668 mut -infected cells at 6, 12, 18, and 24 h p.i.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Infection, Multiplex sample analysis, Control, Software

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Four-way Venn diagram of the total number of proteins significantly differentially expressed ( P < 0.05) between HeLa cells infected with C. muridarum TC0668 wt or TC0668 mut strains at 6, 12, 18, and 24 h p.i. Numbers of shared or unique proteins are indicated at the intersections of the circles in the Venn diagram.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Infection

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Validation of profiling data with qRT-PCR. mRNA expression levels of seven up-regulated (encoded by SRPRB, JAK1, PMM1, HLA-DQB1, BCAP31, ITPR1, and THBS1) and three down-regulated (encoded by MAPKAPK2, TRAFD1, and IFI16) proteins in C. muridarum TC0668 mut -infected HeLa cells were determined using qRT-PCR at 18 h p.i, and compared with those of C. muridarum TC0668 wt -infected group. mRNA levels from three replicates for each group are expressed as mean and the standard errors. *represents that all copy number differences between TC0668 wt and TC0668 mut were statistically significant ( t test, P < 0.05).

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Biomarker Discovery, Quantitative RT-PCR, Expressing, Infection

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Activation of NF-κB and PI3K/Akt signal pathways as determined using western blotting and IFA. With 1 × 10 6 IFUs/well inoculum (six-well plate) or 2.5 × 10 5 IFUs/well inoculum (24-well plate), expression of PI3K, p-Akt, p53, and NF-κB (p65) representing activation of PI3K/Akt and NF-κB signal pathways were determined using western blotting or IFA, respectively. Gel quantification software was used to calculate the relative intensity of the corresponding signals. The relative value (target protein/GAPDH) differences of PI3K, p53, and NF-κB (p65) molecules and the relative expression level of p-Akt (p-Akt/Akt) between TC0668 wt - and TC0668 mut -infected cells were statistically significant (one-way ANOVA, P < 0.05). (A) Expression of PI3K molecules in TC0668 mut - and TC0668 wt -infected cells were determined using western blotting at 6, 12, 18, and 24 h post-infection. (B) Expression of p-Akt and total Akt in TC0668 mut - and TC0668 wt -infected cells were determined using western blotting at 6, 12, 18, and 24 h post-infection. (C) Expression of p53 molecules in the TC0668 mut - and TC0668 wt -infected cells at 6, 12, 18, and 24 h post-infection. (D) Expression of p65 molecules in the TC0668 mut - and TC0668 wt -infected cells at 6, 12, 18, and 24 h post-infection. (E) NF-κB molecules in the cytoplasm and nuclei of TC0668 mut - and TC0668 wt -infected HeLa cells at 6, 12, 18, and 24 h post-infection. DAPI dye core (blue), NF-κB fluorescence secondary antibody is 488 dye (green).

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Activation Assay, Western Blot, Expressing, Software, Infection, Fluorescence

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Differentially expressed proteins related with inflammation in TC0668 mut - vs. TC0668 wt -infected HeLa cells at 18 h p.i.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Infection, Multiplex sample analysis, Ubiquitin Proteomics, Histone Deacetylase Assay, Membrane, Variant Assay

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Differentially expressed proteins related with fibrosis in TC0668 mut - vs. TC0668 wt -infected HeLa cells at 18 h p.i.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Infection, Multiplex sample analysis, Ubiquitin Proteomics

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Protein-protein interaction network of statistically differentially expressed proteins ( P < 0.05) associated with inflammation and fibrosis at 18 h p.i. The proteins interconnectivity of two categories (inflammation and fibrosis) are shown. The yellow circles marked with symbols combining numbers and letters, represent the protein ID of differentially expressed proteins screened by comparison of TC0668 mut -infected and TC0668 wt -infected HeLa cells. The corresponding information of proteins can be analyzed via UniProt ( https://www.uniprot.org/ ), each protein ID corresponds to a protein. For example, the protein ID “P01023” corresponds to the protein “pha-2-macroglobulin”.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Comparison, Infection

Journal: bioRxiv

Article Title: The ERK1/2-Elk1, JNK-cJun, and JAK-STAT Transcriptional Axes as Potential Bortezomib Resistance Mediators in Prostate Cancer

doi: 10.1101/2024.04.15.589569

Figure Lengend Snippet: Assaying the Effects of Bortezomib and Carfilzomib on the Proliferation of Naïve PC-3 and PC-3 RB40 Cells During Dose Elevation. Equal numbers of cells were cultured for 72 h in 24-well microplates with various concentrations of ( a ) Bortezomib (0-240 nM) following 4 weeks of resistance acquirement; ( b ) Bortezomib (0-240 nM) following 12 weeks of resistance acquirement; and ( c ) Carfilzomib (0-325 nM) following 12 weeks of resistance acquirement; The live cells were measured using the Crystal Violet Assay. Each dot represents the average of three experimental values, and the error bars represent the standard error of the mean (SEM). The fitting line was graphed in Prism 8 using the built-in model for IC 50 determination. Each plot represents one experiment, while the mean IC 50 of three replicated experiments is presented in .

Article Snippet: The PC3 (ATCC, Manassas, VT, USA) cell line was used as a human prostate carcinoma cell model.

Techniques: Cell Culture, Crystal Violet Assay

Journal: bioRxiv

Article Title: The ERK1/2-Elk1, JNK-cJun, and JAK-STAT Transcriptional Axes as Potential Bortezomib Resistance Mediators in Prostate Cancer

doi: 10.1101/2024.04.15.589569

Figure Lengend Snippet: Assaying the Effects of Bortezomib, Carfilzomib, and Doxorubicin on the Proliferation of Naïve PC-3 and PC-3 RB40 Cells. Equal numbers of cells were cultured for 72 h in 24-well microplates with various concentrations of ( a ) Bortezomib (0-240 nM); ( b ) Carfilzomib (0-325 nM); and ( c ) Doxorubicin (0-1000 nΜ), and the live cells were measured using the Crystal Violet Assay. Each dot represents the average of three experimental values, and the error bars represent the standard error of the mean (SEM). The fitting line was graphed in Prism 8 using the built-in model for IC50 determination. The blue lines represent the proliferation curves of naïve PC-3 cells, the red line the proliferation curve of PC-3 RB40 cells during Bortezomib treatment, the green line the curve of PC-3 RB40 cells during Doxorubicin treatment. Each plot represents one experiment, while the mean IC50 of three replicated experiments is presented in .

