Journal:

Article Title: Identification of Acyloxyacyl Hydrolase, a Lipopolysaccharide- Detoxifying Enzyme, in the Murine Urinary Tract

doi: 10.1128/IAI.72.6.3171-3178.2004

Figure Lengend Snippet: Bladder cells take up AOAH precursor. (A) Uptake of pro-AOAH by T24 cells after incubation with pro-AOAH-containing medium for 5 h in the presence or absence of 10 mM M6P, G6P, or mannose. Washed cells were lysed and AOAH protein was assayed by ELISA. Each bar shows the mean and standard error of data from four independent experiments. (B) AOAH specific activity (activity/nanograms of protein) in T24 cell lysates. Compared to the pro-AOAH added in the medium (AOAH Source), cell-associated AOAH had a much greater specific activity, reflecting its activation by the T24 cells. M6P inhibited AOAH binding (A) but did not prevent the activation of the cell-associated AOAH (B).

Article Snippet: LLC-PK1 porcine proximal tubule cells (American Type Culture Collection [ATCC], CL-101), T24 human bladder cells (ATCC, HTB-4), and AOAH-transfected and untransfected BHK570 cells ( 31 ) were cultured in low-glucose Dulbecco modified Eagle medium (Invitrogen, Carlsbad, Calif.), Vitacell McCoy's 5a medium (ATCC), or DMEM with glutamine and 4.5 g of glucose (Fisher Scientific)/liter, respectively.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Activity Assay, Activation Assay, Binding Assay

Journal:

Article Title: Identification of Acyloxyacyl Hydrolase, a Lipopolysaccharide- Detoxifying Enzyme, in the Murine Urinary Tract

doi: 10.1128/IAI.72.6.3171-3178.2004

Figure Lengend Snippet: AOAH confers LPS-deacylating activity to bladder cells. T24 cells were allowed to take up AOAH for 5 h, washed, and then incubated with [3H]LPS (125 ng/ml) for the times indicated. Control cells were incubated with medium that did not contain AOAH. Whereas control and AOAH-containing cells took up similar amounts of [3H]LPS (A), only the AOAH-containing cells removed 3H-labeled fatty acids from the LPS backbone (B). The data represent combined results of three separate experiments; the error bars represent one standard error of the mean. Solid squares and bars, T24 cells with AOAH; open circles and bars, control T24 cells.

Article Snippet: LLC-PK1 porcine proximal tubule cells (American Type Culture Collection [ATCC], CL-101), T24 human bladder cells (ATCC, HTB-4), and AOAH-transfected and untransfected BHK570 cells ( 31 ) were cultured in low-glucose Dulbecco modified Eagle medium (Invitrogen, Carlsbad, Calif.), Vitacell McCoy's 5a medium (ATCC), or DMEM with glutamine and 4.5 g of glucose (Fisher Scientific)/liter, respectively.

Techniques: Activity Assay, Incubation, Control, Labeling

Journal: Cancers

Article Title: Inducing Targeted, Caspase-Independent Apoptosis with New Chimeric Proteins for Treatment of Solid Cancers.

doi: 10.3390/cancers17071179

Figure Lengend Snippet: Figure 4. GnRH-AIF chimeric protein kills target cancer cells in a dose- and time-dependent manner. Colo205 and LNCaP cells were treated with various doses of GnRH-AIF and GnRH-AIFinact (0.5, 0.7, and 1 µM) and incubated for 24, 48, or 72 h demonstrating a dose- and time-dependent cell death (A,B). Simultaneously, Colo205, LNCaP, MCF-7, SW48, Colo320, HEK293, and A549 were treated with GnRH-AIF (1 µM) and GnRH-AIFinact mutant chimera (1 µM) for 72 h and tested under the same conditions (C). A204 cells, normal fibroblasts, and T24P cancer cells lacking GnRH-R expression were treated with GnRH-AIF or PBS (D). Results (A–D) are presented as percentage of cell death calculated for treated cells as compared to control cells (treated with PBS only). All results are the average of 3–6 repeats of the experiments ± SD. p value ≤0.01 = **; p value ≤0.001 = ***; inact = inactive.

Article Snippet: A204 rhabdomyosarcoma cells (ATCC-HTB-82), T24P bladder urinary transitional cell carcinoma (ATCC-HTB-4), normal fibroblasts, (ATCC-PCS-420-013), lung A549 cells (ATCC-CCL-185), and HEK-293 renal adenocarcinoma cells (ATCC-CRL-1573) were grown in DMEM medium (01-055-1A, Sartorius) supplemented with 10% FBS, 2 mM L-glutamine, and 100 units/mL penicillin.

Techniques: Incubation, Mutagenesis, Expressing, Control

Journal: American Journal of Translational Research

Article Title: Gene expression profiles to analyze the anticancer and carcinogenic effects of arsenic in bladder cancer

doi:

Figure Lengend Snippet: The expression of DEGs belonging to different intersections. Human bladder cancer cells T24 were treated with 1 μM ATO for 1 h and 6 h. Expression values were calculated using 2-ΔΔCT method and GAPDH as the endogenous reference gene.

Article Snippet: Cell culture and treatment The T24 human bladder cancer cell line was purchased from Procell Co. Ltd., and mycoplasma testing was conducted.

