Journal: Journal of Oncology

Article Title: Initial Report on Differential Expression of Sprouty Proteins 1 and 2 in Human Epithelial Ovarian Cancer Cell Lines

doi: 10.1155/2012/373826

Figure Lengend Snippet: Expression of Spry1 and Spry2 proteins in a range of ovarian cancer cell lines compared with human ovarian surface epithelial cell line (HOSEpiC). (a) Western blot analysis of Spry1 and Spry2 expression in HOSEpiC, OVCAR-3, SKOV-3, 1A9, A2780, CAOV-3, OV-90, and IGROV-1 cell lines using GAPDH as a loading control. (b)-(c) Quantification of Spry expression in the cell lines yielded from at least three independent immunoblots similar to (a), using ImageQuant software. Loading amounts and quantification were normalized against GAPDH values. Moreover, the expression levels in cancer cell lines were normalized against those in HOSEpiC cells used as the normal control. The graphs represent the expression of Spry1 (b) and Spry2 (c) in arbitrary units after normalization. Altered expression of both isoforms was observed in the cancer cells as compared to the control. Data are shown as means ± SE. Significant values ( P < 0.05) are indicated with asterisks.

Article Snippet: Cells were then incubated overnight at 4°C with monoclonal anti-Spry1 and anti-Spry2 antibodies (1 : 20 in 1% BSA/1x PBS) (Abnova, Taiwan), with the exception of the negative control samples to which no primary antibody was applied.

Techniques: Expressing, Western Blot, Control, Software

Journal: Journal of Oncology

Article Title: Initial Report on Differential Expression of Sprouty Proteins 1 and 2 in Human Epithelial Ovarian Cancer Cell Lines

doi: 10.1155/2012/373826

Figure Lengend Snippet: The presence of Spry1 and 2 mRNAs evaluated by RT-PCR in OVCAR-3 (1), SKOVE-3 (2), 1A9 (3), A2780 (4), CAOV-3 (5), OV-90 (6), and IGROV-1 (7) cells compared to HOSEpiC. As seen above, Spry1 and Spry2 were differently expressed in the cancerous cells. While Spry1 mRNA was almost undetectable in CAOV-3 cells, it was expressed by other cells, being more prominent in 1A9. Spry2 mRNA was identified in all cell lines with more prominent expression in cancerous ones. The product size for Spry1 and Spry2 was 438 bases and 471 bases, respectively. β -actin was used as a reference.

Article Snippet: Cells were then incubated overnight at 4°C with monoclonal anti-Spry1 and anti-Spry2 antibodies (1 : 20 in 1% BSA/1x PBS) (Abnova, Taiwan), with the exception of the negative control samples to which no primary antibody was applied.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of Oncology

Article Title: Initial Report on Differential Expression of Sprouty Proteins 1 and 2 in Human Epithelial Ovarian Cancer Cell Lines

doi: 10.1155/2012/373826

Figure Lengend Snippet: The subcellular localization of Spry1 and Spry2 proteins in primary human ovarian surface epithelial cell line HOSEpiC and human epithelial ovarian cancer cell lines OVCAR-3 and SKOV-3 using confocal immunofluorescence microscopy (60X oil immersion lense). Affinity-purified antibodies were used to specifically detect Spry1 and Spry2 (green). Propidium iodide staining was performed for nuclei (red). Two top rows represent HOSEpiC cells, two middle rows show SKOV-3 cells, and the bottom rows indicate OVCAR-3 cells. As seen, the staining intensity of Spry1 and Spry2 proteins was stronger in OVCAR-3 cells compared with HOSEpiC and SKOV-3 cells. Moreover, whereas Spry2 was predominantly distributed within the whole cytoplasm in vesicular structures, Spry1 was found in both cytoplasm and nucleus. Scale bar: 50 μ m.

Article Snippet: Cells were then incubated overnight at 4°C with monoclonal anti-Spry1 and anti-Spry2 antibodies (1 : 20 in 1% BSA/1x PBS) (Abnova, Taiwan), with the exception of the negative control samples to which no primary antibody was applied.

Techniques: Immunofluorescence, Microscopy, Affinity Purification, Staining

Journal: Journal of Oncology

Article Title: Initial Report on Differential Expression of Sprouty Proteins 1 and 2 in Human Epithelial Ovarian Cancer Cell Lines

doi: 10.1155/2012/373826

Figure Lengend Snippet: Immunohistochemical staining of Spry1 and Spry2 in paraffin-embedded slides from three different patients with epithelial ovarian cancer observed by Leica DMLB microscope (magnification ×40) and photographed using Leica DC200 digital imaging system. The blue color represents unstained parts and the yellow-brown color shows stained Spry with different intensity. As seen, Spry2 is expressed in cytoplasm (a), (c), and (e), while Spry1 staining is observed in both cytoplasm and nucleus (b), (d), and (f).