Article Snippet: The PC3 (ATCC, Manassas, VT, USA) cell line was used as a human prostate carcinoma cell model.

Techniques: Cell Culture, Crystal Violet Assay

Journal: bioRxiv

Article Title: The ERK1/2-Elk1, JNK-cJun, and JAK-STAT Transcriptional Axes as Potential Bortezomib Resistance Mediators in Prostate Cancer

doi: 10.1101/2024.04.15.589569

Figure Lengend Snippet: Cell Cycle Analysis of Naïve PC-3 and PC-3 RB40 Cells Following Treatment with Bortezomib. Equal numbers of cells were cultured inside 100-mm dishes, and 24 h before analysis, the media were replaced. ( a ) Naïve PC-3 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS (experimental control of baseline cell cycle progression); ( b ) Naïve PC-3 cells were cultured in medium containing 40 nM of Bortezomib for 24 h; ( c ) PC-3 RB40 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS and; ( d ) PC-3 RB40 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS and 40 nM of Bortezomib (basal culture conditions for this cell line). The cells were fixed and permeabilized with methanol, treated with RNAase, and stained with propidium iodide. Equal numbers of events were acquired using a FACS Calibur flow cytometer by measuring the propidium iodide, which is correlated to the DNA content, so the different cell cycle phases (G1/G0, S, and G2/M) could be shown in histograms using the FlowJo software. The figure presents a representative experiment. The same procedure was replicated three times.

Article Snippet: The PC3 (ATCC, Manassas, VT, USA) cell line was used as a human prostate carcinoma cell model.

Techniques: Cell Cycle Assay, Cell Culture, Control, Staining, Flow Cytometry, Software

Journal: bioRxiv

Article Title: The ERK1/2-Elk1, JNK-cJun, and JAK-STAT Transcriptional Axes as Potential Bortezomib Resistance Mediators in Prostate Cancer

doi: 10.1101/2024.04.15.589569

Figure Lengend Snippet: Apoptosis Assay of Naïve PC-3 and PC-3 RB40 Cells Following Treatment with Bortezomib. Equal numbers of cells were cultured inside 100-mm dishes, and 24 h before analysis, the media were replaced. ( a ) Naïve PC-3 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS (blue coloring)(experimental control of baseline cell cycle progression); ( b ) naïve PC-3 cells were cultured in medium containing 40 nM of Bortezomib for 24 h (red coloring); ( c ) PC-3 RB40 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS (yellow coloring); ( d ) PC-3 RB40 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS and 40 nM of Bortezomib (magenta coloring)(basal culture conditions for this cell line); ( e ) PC-3 RB40 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS and 20 nM of Bortezomib (purple coloring); and ( f ) PC-3 RB40 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS and 80 nM of Bortezomib (lilac coloring) The cells were stained with an Annexin-V/propidium iodide (PI) kit inside a Calcium-containing buffer to measure the levels of apoptosis. Equal numbers of events were acquired using a FACS Calibur flow cytometer by measuring the fluorescence emitted by FITC, which is bound to Annexin-V and PI, and the data were analyzed using the FlowJo software. The graphs display the density plots of Annexin-V and PI, and the four different quartiles correspond to Q1: necrotic cells; Q2: cells in late apoptosis; Q3: cells in early apoptosis; and Q4: live cells. The figure presents a representative experiment. The same procedure was replicated three times.

Article Snippet: The PC3 (ATCC, Manassas, VT, USA) cell line was used as a human prostate carcinoma cell model.

Techniques: Apoptosis Assay, Cell Culture, Control, Staining, Flow Cytometry, Fluorescence, Software

Journal: bioRxiv

Article Title: The ERK1/2-Elk1, JNK-cJun, and JAK-STAT Transcriptional Axes as Potential Bortezomib Resistance Mediators in Prostate Cancer

doi: 10.1101/2024.04.15.589569

Figure Lengend Snippet: Assaying the Effects of Bortezomib on the Migration of Naïve PC-3 and PC-3 RB40 Cells. ( a-d ) Scratch Test/Wound Healing Assay; Cells were cultured in 6-well plates until confluency, and then scratches were made. Various concentrations of Bortezomib (0, 20, 40, 80 nM) were added, and photographs were taken at the key time points of 0, 24, 48, and 72 h using a camera mounted on an inverted microscope at 100X magnification. The photographs showing wound closure were then analyzed using an ImageJ plug-in, and the healing rates were determined. The data were plotted in Prism 8. ( a ) Naïve PC-3 and PC-3 RB40 cells untreated for 72h; ( b ) Naïve and PC-3 RB40 cells, both treated with 20 nM Bortezomib. ( c ) Naïve and PC-3 RB40 cells, both treated with 40 nM Bortezomib. ( d ) RB40 cells treated with 80 nM Bortezomib. Each wound healing experiment was conducted in triplicate, and the values on the plots are the averages. The error bars correspond to the standard error of the mean (SEM) from the three experiments. ( e, f ) Migration Assay; Equal numbers of cells were placed in Transwell/Boyden Chambers in serum-free RPMI 1640 medium containing increasing concentrations of Bortezomib (0, 20, 40, and 80 nM), and the inserts were placed in microwells containing FBS-supplemented medium. The cells were left for 24 h to migrate, and afterwards, the cells crossing the filters were fixed and stained with crystal violet. Photographs were taken using a 10x objective and the fixed cells were counted using the multipoint tool by ImageJ; ( e ) The percentage of migrated cells were compared to the naïve untreated sample; ( f ) The percentages of total migrated cells, compared to each clone’s baseline conditions (untreated for the naïve cells, and 40 nM Bortezomib for the RB40 cells); ( g, h ) The migration experiments were also conducted following a 48-hour Bortezomib withdrawal/ clearance period of the RB40 clone. ( i, j ) Chemotaxis Assay; Equal numbers of cells were placed in Transwell/Boyden Chambers in serum-free RPMI 1640 medium, and the inserts were placed in microwells with FBS-supplemented medium containing increasing concentrations of Bortezomib (0, 20, 40, and 80 nM). The cells were left for 24 h to migrate, and afterwards, the cells crossing the filters were fixed and stained with crystal violet. Photographs were taken using a 10x objective, and the fixed cells were counted using the multipoint tool by ImageJ; ( i ) The percentage of migrated cells were compared to the naïve untreated sample; ( j ) The percentages of total migrated cells, compared to each clone’s baseline conditions (untreated for the naïve cells, and 40 nM Bortezomib for the RB40 cells). The results were analyzed using multiple comparisons of one-way ANOVA in the Prism 8 software. (* corresponds to P =0,01; ** corresponds to P =0,001; *** corresponds to P =0,0001; and **** corresponds to P <0,0001). The error bars represent the standard error of the mean (SEM).

