Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: (A) Schematic depicts bioinformatics approach to compare Egr2 and Myrf binding sites in Schwann cells versus oligodendrocytes, respectively. Egr2 binding sites in Schwann cell-rich P15 rat sciatic nerve and Myrf sites in cultured oligodendrocytes were recently published (Lopez-Anido et al. 2015, Srinivasan et al. 2012, Bujalka et al. 2013). (B) Overlap analysis of genes with Egr2 and Myrf binding sites nearby identifies 304 genes that may be shared targets in Schwann cells and oligodendrocytes. (C) Gene ontology (GO) terms clustering and Kegg pathway analysis on Egr2/Myrf shared target genes reveals enrichment in signaling pathways and other processes. (D) Scatter plot represents gene expression for a given gene (represented by dots) in control versus Egr2-deficient sciatic nerve. Gene expression values were obtained from a previous microarray study on mice with a hypomorphic allele of Egr2 (Le et al. 2005a, Le et al. 2005b). Dark grey dots are either upregulated in Egr2-deficient nerve by >1.25-fold or downregulated by <0.8-fold. The black dots represent genes that are expressed in control nerve and also have an Egr2/Myrf binding event nearby, and these include (highlighted in red) the well-studied myelin gene Myelin-associated glycoprotein (Mag) as well as Dual specificity phosphatase 15 (Dusp15), a gene with an unknown role in peripheral nerve myelination. (E) ChIP-Seq analysis profiles depict genomic regions enriched with transcriptional regulators and enhancers in P15 rat sciatic nerve and spinal cord. Included are binding profiles of Sox10 and Egr2 (Lopez-Anido et al. 2015, Srinivasan et al. 2012). Profiles of H3K27ac and the oligodendrocyte-specific regulator Myrf in cultured oligodendrocytes were also obtained from published datasets (Yu et al. 2013, Bujalka et al. 2013, Lopez-Anido et al. 2015). Egr2- versus Myrf-enriched enhancers specific to Schwann cells versus oligodendrocytes are indicated by grey boxes.

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: Binding Assay, Cell Culture, Protein-Protein interactions, Gene Expression, Control, Microarray, ChIP-sequencing

Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: (A) ChIP-Seq analysis of P15 sciatic nerve reveals Sox10 and Egr2 enrichment at the Dusp15 gene, with a grey box highlighting the promoter region. (B) ChIP-Seq analysis on P25 rat sham sciatic nerve show the H3K27ac-marked enhancer (grey box), which is diminished in cut nerve (3 d post-injury). (C) Luciferase assays were performed in S16 Schwann cells with constructs containing the promoter region of Dusp15 (Pro_Dusp15) and treated with Sox10 siRNA. Relative light units (RLU) are shown relative to siControl treatment after normalizing to β-galactosidase activity from a co-transfected CMV-lacZ plasmid. Quantitative RT-PCR was used to determine relative mRNA expression levels in (D) S16 rat Schwann cells treated with siRNA against Sox10. (E) S16 Schwann cells were treated with siRNA against Egr2 and quantitative RT-PCR was used to measure mRNA levels of Sox10, Egr2 and Dusp15. (F) Dusp15 levels were measured in uncut (sham) or cut sciatic nerve (1 and 4 d post-injury) using quantitative RT-PCR. 18S housekeeping gene was used to normalize all quantitative RT-PCR assays and error bars indicate the standard deviation of three biological replicates (n=7 for 1 d post-injury and 3 for 4 d post-injury). (*P<0.05)

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: ChIP-sequencing, Luciferase, Construct, Activity Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: (A) Dusp15 levels were measured using quantitative RT-PCR in RT4 Schwann cells treated with siRNA against Dusp15. (B) Western Blot analysis of Erk1/2 phosphorylation was performed and (C) quantified in RT4 Schwann cells treated with siRNA against Dusp15. (D) Dusp15 mRNA levels were compared between S16 and RT4 Schwann cells using quantitative RT-PCR. (E) Diagram depicting guide RNA (red) target sites for CRISPR/Cas9 deletion of the Dusp15 gene. (F) Genotyping of Cas9 treated cells was determined by PCR using primers (orange pair) flanking the deleted genomic and the uncut 3′UTR region in both lines (green). (G) Dusp15 mRNA levels in WT and KO cell lines were determined by quantitative RT-PCR. (H) Erk1/2 phosphorylation was measured in WT versus KO lines and compared to total levels of Erk1/2 by western blot analysis. (I) Band signals obtained from western blot analysis were normalized to Total-Erk (T-Erk) levels. All experiments represent average values of biological triplicates. (*P<0.05)