Article Snippet: Cells were then incubated overnight at 4°C with monoclonal anti-Spry1 and anti-Spry2 antibodies (1 : 20 in 1% BSA/1x PBS) (Abnova, Taiwan), with the exception of the negative control samples to which no primary antibody was applied.

Techniques: Immunohistochemical staining, Staining, Microscopy, Imaging

Journal: Cell Genomics

Article Title: Mutant FOXO1 controls an oncogenic network via enhancer accessibility

doi: 10.1016/j.xgen.2024.100537

Figure Lengend Snippet: Degradation of FOXO1 disrupts the DZ gene expression program (A) MA plots of mRNA levels measured by RNA-seq 6 and 24 h after FOXO1 degradation. Differentially expressed genes (DEGs) are labeled in blue (down) or red (up) (padj <0.05 and fold change ≥|1.5|) (CuffDiff). (B) Heatmap of the log2(FPKM dTAG /avg FPKM CTR ) for each DTG. (C) Boxplots show average log2(FoldChange) in mRNA levels separated by PRO-Seq regulatory group and cell line, 6 h (top) and 24 h (bottom) after dTAG-47 treatment (Wilcoxon). (D) Average fold change of mRNA levels of CXCR4 (top) and IL-7R (bottom) after dTAG-47 treatment with SEM from two biological replicates. (E) Average fold change of cell surface protein levels measured by flow cytometry (median fluorescence intensity) for CXCR4 (top) and IL-7R (bottom) 24 h after DMSO or dTAG-47 treatment with SEM from three biological replicates (t test). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. See also Figure S3 .

Article Snippet: Antibodies were used at the following concentrations per 1 x 10 6 cells: CXCR4 (BD Biosciences, clone 12G5): 5 μL and IL7R (BD Biosciences, clone HIL-7R-M21): 15 μL.

Techniques: Expressing, RNA Sequencing Assay, Labeling, Flow Cytometry, Fluorescence

Journal: Cell Genomics

Article Title: Mutant FOXO1 controls an oncogenic network via enhancer accessibility

doi: 10.1016/j.xgen.2024.100537

Figure Lengend Snippet: AKT signaling prevents WT FOXO1 from rescuing mFOXO1 transcriptional activity (A) Schematic depicting point mutations and domains targeted to assess FOXO1 transcriptional function. ARM, AKT regulatory motif; FHD, Forkhead domain (DNA-binding domain); NES, nuclear export sequence; CR3, conserved region 3 (transcriptional activation domain). (B) Western blot showing expression levels WT FOXO1 and specified point mutants or domain deletions in 293FT cells. (C) Quantification of IL-7R protein levels in the OCI-LY1-FOXO1-FKBP cell line 24 h after dTAG-47 treatment. Data are presented as average median fluorescence intensity (MFI) with SEM from three biological replicates (ANOVA). (D) Quantification of CXCR4 protein levels in the OCI-LY1-FOXO1-FKBP cell line 24 h after dTAG-47 treatment. Data are presented as average MFI with SEM from three biological replicates (ANOVA). (E) Quantification of IL-7R protein levels in the OCI-LY1-FOXO1-FKBP cell line 24 h after treatment with dTAG-47 alone or in combination with capivasertib (AKT inhibitor). Data are presented as average MFI with SEM from 4–6 biological replicates (ANOVA). (F) Quantification of CXCR4 protein levels in the OCI-LY1-FOXO1-FKBP cell line 24 h after treatment with dTAG-47 alone or in combination with capivasertib (AKT inhibitor). Data are presented as average MFI with SEM from 4 to 6 biological replicates (ANOVA). (G) Average fold change with SEM of IL-7R protein levels 24 h after treatment with the specified drug from three biological replicates (ANOVA). (H) Average fold change with SEM of CXCR4 protein levels 24 h after treatment with the specified drug from three biological replicates (ANOVA). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. See also Figure S7 .

Article Snippet: Antibodies were used at the following concentrations per 1 x 10 6 cells: CXCR4 (BD Biosciences, clone 12G5): 5 μL and IL7R (BD Biosciences, clone HIL-7R-M21): 15 μL.

Techniques: Activity Assay, Binding Assay, Sequencing, Activation Assay, Western Blot, Expressing, Fluorescence

Journal: Cell Genomics

Article Title: Mutant FOXO1 controls an oncogenic network via enhancer accessibility

doi: 10.1016/j.xgen.2024.100537

Figure Lengend Snippet:

Article Snippet: Antibodies were used at the following concentrations per 1 x 10 6 cells: CXCR4 (BD Biosciences, clone 12G5): 5 μL and IL7R (BD Biosciences, clone HIL-7R-M21): 15 μL.

Techniques: Recombinant, Clone Assay, Mutagenesis, CRISPR, Software, RNA Sequencing Assay