Article Snippet: The PC3 (ATCC, Manassas, VT, USA) cell line was used as a human prostate carcinoma cell model.

Techniques: Migration, Wound Healing Assay, Cell Culture, Inverted Microscopy, Staining, Clone Assay, Chemotaxis Assay, Software

Journal: bioRxiv

Article Title: The ERK1/2-Elk1, JNK-cJun, and JAK-STAT Transcriptional Axes as Potential Bortezomib Resistance Mediators in Prostate Cancer

doi: 10.1101/2024.04.15.589569

Figure Lengend Snippet: Scratch Test/Wound Healing Assay; Cells were cultured in 6-well plates until confluency, and then scratches were made. Various concentrations of Bortezomib (0, 20, 40, 80 nM) were added, and photographs were taken at the key time points of 0, 24, 48, and 72 h using a camera mounted on an inverted microscope at 100X magnification. The photographs showing wound closure were then analyzed using an ImageJ plug-in, and the healing rates were determined. The data were then plotted in Prism 8. ( a ) Naïve PC-3; and ( b ) PC-3 RB40.

Article Snippet: The PC3 (ATCC, Manassas, VT, USA) cell line was used as a human prostate carcinoma cell model.

Techniques: Wound Healing Assay, Cell Culture, Inverted Microscopy

Journal: bioRxiv

Article Title: The ERK1/2-Elk1, JNK-cJun, and JAK-STAT Transcriptional Axes as Potential Bortezomib Resistance Mediators in Prostate Cancer

doi: 10.1101/2024.04.15.589569

Figure Lengend Snippet: Western Analysis of N-cadherin, E-cadherin, ανβ3-integrin, β-catenin PSMB5, p62/SQSTM1, Atg5, Beclin-1, LC3A/B, and ubiquitinated proteins. Cells (Naïve PC-3 and PC-3 RB40) were cultured inside 100 mm dishes, and 24 h before confluency, the media were changed and fresh RPMI 1640 supplemented with 10% with or without the designated bortezomib doses (20, 40, 80 nM) was added. Following 24 h of incubation, the cells were lysed using RIPA buffer, and equal quantities of total proteins were loaded onto 12% polyacrylamide gels and analyzed with SDS-PAGE. The protein content was determined using the Bradford assay, and as a validation of successful transfer, the total proteins on the gel and the membrane were stained with a Coomassie Brilliant Blue Solution. Western analysis of β-actin was used as a reference protein after it was validated that its accumulation remained stable during all procedures. ( a ) Using specific polyclonal antibodies, the accumulation of N-cadherin, E-cadherin, ανβ3-integrin, β-catenin, PSMB5, p62/SQSTM1, Atg5, Beclin-1 and LC3A/B was detected using the SuperSignal™ West Femto Maximum chemiluminescence Kit. The chemiluminescence was developed on autoradiography films, which were subsequently scanned. Molecular weight markers were used during SDS-PAGE and the approximate molecular weight of each detected polypeptide is annotated next to the band. ( b-l ) Quantification of the scanned blots was followed using the plug-in “Gels” in ImageJ after conversion to grayscale images, and the band intensities and bit depth were calculated. The data were retrieved from triplicate experiments and after normalization using β-actin, bar charts were created. Each bar represents the average relative accumulation of the target protein compared to the untreated naïve PC-3 cells (first blot lane). The results were analyzed using multiple comparisons of one-way ANOVA in the Prism 8 software. (* corresponds to P =0,01; ** corresponds to P =0,001; *** corresponds to P =0,0001; and **** corresponds to P <0,0001). The error bars represent the standard error of the mean (SEM). ( m ) To obtain information about the ubiquitination, a mouse polyclonal antibody was used, and a membrane was appropriately probed. The resulting film was scanned, and the results were presented as they were.

Article Snippet: The PC3 (ATCC, Manassas, VT, USA) cell line was used as a human prostate carcinoma cell model.

Techniques: Western Blot, Cell Culture, Incubation, SDS Page, Bradford Assay, Biomarker Discovery, Membrane, Staining, Autoradiography, Molecular Weight, Software, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: The ERK1/2-Elk1, JNK-cJun, and JAK-STAT Transcriptional Axes as Potential Bortezomib Resistance Mediators in Prostate Cancer

doi: 10.1101/2024.04.15.589569

Figure Lengend Snippet: Autophagy Assay of Naïve PC-3 and PC-3 RB40 Cells Following Treatment with Bortezomib. Equal numbers of cells were cultured inside 100 mm dishes, and 24 h before analysis, the media were replaced. ( a ) Naïve PC-3 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS (blue coloring)(experimental control of baseline cell cycle progression); ( b ) Naïve PC-3 cells were cultured in medium containing 40 nM of Bortezomib for 24 h (magenta coloring); ( c ) PC-3 RB40 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS and (yellow coloring); ( d ) PC-3 RB40 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS and 40 nM of Bortezomib (red coloring)(basal culture conditions for this cell line). The cells were then stained with the LIVE/DEAD kit and with Lysotracker RED. Equal numbers of events were acquired using a FACS Calibur flow cytometer by measuring the fluorescence of the LIVE/DEAD stain and Lysotracker RED, and the data were analyzed using the FlowJo software. The histograms display the median fluorescence intensity (MFI) of the Lysotracker RED channel. The figure presents a representative experiment. The same procedure was replicated three times.