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: Quantitative RT-PCR, Western Blot, Phospho-proteomics, CRISPR

Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: Quantitative RT-PCR was used to determine Ccl2, Vegfc and Pmp2 mRNA levels in (A) RT4 Schwann cells treated with siRNA against Dusp15 and (B) Dusp15-KO-RT4 Schwann cell line. (C) Ccl2 mRNA levels were measured in Dusp15-KO Schwann cells transfected with an expression plasmid for Dusp15 using quantitative RT-PCR. (*P<0.05)

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: Quantitative RT-PCR, Transfection, Expressing, Plasmid Preparation

Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: Mag, Mbp, Cx32, Pmp22 and Srebp-1c mRNA levels were measured by quantitative RT-PCR in (A) RT4 Schwann cells, Dusp15 and Mag in (B) Primary Schwann cells treated with siRNA against Dusp15 and in (C and D) Dusp15-KO Schwann cell line compared to parent RT4 Schwann cells. (E) Mbp and (F) Pmp22 mRNA levels were measured in Dusp15-KO Schwann cells treated with ectopic expression levels of Dusp15. All experiments represent biological triplicates, and expression levels were normalized to 18S rRNA. (*P<0.05)

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: Quantitative RT-PCR, Expressing

Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: Mbp, Mag, Pmp22, Mpz, Pmp2, Vegfc and Ccl2 mRNA levels were measured in RT4 Schwann cells treated with (A and B) Trametinib 18nM, or with (C, D and F) PMA using quantitative RT-PCR. Samples were normalized to the level of 18S rRNA. Error bars represent the standard deviation from biological triplicates.

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: Quantitative RT-PCR, Standard Deviation

Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: Mag, Mpz, Mbp, Pmp22, Sox10, Egr2, Dusp15, Pmp2 and Ccl2 were measured in Primary Schwann cells when these were treated with Trametinib (A) and PMA (B) as before. Genes were measured using quantitative RT-PCR and normalized to 18S rRNA. Error bars represent the standard deviation from biological triplicates. (*P<0.05)

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: Quantitative RT-PCR, Standard Deviation

Journal: BioMed Research International

Article Title: Comparison of Prostate-Specific Promoters and the Use of PSP-Driven Virotherapy for Prostate Cancer

doi: 10.1155/2013/624632

Figure Lengend Snippet: Conditional oncolytic effects of AdPSAE1 in prostate cancer cells. The design of a prostate-specific conditional replication-competent adenovirus AdPSAE1. The native Ad5 early region 1 (E1) gene that is required for adenoviral replication is replaced by an expression cassette that contains an Ad5 E1 gene under the control of an 860 bp PSA promoter. Human prostate cancer cell lines PPC-1 (a) and LNCaP (b), human bladder cancer cell line RT4 (c), human breast cancer cell line MCF-7 (d), and human glioma cell line 9L (e) were transduced with AdPSAE1 or AdPSAlacZ at moi of 1. Attached viable cells were stained with crystal violet 6 days after viral infection and were compared to the untreated controls.

Article Snippet: PPC-1 cells (1 × 10 7 cells in 0.2 mL of PBS) or RT4 cells (5.7 ×10 6 cells in 0.2 mL of PBS) were injected subcutaneously into the flank of male Balb/c athymic nude mice (Harlan Sprague Dawley, Indianapolis, IN, USA).

Techniques: Expressing, Control, Transduction, Staining, Infection

Journal: BioMed Research International

Article Title: Comparison of Prostate-Specific Promoters and the Use of PSP-Driven Virotherapy for Prostate Cancer

doi: 10.1155/2013/624632

Figure Lengend Snippet: Time course of the growth inhibition effects of AdPSAE1 on prostate cancer cells. Prostate cancer cells PPC-1 (a) and LNCaP (b) and nonprostate cancer cells (RT4, MCF-7, and 9L) (c) were transduced with AdPSAE1 at moi of 1. Cell numbers were determined daily from day 1 to 6 after viral transduction. Untreated and AdPSAlacZ transduced cells were used as controls. The data represent the results from two independent experiments, each performed in duplicate. Some error bars are too small to show.

Article Snippet: PPC-1 cells (1 × 10 7 cells in 0.2 mL of PBS) or RT4 cells (5.7 ×10 6 cells in 0.2 mL of PBS) were injected subcutaneously into the flank of male Balb/c athymic nude mice (Harlan Sprague Dawley, Indianapolis, IN, USA).

Techniques: Inhibition, Transduction

Journal: BioMed Research International

Article Title: Comparison of Prostate-Specific Promoters and the Use of PSP-Driven Virotherapy for Prostate Cancer

doi: 10.1155/2013/624632

Figure Lengend Snippet: Specific transgene expression driven by a PSA promoter in prostate cancer cells. Xenograft tumors were established by subcutaneous injection of cancer cells into the flank of nude mice. When tumors reached about 50 mm 3 , each of the adenoviral constructs was injected directly into the tumor. The tumors were harvested 72 hr later and processed to cryosections. Shown is X-gal staining of tumor sections derived from prostate cancer PPC-1 cells ((a), (c), and (e)) and bladder cancer RT4 cells ((b), (d), and (f)). (a) and (b) are untreated control tumors to serve as negative controls. (c) and (d) are tumors transduced by AdPSAlacZ (1 × 10 10 pfu). (e) and (f) are tumors transduced by AdRSVlacZ (5 × 10 9 pfu) to serve as positive controls.

Article Snippet: PPC-1 cells (1 × 10 7 cells in 0.2 mL of PBS) or RT4 cells (5.7 ×10 6 cells in 0.2 mL of PBS) were injected subcutaneously into the flank of male Balb/c athymic nude mice (Harlan Sprague Dawley, Indianapolis, IN, USA).

Techniques: Expressing, Injection, Construct, Staining, Derivative Assay, Control

Journal: BioMed Research International

Article Title: Comparison of Prostate-Specific Promoters and the Use of PSP-Driven Virotherapy for Prostate Cancer

doi: 10.1155/2013/624632

Figure Lengend Snippet: AdPSAE1 specifically inhibits prostate tumor growth in vivo . (a) The human prostate cancer line PPC-1 and (b) human bladder cancer line RT4 were injected subcutaneously into the flank of nude mice. When tumors reached an average volume of 200 mm 3 , tumors were either untreated (control) or treated with intratumoral injection (day 0) with 5 × 10 6 pfu of AdPSAlacZ (control virus) or 5 × 10 6 pfu AdPSAE1. The tumor sizes were periodically measured after viral injection. Each point represents the average tumor volume from 8 mice. Some error bars are too small to show.

Article Snippet: PPC-1 cells (1 × 10 7 cells in 0.2 mL of PBS) or RT4 cells (5.7 ×10 6 cells in 0.2 mL of PBS) were injected subcutaneously into the flank of male Balb/c athymic nude mice (Harlan Sprague Dawley, Indianapolis, IN, USA).

Techniques: In Vivo, Injection, Control, Virus

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: Farnesoid X receptor (FXR) expression and the effects on cell viability in human bladder cancers. ( A , B ) Overall and disease-free survival rate in differential expression of NR1H4 (FXR) gene in TCGA database of bladder cancer patients. ( C ) Scatter plots in differential expression of FXR gene in TCGA database of bladder cancer tissues (red plot) and adjacent normal tissues (blue plot). * p < 0.05 compared with the bladder cancer tissues group. ( D ) The expression levels of FXR and SHP in the bladder cancer cell lines were analyzed by Western blotting. GAPDH was a loading control. * p < 0.05 compared with the T24 group. ( E ) The survival rate of TSGH8301 and T24 were analyzed after doxycycline induced for 72 h in vector control and FXR overexpressed groups using MTT assay. ( F ) Colony formation assay of TSGH8301 and T24 were analyzed after 9 days of doxycycline induction in vector control and FXR overexpressed groups. Wells were visualized and quantified. ** p < 0.01; *** p < 0.001 compared with the control group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Expressing, Quantitative Proteomics, Western Blot, Control, Plasmid Preparation, MTT Assay, Colony Assay