Article Snippet: The PC3 (ATCC, Manassas, VT, USA) cell line was used as a human prostate carcinoma cell model.

Techniques: Cell Culture, Control, Staining, Flow Cytometry, Fluorescence, Software

Journal: bioRxiv

Article Title: The ERK1/2-Elk1, JNK-cJun, and JAK-STAT Transcriptional Axes as Potential Bortezomib Resistance Mediators in Prostate Cancer

doi: 10.1101/2024.04.15.589569

Figure Lengend Snippet: Western analysis of Hsp70, MEK4, p-p38 MAPK, p-JNK1, and SOD1. Cells (Naïve PC-3 and PC-3 RB40) were cultured inside 100 mm dishes, and 24 h before confluency, the media were changed, and fresh RPMI 1640 supplemented with 10% with or without the designated bortezomib doses (20, 40, 80 nM) was added. ( a ) The samples were prepared as previously noted, and by using specific polyclonal antibodies, the accumulation of Hsp70, MEK4, p-p38 MAPK, p-JNK1, and SOD1 was detected and developed on autoradiography films, which were subsequently scanned. Molecular weight markers were used during SDS-PAGE, and the approximate molecular weight of each detected polypeptide is annotated next to the band. ( b-f ) Quantification of the scanned blots was followed using the plug-in “Gel Blots” in ImageJ after conversion to grayscale images, and the band intensities and bit depth were calculated. The data were retrieved from triplicate experiments and after normalization using β-actin, bar charts were created. Each bar represents the average relative accumulation of the target protein compared to the untreated naïve PC-3 cells (first blot lane). The results were analyzed using multiple comparisons of one-way ANOVA in the Prism 8 software. (* corresponds to P =0,01; ** corresponds to P =0,001; *** corresponds to P =0,0001; and **** corresponds to P <0,0001). The error bars represent the standard error of the mean (SEM).

Article Snippet: The PC3 (ATCC, Manassas, VT, USA) cell line was used as a human prostate carcinoma cell model.

Techniques: Western Blot, Cell Culture, Autoradiography, Molecular Weight, SDS Page, Software

Journal: bioRxiv

Article Title: The ERK1/2-Elk1, JNK-cJun, and JAK-STAT Transcriptional Axes as Potential Bortezomib Resistance Mediators in Prostate Cancer

doi: 10.1101/2024.04.15.589569

Figure Lengend Snippet: Intracellular Reactive Oxygen Species Assay of Naïve PC-3 and PC-3 RB40 Cells Following Treatment with Bortezomib. Equal numbers of cells were cultured inside 100-mm dishes, and 24 h before analysis, the media were replaced. ( a ) Naïve PC-3 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS (blue coloring) (experimental control of baseline cell cycle progression); ( b ) Naïve PC-3 cells were cultured in medium containing 40 nM of Bortezomib for 24 h (magenta coloring); ( c ) PC-3 RB40 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS (yellow coloring); and ( d ) PC-3 RB40 cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS and 40 nM of Bortezomib (red coloring) (basal culture conditions for this cell line). The cells were then stained with the LIVE/DEAD kit and with H2DCFDA. Equal numbers of events were acquired using a FACS Calibur flow cytometer by measuring the fluorescence of the LIVE/DEAD stain and H2DCFDA, and the data were analyzed using the FlowJo software. The histograms display the median fluorescence intensity (MFI) of the H2DCFDA channel. The figure presents a representative experiment. The same procedure was replicated three times.

Article Snippet: The PC3 (ATCC, Manassas, VT, USA) cell line was used as a human prostate carcinoma cell model.

Techniques: Cell Culture, Control, Staining, Flow Cytometry, Fluorescence, Software

Journal: bioRxiv

Article Title: The ERK1/2-Elk1, JNK-cJun, and JAK-STAT Transcriptional Axes as Potential Bortezomib Resistance Mediators in Prostate Cancer

doi: 10.1101/2024.04.15.589569

Figure Lengend Snippet: Western analysis of JAK1, ERK1/2, p-ERK1/2, PI3K, p-PI3K, Akt, p-Akt, c-Src, p21, p27, and p53. Cells (Naïve PC-3 and PC-3 RB40) were cultured inside 100 mm dishes, and 24 h before confluency, the media were changed and fresh RPMI 1640 supplemented with 10% with or without the designated bortezomib doses (20, 40, 80 nM) was added. ( a ) The samples were prepared as previously noted, and by using specific polyclonal antibodies the accumulation of JAK1, ERK1/2, p-ERK1/2, PI3K, p-PI3K, Akt, p-Akt, c-Src, p21, p27, and p53 was detected and developed on autoradiography films, which were subsequently scanned. Molecular weight markers were used during SDS-PAGE and the approximate molecular weight of each detected polypeptide is annotated next to the band. ( b-l ) Quantification of the scanned blots was followed using the plug-in “Gels” in ImageJ after conversion to grayscale images, and the band intensities and bit depth were calculated. The data were retrieved from triplicate experiments and after normalization using β-actin, bar charts were created. Each bar represents the average relative accumulation of the target protein compared to the untreated naïve PC-3 cells (first blot lane). The results were analyzed using multiple comparisons of one-way ANOVA in the Prism 8 software. (* corresponds to P =0,01; ** corresponds to P =0,001; *** corresponds to P =0,0001; and **** corresponds to P <0,0001). The error bars represent the standard error of the mean (SEM).

Article Snippet: The PC3 (ATCC, Manassas, VT, USA) cell line was used as a human prostate carcinoma cell model.

Techniques: Western Blot, Cell Culture, Autoradiography, Molecular Weight, SDS Page, Software

Journal: bioRxiv

Article Title: The ERK1/2-Elk1, JNK-cJun, and JAK-STAT Transcriptional Axes as Potential Bortezomib Resistance Mediators in Prostate Cancer

doi: 10.1101/2024.04.15.589569

Figure Lengend Snippet: Western analysis of Nf-kB, p-Nf-κΒ, STAT1, p-STAT1, STAT3, p-STAT3, Elk1, p-Elk1, and cJun. Cells (Naïve PC-3 and PC-3 RB40) were cultured inside 100 mm dishes, and 24 h before confluency, the media were changed, and fresh RPMI 1640 supplemented with 10% with or without the designated bortezomib doses (20, 40, 80 nM) was added. ( a ) The samples were prepared as previously noted, and by using specific polyclonal antibodies the accumulation of Nf-kB, p-Nf-κΒ, STAT1, p-STAT1, STAT3, p-STAT3, Elk1, p-Elk1, and cJun was detected and developed on autoradiography films, which were subsequently scanned. Molecular weight markers were used during SDS-PAGE, and the approximate molecular weight of each detected polypeptide is annotated next to the band. ( b-j ) Quantification of the scanned blots was followed using the plug-in “Gel Blots” in ImageJ after conversion to grayscale images, and the band intensities and bit depth were calculated. The data were retrieved from triplicate experiments and after normalization using β-actin, bar charts were created. Each bar represents the average relative accumulation of the target protein compared to the untreated naïve PC-3 cells (first blot lane). The results were analyzed using multiple comparisons of one-way ANOVA in the Prism 8 software. (* corresponds to P =0,01; ** corresponds to P =0,001; *** corresponds to P =0,0001; and **** corresponds to P <0,0001). The error bars represent the standard error of the mean (SEM).

Article Snippet: The PC3 (ATCC, Manassas, VT, USA) cell line was used as a human prostate carcinoma cell model.

Techniques: Western Blot, Cell Culture, Autoradiography, Molecular Weight, SDS Page, Software

Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet: REIMS Analysis Predicts Breast Cancer Molecular Markers Including Oncogenic Mutations in PIK3CA (A) Schematic overview of sample preparation for REIMS analysis. (B) Area under the curve (AUC) classification accuracies for ER, PR, HER2 receptor, and triple negative status of 43 breast cancer (BC) cell lines (median intensity of n = 3 biological replicates) following feature selection for phospholipids in the m/z range 600–900, and leave-one-out cross validation. (C) Immunoblot analysis of estrogen inducible protein pS2 and predicted ESR1 expression in ER +ve MCF7 cells following treatment with 0.1% DMSO or indicated concentrations of 4-OHT for 72 h. (D) Unsupervised hierarchical clustering of 872 lipid species detected by REIMS across 43 BC cell lines. (E) Dendrogram of BC cell lines and isogenic MCF10A cells harboring either WT or MUT (E545K or H1047R) PIK3CA. (F) Immunoblot analysis of mature SREBP1 transcription factor expression in nuclear extracts of the MCF10A PIK3CA isogenic panel. (G) Relative exogenous fatty acid uptake in MCF10A PIK3CA WT and MUT cells following serum starvation for 1 h and supplementation with fluorescently labeled dodecanoic acid (n = 5 replicates). (H and I) Unsupervised hierarchical clustering of 9 PIK3CA WT and 9 MUT breast PDX tumors (H) and (I) 5 WT and 7 MUT primary breast tumors. Individual rows in the heatmaps in (D), (H) and (I) correspond to scaled Z score phospholipid intensities (n = 3 biological replicates). Error bars represent ± SEM. n.s., not significant; ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001. p values in (C, bottom panel) and (G) were calculated with one-way ANOVA, followed by unpaired, two-tailed Student’s t test with Bonferroni correction.

Article Snippet: MCF7 (human breast carcinoma) , ATCC , Cat# HTB-22; RRID: CVCL_0031.

Techniques: Sample Prep, Selection, Biomarker Discovery, Western Blot, Expressing, Labeling, Two Tailed Test

Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet: Related to Figure 2 (A) Cell viability of MCF10A PIK3CA MUT cells following treatment with increasing concentrations of rapamycin, torin 1, BYL-719, BKM120, MK2206 or GSK690693 for 72 hours. (B) Unsupervised hierarchical clustering of the median phospholipid intensities of 5 PIK3CA MUT breast cancer cell lines (MCF7, T47D, MDAMB361, MDAMB453 and BT474) treated with 20 nM rapamycin, 100 nM BYL-719 and 150 nM MK2006 for 72 hours. (C) Immunoblot analysis of mTORC1 and mTORC2 signaling in the PIK3CA MUT isogenic panel. Cells were serum and growth-factor starved for 16 hours and subsequently stimulated with 5% horse serum, 20 ng/ml EGF, 0.5 mg/ml hydrocortisone and 10 μg/ml insulin for 30 min. Data in (A) are presented as the mean ± SEM of n = 4 biological replicates and are representative of at least two independent experiments. n.s., not significant; ∗ p ≤ 0.05; ∗∗ p ≤ 0.01. P value s in (A) were calculated with unpaired, two-tailed Student’s t test.

Article Snippet: MCF7 (human breast carcinoma) , ATCC , Cat# HTB-22; RRID: CVCL_0031.

Techniques: Western Blot, Two Tailed Test

Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet:

Article Snippet: MCF7 (human breast carcinoma) , ATCC , Cat# HTB-22; RRID: CVCL_0031.