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect on migration and adhesion abilities after FXR overexpression. ( A ) Wound healing migration assays were performed in FXR-overexpressed (FXR-O) TSGH8301 and T24 human bladder cancer cells after 6 h scratch. The right panels display the relative rate of the wound healing migratory ability. ** p < 0.01; *** p < 0.001 compared to the control group. ( B ) Adhesion assays were performed in FXR-O TSGH8301 and T24 cells after 50 min of incubation. Thereafter, the adhered cells were stained and captured. The right panels display the relative rate of the adhesive ability. ** p < 0.01 compared to the control group. Scale bar = 200 μm.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Migration, Over Expression, Relative Rate, Control, Incubation, Staining, Adhesive

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect on focal adhesion complex expression after FXR overexpression. ( A ) Focal adhesion kinase (FAK), phospho-focal adhesion kinase (p-FAK), integrin β1, integrin β3, myosin light chain (MLC) and phospho-myosin light chain (p-MLC) were analyzed by Western blotting in the TSGH8301 and T24 cells after FXR overexpression for 48 and 72 h. GAPDH was used as the loading control. ( B ) The bar graphs show the relative quantitative analysis of the aforementioned proteins. * p < 0.05; ** p < 0.01 compared with the control group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Expressing, Over Expression, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect of proteosome inhibitor MG132 and lysosome inhibitor NH4Cl on the migratory and adhesive abilities after FXR overexpression. ( A ) Wound healing migration assays were performed with or without MG132 or NH4Cl in TSGH8301 and T24 cells after 6 h scratch. The right panels displayed quantitative results of the relative migration rate. ** p < 0.01 compared to the control group; ## p < 0.01 compared to the FXR-O group. ( B ) Adhesion assays were performed in TSGH8301 and T24 cells for 50 min. The cells were stained and captured. The right panels displayed quantitative results of the relative adhesive rate. ** p < 0.01 compared to the control group; # p < 0.05 compared to the FXR-O group. Scale bar = 200 μm.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Adhesive, Over Expression, Migration, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect of MG132 and NH4Cl on integrin expression after FXR overexpression. ( A ) Integrin β1, integrin β3, MLC and p-MLC were analyzed by Western blotting in the TSGH8301 and T24 cells with or without MG132 or NH4Cl. GAPDH was used as the loading control. ( B ) The bar graphs show the relative quantitative analysis of the aforementioned proteins. ** p < 0.01 compared with the control group; # p < 0.05 compared to the FXR-O group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Expressing, Over Expression, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect of invasive abilities after FXR overexpression. ( A ) Transwell invasion assays were performed for 16 h incubation in the T24 cells after FXR overexpression. The invasive cells were stained and captured. The right panel displayed the quantitative result. ** p < 0.01 compared to the control group. Scale bar = 200 μm. ( B ) The expression of matrix metalloproteinases-2 (MMP2) and matrix metalloproteinases-9 (MMP9) were analyzed by Western blotting in the T24 cells. GAPDH was used as the loading control. ( C ) The concentration levels of MMP2 in the CM of T24 cells were analyzed by ELISA. ( D ) The protein activity of MMP9 in the CM of T24 cells were analyzed by the Fluorokine assay. * p < 0.05; ** p < 0.01 compared with the control group. # p < 0.05 compared to the FXR-O group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Over Expression, Incubation, Staining, Control, Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect of the overexpression of FXR on angiogenesis. ( A ) The human umbilical vein endothelial cells (HUVECs) were cultured with an FXR overexpression (72 h) conditioned medium (CM) and control CM of bladder cancer cells for 6 h. The formation of endothelial cell networks was observed and the number of branch points and tube length in the TSGH8301 and T24 CM were analyzed. The bar graphs show the quantitative results of relative branch points and tube lengths. Scale bar = 200 μm. ( B ) The concentration levels of vascular endothelial growth factor (VEGF) in the CM of TSGH8301 and T24 cells were analyzed by ELISA. ( C ) RT-PCR was used to analyze the mRNA expression of VEGF after FXR overexpression in TSGH8301 and T24 cells. * p < 0.05; ** p < 0.01; *** p < 0.001 compared with the control group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Over Expression, Cell Culture, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect of the overexpression of FXR on angiogenic related protein expression. ( A ) The expression of VEGFA, signal transducer and activator of transcription 3 (STAT3), phospho-Stat3 (p-STAT3), nitric oxide synthase 2 (NOS2) and hypoxia-inducible factors-1α (HIF-1α) were analyzed by Western blotting in TSGH 8301 and T24 cells after FXR overexpression. GAPDH was used as the loading control. ( B ) The bar graphs show the relative quantitative analysis of the aforementioned proteins. * p < 0.05; ** p < 0.01 compared with the control group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Over Expression, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect of proteosome inhibitor MG132 and lysosome inhibitor NH4Cl on the angiogenic abilities after FXR overexpression. ( A ) The human umbilical vein endothelial cells (HUVECs) were cultured with FXR overexpressed conditioned medium (CM) and control CM with or without MG132 or NH4Cl of bladder cancer cells for 6 h. The formation of an endothelial cell network was observed and the number of branch points and tube length in the T24 CM were analyzed. Scale bar = 200 μm. ( B ) VEGFA, p-STAT3, NOS2 and HIF-1α were analyzed by Western blotting in T24 cells with or without MG132 or NH4Cl. GAPDH was used as the loading control. The bar graphs show the relative attenuation of branch points and tube lengths. * p < 0.05, ** p < 0.01 compared with control group; # p < 0.05, ## p < 0.01; ### p < 0.001 compared with FXR overexpression group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Over Expression, Cell Culture, Control, Western Blot