Techniques: Produced, Virus, Recombinant, Transfection, Protease Inhibitor, Lysis, Mutagenesis, Proliferation Assay, In Situ, Calcium Assay, Reverse Transcription, Kinase Assay, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Proximity Ligation Assay, CRISPR, Control, shRNA, Amplification, Plasmid Preparation, Positive Control, Software, Modification, Targeted Proteomics, Mass Spectrometry, Western Blot

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: ATR Target Activation by the CHK1 Inhibitor AZD7762 in U2OS Cancer Cells (A) Western blot showing activation of ATR targets. U2OS cells were treated with the indicated concentrations for 30 and 60 min, lysed, and probed with anti-phospho (Serine 345) CHK1 and β-actin antibodies. (B) Induction of pre-apoptotic pan-nuclear γ-H2AX by ATR and CHK1 inhibitor in combination in cancer cells. U2OS cells were treated with the indicated drug concentrations for 24 hr. Cells were probed with anti-phospho (Serine 139) H2AX antibody. Scale bar, 20 μm. (C) Quantitative data of γH2AX- (nine or more foci per cells) positive cells or pan-nuclear γH2AX signal after indicated treatments are shown (n = 3, mean ± SEM). (D) Western blot showing increased phosphorylation of H2AX after combination treatment. U2OS cells were treated with the indicated concentrations for 24 hr. At the end of incubation time, western blotting was performed using anti-phospho (Serine 139) H2AX, anti-phospho (Serine 345) CHK1, cleaved PARP, anti-phospho (Serine 10) H3, and β-actin antibodies. (E) Comet assay showing DNA damage induction by ATR and CHK1 inhibitor in combination. U2OS cells were treated with the indicated drug concentrations for 24 hr. At the end of incubation, cells were harvested and alkaline comet assay was performed. (F) Quantitative data of the tail moment are shown (n = 3, mean ± SEM, in each experiment ≥100 comets were measured). (G) Cancer-specific ssDNA formation by VE-821 and AZD7762, either alone or in combination. U2OS cells were treated with the indicated drug concentrations for 24 hr and pre-extracted using CSK buffer before fixation. Cells were stained with anti-RPA32 antibody; images were taken using a confocal microscope and were analyzed using ImageJ software. A mean intensity of ≥70 a.u. per cell was considered as positive. Quantitative data are presented as mean ± SEM from three independent experiments. (H) ssDNA formation in normal fibroblast VH-10 cells is shown. (I) Pre-apoptotic pan-nuclear γH2AX induction by combination treatment of ATR and CHK1 inhibitors in U2OS is mediated through the JNK pathway. U2OS cells were treated with the indicated drug concentrations for 24 hr. Cells were probed with anti-phospho (Serine 139) H2AX antibody, and high-throughput microscopy was used to determine the percentage of γH2AX-positive cells (nine or more γH2AX foci per cell) or an average intensity of ≥2,000 a.u. for pan-nuclear γH2AX-positive cells (n = 2 with multiple wells, mean ± SEM). Statistical significance was determined using one-way ANOVA ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001).

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Incubation, Single Cell Gel Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Staining, Microscopy, Software, High Throughput Screening Assay

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: Combination of the ATR Inhibitor VE-821 and the CHK1 Inhibitor AZD7762 Synergistically Kills Cancer Cells (A) Clonogenic survival of U2OS, VH-10, and MCF-7 cells. The 500 (U2OS and MCF-7) or 1,000 (VH-10) cells were seeded in 10-cm 2 dishes, and, after 5-hr incubation, the inhibitors were added directly to the media. After 72-hr incubation, drug-containing media were replaced with fresh media and cells were kept for another 5–8 days before colonies were stained with methylene blue. Quantitative data: n = 3, mean ± SEM. (B) Parental and cMYC-transformed cells were treated with the indicated doses for 72 hr. At the end of the incubation period, resazurin was added and cell viability was measured. Quantitative data: n = 3, mean ± SEM. (C) BJ-hTERT, BJ-hTERT SV40, and BJ SV40 RAS cells were treated with the indicated doses for 72 hr. At the end of the incubation period, resazurin was added and cell viability was measured. Quantitative data: n = 3, mean ± SEM. (D) CHK1 functionally compromised cells are sensitive to ATR inhibitor. Clonogenic survival of DLD-1, DLD-1 CHK1 S317A/− , DLD-1 CHK1 +/− , and DLD-1 ATR S/S after ATR inhibitor VE-821 treatment is shown. A similar protocol was used as for U2OS and VH-10 cells. Quantitative data: n = 3, mean ± SEM. (E) Therapeutic efficacy of combined inhibition of ATR and CHK1 in mouse tumor models. Therapeutic efficacy of VX-970 and AZD7762 in H460 lung cancer xenografted mice is shown. BALB/c nude mice bearing H460 xenograft were divided in four groups (five animals in each group) with a tumor volume of ∼130 mm 3 in each group. The first control group of animals was treated with vehicle (orally and intraperitoneally). The second group of animals was treated with 25 mg/kg body weight of CHK1 inhibitor AZD7762 (intraperitoneal route). The third group of animals was treated with 60 mg/kg body weight of ATR inhibitor VE-822 (oral administration), and the fourth group received a combination of both CHK1 and ATR inhibitors. Vehicle and drugs were administered on days 0–3, 10–12, and 18–20 irrespective of no mice survival in each group. Tumor volume was measured with calipers and is shown here as mean ± SEM. Statistical significance was determined using two-way ANOVA with repeated measurement ( ∗ p < 0.05 and ∗∗ p < 0.01). (F) Kaplan-Meier survival curve of H460-xenografted mice. When tumor size reached 1,000 mm 3 , the animal was sacrificed.

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Incubation, Staining, Transformation Assay, Drug discovery, Inhibition, Control

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: ATR and CHK1 Inhibitors, Alone or in Combination, Decrease Replication Fork Speed Only in Cancer Cells (A) Treatment regimen is shown. U2OS and VH-10 cells were treated for 60 min with the indicated drug concentrations and sequentially labeled with 5-chlorodeoxyuridine (CldU) and 5-iododeoxyuridine (IdU) for 30/20 min each in the presence of the inhibitors. DNA fibers were stained and replication speed was measured by IdU labeling. (B and C) Representative images show stained replication fork tracts for each treatment group. (D) Quantitative data of replication fork speed (kb/min), mean ± SEM, and p values were analyzed with one-way ANOVA for each condition and cell line. (E and F) Average distribution of replication fork rates. A minimum of 450 forks per condition were analyzed from at least three independent repeats.

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Labeling, Staining

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: Combination Treatment of VE-821 and AZD7762 Results in S Phase Arrest in U2OS Cells (A) U2OS cells were treated with the indicated drug concentrations for 24 hr and propidium iodide (PI) staining was carried out to measure cell-cycle profile using flow cytometry. (B) Quantitative data were obtained using Modfit software. (C) ATR and CHK1 inhibitors in combination decrease EdU incorporation in U2OS cells. U2OS cells were treated for 24 hr with the indicated doses. Images were taken with a confocal microscope and analyzed using ImageJ software. A mean intensity of ≥80 a.u. per cell was considered as EdU-positive cells. Quantitative data: n = 3, mean ± SEM. (D) No significant decrease in EdU incorporation in normal fibroblast VH-10 cells treated with the ATR and CHK1 inhibitors either alone or in combination. VH-10 cells were treated for 24 hr with the indicated doses. Quantitative data: n = 3, mean ± SEM. Statistical significance was determined using one-way ANOVA ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Staining, Flow Cytometry, Software, Microscopy

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: Synergistic Cytotoxic Effect in U2OS Cancer Cells by Combination Treatment of AZD7762/VE-821 Is Mainly Due to CDK-Mediated Excess Origin Firing (A) U2OS cells were pretreated with the indicated concentrations of the CDK inhibitor Roscovitine for 1 hr prior to the addition of VE-821 and AZD7762 for 24 hr. Cells were probed with anti-phospho (Serine 139) H2AX antibody and anti-53BP1, and DNA was counterstained with ToPro. (B) Quantitative data of pan-nuclear γH2AX are shown (mean ± SEM from two independent experiments). (C) Treatment regimen for DNA fiber assay in U2OS cells is shown. (D) CDK inhibitors Roscovitine and PHA-767491 enhance the replication fork speed of U2OS cells treated with VE-821 and AZD7762 in combination. U2OS cells were pre-treated with Roscovitine or PHA-767491 for 1 hr prior to the addition of VE-821 and AZD7762. Representative images show stained replication fork tracts for each treatment group. (E) Average distribution of replication fork rates. A minimum of 450 forks per condition were analyzed from at least three independent repeats. (F) Roscovitine abolishes the synergistic cytotoxic effect of combination treatment of AZD7762 and VE-821 in U2OS cancer cells. U2OS cells were individually treated with VE-821, AZD7762, or the combination with our without Roscovitine for 24 hr, followed by recovery for another 48 hr. Cell viability was measured by using resazurin at 72 hr. (G) Model for ATR/CHK1 synthetic lethality. CHK1 is activated by replication stress both by ATR-dependent and -independent pathways ( Yang et al., 2008 ) to suppress replication stress in cancer, promoting restart and survival. CHK1 inhibitors increase oncogene-activated CDK activity and origin firing, leading to replication stress and accumulation of stalled replication forks, requiring ATR activity to prevent replication collapse. Red arrows indicate primary route in the presence of both ATR and CHK1 inhibitors.

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Staining, Activity Assay

Journal: Cell Reports

Article Title: Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

doi: 10.1016/j.celrep.2015.12.032

Figure Lengend Snippet: HU-Induced Replication Stress in Combination with VE-821 and AZD7762 Causes Fragmented Nuclei and the Early Onset of Apoptosis Only in U2OS Cells (A) U2OS cells were treated for 24 hr with the indicated drug concentrations and stained with anti-cleaved caspase 3 and β-actin antibodies. Representative confocal images are shown. (B) Quantitative data of fragmented nuclei and cleaved caspase-3 positive cells presented as mean ± SEM from three independent experiments. (C) Western blot showing apoptosis in U2OS cells treated with ATR and CHK1 inhibitors alone or in combination. U2OS cells were treated with the indicated concentrations for 24 hr; lysed; protein extracted; and western blotting was performed with anti-Cleaved PARP, anti-phospho (Serine 10) Histone H3, and anti-β-actin antibodies. (D) HU-induced replication stress does not cause fragmentation of nuclei or apoptosis in combination with VE-821 and AZD7762 in VH-10 normal fibroblast cells in 24 hr. Etoposide treatment (4 μM) was used to induce apoptosis as a positive control. Scale bar represents 20 μM.

Article Snippet: U2OS (human bone osteosarcoma cells); VH-10 (human foreskin fibroblast cells); MCF-7 (human breast cancer cells) (ATCC); CCD841 (human colon epithelial cells) (ATCC); HA1EB-GFP (GFP-expressing HA1EB, human immortalized kidney epithelial cells); HA1EB-GFP-cMYC (GFP-cMYC-expressing HA1EB cells); and genetically modified cell lines BJ-hTERT (hTERT-immortalized BJ cells), BJ-SV40T (SV40T-transformed BJ-hTERT cells), and BJ-RASV12 (H-RAS V12-transformed BJ-SV40T cells) ( ) were grown in DMEM Glutamax.