Journal: Scientific Reports

Article Title: Amyloids in bladder cancer hijack cancer-related proteins and are positive correlated to tumor stage

doi: 10.1038/s41598-025-88307-7

Figure Lengend Snippet: Determination of amyloid presence in bladder cancer cell lines by spectrofluorimetry ( a - c ) and confocal microscopy ( d - g ). ( a ) ThT fluorescence fold change (mean ± SD) was plotted versus ThT concentration (µM) for serial dilutions (1.6 to 0.1 mg/mL) of protein homogenates from RT4, EJ138 and HT1197 cell lines. The scale of the x-axis is expressed as Log2. ( b ) ThT fluorescence fold change (mean ± SD) was plotted against protein concentration (mg/mL) for ThT serial dilutions (200 to 3.125 µM) for RT4, EJ138 and HT1197 cell lines. ( c ) Spectrofluorometric analysis of ThT (50 µM final concentration) fluorescence fold change of cell homogenates (at 1.6 mg/mL) of each indicated cell type was measured. Letters denote statistical differences between groups. ( d ) RT4, EJ138 and HT1197 cells were stained with ThT (green) to visualize amyloids. F-actin (red) was stained with fluorescent phalloidin to visualize the cell border. Images are representative areas of maximum intensity Z-projections of serial sections at 0.25 μm. Arrows: intracellular/intercellular droplet-like ThT staining. Asterisk: fibril extracellular ThT staining. ( e ) Single focal plane of primary urothelial cells stained with ThT (green) and fluorescent phalloidin (red). ( f ) Single focal plane of blood cells from healthy donors stained with ThT (green), fluorescent phalloidin (red) and TO-PRO-3 iodide (white). g: granulocyte. e: erythrocyte. ( g ) Single focal plane of RT4, EJ138, HT1197 and primary urothelial cells stained with ThT (green), anti-oligomer antibody (red) and fluorescent phalloidin (white) to visualize the cell border. Images shown were taken at identical microscope settings. Bar: 10 μm.