Techniques: Staining, Western Blot, Positive Control

Journal: Advanced Science

Article Title: Discovery of Natural Compound α‐Hederin via Large‐Scale Screening as a Targeted JAK/STAT3 Inhibitor for Ovarian Cancer Therapy

doi: 10.1002/advs.202417278

Figure Lengend Snippet: Multiscale analysis identifies α‐Hederin as a JAK/STAT3‐targeting compound for OC. (A) 3D Principal component analysis (PCA). PCA plot was generated using the 2500 genes with the highest variance across samples. Normal tissues are represented by orange points, while OC samples are indicated by blue points. (B) Volcano plot showing differentially expressed genes between OC and normal tissues. (C) Boxplot showing increased mRNA expression of IL‐6 in OC tissues compared to normal tissues, based on TCGA and GTEx datasets. (D) GSEA indicating significant enrichment of the IL‐6/JAK/STAT3 signaling pathway in OC. (E) Uniform Manifold Approximation and Projection (UMAP) plot of 51 786 single cells from 11 epithelial ovarian cancer (EOC) patients ( GSE165897 ), color‐coded by patient identity. (F) Expression levels of IL6, IL6ST, JAK1, and STAT3 across single‐cell populations. (G) UMAP plots show the distribution of cells before and after NACT treatment. (H) JAK1 expression levels before and after NACT treatment. (I) Workflow of structure‐based virtual screening of 2908 natural compounds targeting JAK1 and JAK2, followed by cytotoxicity validation in OC and normal ovarian epithelial cells. (J) Dose‐response curves quantifying viability of OC cells upon drug treatment for 48 h. The code names of drugs are listed on the right. (K) Dose‐response curves quantifying viability of ovarian epithelial cells upon drug treatment for 48 h. (L) Binding affinity measurements of α‐Hederin and JAK1 as measured via MST thermophoresis curve analysis. (M) Binding affinity measurements of α‐Hederin and JAK2 as measured via MST thermophoresis curve analysis. (N) Schematic structures of JAK1. Molecular docking results of α‐Hederin (green) with JAK1 (blue). The docking sites of α‐Hederin on JAK1 were highlighted in magenta. (O) Schematic structures of JAK2. Molecular docking results of α‐Hederin (green) with JAK2 (blue). The docking sites of α‐Hederin on JAK2 were highlighted in magenta. (P) Venn diagram displaying α‐Hederin targets (pink) and OC‐associated genes (yellow). The overlapping regions indicate common targets. (Q) KEGG analysis highlighted the top 20 pathways with significant enrichment. Then red box indicated the JAK/STAT3 signaling pathway. (R)The schematic diagram of the drug‐target gene network was visualized using Cytoscape software.

Article Snippet: Active human JAK1 and JAK2 kinase, obtained from Signal Chem (Canada), was diluted to a final concentration of 0.1 μg mL −1 in Kinase Dilution Buffer III (Signal Chem, K23‐09).

Techniques: Generated, Expressing, Biomarker Discovery, Binding Assay, Software

Journal: Advanced Science

Article Title: Discovery of Natural Compound α‐Hederin via Large‐Scale Screening as a Targeted JAK/STAT3 Inhibitor for Ovarian Cancer Therapy

doi: 10.1002/advs.202417278

Figure Lengend Snippet: α‐Hederin directly binds to JAK1/2 and inhibits STAT3 phosphorylation and nuclear translocation. (A,B) IF staining of p‐STAT3 and statistical analysis of fluorescence intensity. Scale bar: 10 µ m . (C) Heatmap showing relative mRNA expression of STAT3 downstream targets (MYC, CCND1, BIRC5, BCL2, VEGFA, TWIST1, MMP2, and MMP9) following α‐Hederin treatment, measured by qRT‐PCR and normalized to GAPDH. (D) Western blot analysis of total and phosphorylated STAT3, JAK1, JAK2, JAK3, and SRC in SKOV‐3 cells treated with α‐Hederin (5 or 10 µ m ) or DMSO. Statistical analysis is presented. (E,F) Kinase assay to examine the effect of α‐Hederin on JAK1 (E) and JAK2 (F) kinase activity. (G,H) The competitive binding relationship between α‐Hederin and ATP was confirmed using a pull‐down assay. (I,J) In vitro kinase assays were performed using bacterial‐purified His‐STAT3 and the active JAK1 (I) and JAK2 (J) kinase. The amount of α‐Hederin in the reaction is indicated. (K) DARTS (drug affinity responsive target stability) assay showing α‐Hederin‐mediated stabilization of JAK1 and JAK2 proteins in SKOV‐3 lysates. (L) Western blot analysis of p‐STAT3 and total STAT3 in SKOV‐3 cells with sgCtrl, sgJAK1, sgJAK2, or sgJAK1+sgJAK2, treated or not with 5 µ m α‐Hederin. Bottom panel: quantification of p‐STAT3/STAT3 ratio. Data are presented as mean ± SD from at least three independent experiments. Statistical significance was determined by unpaired two‐tailed Student's t ‐test for two‐group comparisons and one‐way ANOVA for comparisons among multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: Active human JAK1 and JAK2 kinase, obtained from Signal Chem (Canada), was diluted to a final concentration of 0.1 μg mL −1 in Kinase Dilution Buffer III (Signal Chem, K23‐09).

Techniques: Phospho-proteomics, Translocation Assay, Staining, Fluorescence, Expressing, Quantitative RT-PCR, Western Blot, Kinase Assay, Activity Assay, Binding Assay, Pull Down Assay, In Vitro, Purification, Stability Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Discovery of Natural Compound α‐Hederin via Large‐Scale Screening as a Targeted JAK/STAT3 Inhibitor for Ovarian Cancer Therapy

doi: 10.1002/advs.202417278

Figure Lengend Snippet: Proposed schematic model of the anti‐tumor mechanism of α‐Hederin in OC. α‐Hederin directly binds to JAK1/2 kinases, inhibiting JAK‐mediated phosphorylation of STAT3. This blocks STAT3 activation and its subsequent nuclear translocation, leading to downregulation of target gene transcription involved in proliferation, migration, invasion, and EMT in OC cells.

Article Snippet: Active human JAK1 and JAK2 kinase, obtained from Signal Chem (Canada), was diluted to a final concentration of 0.1 μg mL −1 in Kinase Dilution Buffer III (Signal Chem, K23‐09).

Techniques: Phospho-proteomics, Activation Assay, Translocation Assay, Migration