Article Snippet: RT4 (ECACC 91091914), EJ138 (ECACC 85061108) and HT1197 (ECACC 87032403) cell lines were obtained from the European Collection of Authenticated Cell Cultures (ECCAC).

Techniques: Confocal Microscopy, Fluorescence, Concentration Assay, Protein Concentration, Staining, Microscopy

Journal: Scientific Reports

Article Title: Amyloids in bladder cancer hijack cancer-related proteins and are positive correlated to tumor stage

doi: 10.1038/s41598-025-88307-7

Figure Lengend Snippet: Electrophoretic and size exclusion chromatographic analyses of cell line amyloids. ( a ) ThT staining of a 1.5% agarose gel native electrophoresis of protein homogenates treated with proteinase K (+ PK) or not treated (-PK). Protein homogenates from blood cells, RT4, EJ138, and HT1197 cell lines were included. The loaded sample in each well is indicated above each lane. Asterisks indicate the presence of positive ThT bands, which appear more evident in the signal on the weaker fluorescent background smear. ( b ) The same gel as in a , stained with Coomassie Blue. ( c ) Chromatogram obtained during gel filtration of HT1197 protein homogenate mixed with ThT, using Superdex matrix 200 (range: 3-600 kDa), recorded with in-line PDA (absorbance at 280 nm, 10 − 3 AU, blue line) and FD (Ex350 nm and Em472 nm, mV, black line). X-axis is elution volume (mL). Arrows indicate void volume (Vo) and total volume (Vt). Asterisks indicate ThT-positive peaks.

Article Snippet: RT4 (ECACC 91091914), EJ138 (ECACC 85061108) and HT1197 (ECACC 87032403) cell lines were obtained from the European Collection of Authenticated Cell Cultures (ECCAC).

Techniques: Staining, Agarose Gel Electrophoresis, Electrophoresis, Filtration

Journal: Scientific Reports

Article Title: Amyloids in bladder cancer hijack cancer-related proteins and are positive correlated to tumor stage

doi: 10.1038/s41598-025-88307-7

Figure Lengend Snippet: Analysis of cell line amyloids purified by ultracentrifugation. ( a ) Microscopic analysis of purified amyloids from RT4, EJ138 and HT1197, stained with ThT and visualized by fluorescence microscopy, Congo red and visualized by polarization microscopy or uranyl acetate and visualized by transmitted electron microscopy. The arrows in each image show examples of the green-yellow colour of the amyloids when they are stained with Congo red and observed under polarized light. Blood samples from healthy donors subjected to the same ultracentrifugation protocol were included as a negative control for each staining. Bars: 1 μm. ( b ) Far-UV CD analysis of amyloids derived from EJ138 and HT1197. The black line represents the data obtained, while the red trace corresponds to the best fit from the analysis performed with the BeStSel web server ( https://bestsel.elte.hu/ ), which indicates that EJ138 and HT1197 have β-sheet contents of 34.2% and 35.5%, respectively. The inset shows the residuals of the fit. ( c ) Dynamic Light Scattering (DLS) analysis of amyloids derived from RT4, EJ138 and HT1197 cell lines. The left panel illustrates the highly reproducible autocorrelation functions obtained in triplicate for each batch, while the right panel depicts representative intensity-weighted size distributions. The hydrodynamic radius (RH) is plotted on the x-axis, and the Z-average (Zave) and polydispersity index (PdI), calculated as the mean of three replicates, are indicated alongside each distribution. ( d ) Differential Scanning Fluorimetry (DSF) obtained with amyloids from RT4, EJ138 and HT1197 cell lines. The ratio of intrinsic fluorescence measured at 350 and 330 nm upon heating and cooling scans is displayed in the left and right panels, respectively.

Article Snippet: RT4 (ECACC 91091914), EJ138 (ECACC 85061108) and HT1197 (ECACC 87032403) cell lines were obtained from the European Collection of Authenticated Cell Cultures (ECCAC).

Techniques: Purification, Staining, Fluorescence, Microscopy, Electron Microscopy, Negative Control, Derivative Assay