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: Gene expression profiles to analyze the anticancer and carcinogenic effects of arsenic in bladder cancer

doi:

Figure Lengend Snippet: Expression of putative downstream genes of arsenic binding proteins in each expression matrix (A) or T24 cells examined by real-time PCR (B). Human T24 bladder cancer cells were treated with 1 μM ATO for 1 h and 6 h. Expression values were calculated using the 2-ΔΔCT method and GAPDH as the endogenous reference gene.

Article Snippet: Cell culture and treatment The T24 human bladder cancer cell line was purchased from Procell Co. Ltd., and mycoplasma testing was conducted.

Techniques: Expressing, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Cancer Research

Article Title: Aberrant Nuclear Export of circNCOR1 Underlies SMAD7-Mediated Lymph Node Metastasis of Bladder Cancer

doi: 10.1158/0008-5472.CAN-21-4349

Figure Lengend Snippet: circNCOR1 inhibits LN metastasis of bladder cancer by inhibiting the TGFβ–SMAD signaling pathway. A – C, Representative images ( A ) and quantification of the tube formation ( B ) and Transwell migration ( C ) by HLECs cocultured with UM-UC-3 cells. Scale bars, 100 μm. D, The popliteal LN metastatic rate in the nude mice ( n = 12 per group). E and F, Bioluminescence ( E ) and LN volume ( F ) of popliteal LN metastasis in the nude mice ( n = 12 per group). G, Luciferase activity of the SMAD7 promoter in indicated UM-UC-3 cells. H, qRT-PCR analysis of SMAD7 expression in indicated UM-UC-3 cells. I and J, Western blotting of SMAD7 expression in indicated UM-UC-3 ( I ) and T24 ( J ) cells. The statistical difference was assessed by the two-tailed Student t test in B , C , E , and F , and the χ 2 test in D ; and one-way ANOVA followed by Dunnett tests in G and H . Error bars show the SD from three independent experiments. **, P < 0.01.

Article Snippet: The human urinary bladder transitional cell carcinoma cell lines T24 (RRID: CVCL_0554), UM-UC-3 (RRID: CVCL_1783) were purchased from ATCC, RT112 (RRID: CVCL_1670) was purchased from German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) and UM-UC-1 (RRID: CVCL_2743) was purchased from European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Migration, Luciferase, Activity Assay, Quantitative RT-PCR, Expressing, Western Blot, Two Tailed Test

Journal: Cancer Science

Article Title: Role of galectin‐1 in urinary bladder urothelial carcinoma cell invasion through the JNK pathway

doi: 10.1111/cas.13016

Figure Lengend Snippet: Effects of galectin‐1 (Gal‐1) knockdown on the malignant characteristics of urinary bladder urothelial carcinoma T24 and J82 cells. (a) Cell survival assays of T24 cell clones with knockdown at +120 nt (sh‐Gal‐1(+120)) and +380 nt (sh‐Gal‐1(+380)). (b) Cell number counting assays of sh‐Gal‐1(+120) T24 cells. (c–e) Soft agar (c), cell migration (d), and cell invasion (e) assays of sh‐Gal‐1(+120) and sh‐Gal‐1(+380) T24 cells. (f,g) Cell migration (f) and cell invasion (g) assays of sh‐Gal‐1(+120) J82 cells. The results of MTT , cell number counting, soft agar, migration, and invasion assays show representative data of three independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: Human urinary bladder urothelial carcinoma T24 cell, which is recognized as high grade and invasive, was purchased in 2010 from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and cultured at 37°C in McCoy's 5A (Gibco, Life Technologies, Grand Island, NY, USA), supplemented with 10% FBS (Gibco).

Techniques: Knockdown, Clone Assay, Migration

Journal: Cancer Science

Article Title: Role of galectin‐1 in urinary bladder urothelial carcinoma cell invasion through the JNK pathway

doi: 10.1111/cas.13016

Figure Lengend Snippet: Impacts of galectin‐1 (Gal‐1) silencing on the Gal‐1‐mediated signaling pathway. (a) MMP 9 protein (left panel) and mRNA (middle panel) amounts in T24 cell clones with knockdown at +120 nt (shGal‐1(+120)) measured by Western immunoblotting and RT ‐ PCR , respectively. MMP 9 activities (right panel) in shGal‐1(+120) T24 cells were evaluated by zymography. The blot in each figure was the typical result of at least three independent studies. The diagram (ratio [mean ± SD]) under each blot shows the differential protein expression that was expressed as the ratio of normalized intensity (observed protein/actin) of sh‐Gal‐1(+120) cells divided by that of scrambled sh‐Sc(+120) cells. The number above each bar is the average ratio. (b) MMP 9 protein level in shGal‐1(+120) J82 cells. (c) Confocal microscopic image of shGal‐1(+120) T24 cells, typical of three independent experiments. shGal‐1(+120) and sh‐Sc(+120) T24 cells were transfected with H‐Ras expression vector ( pMSCV ‐H‐Ras(12V)‐ GFP ). H‐Ras protein is shown in green and the cell membrane is stained in red in T24 sh‐Sc(+120) cells. Yellow (arrow), shown on the cell membrane in the merged image, indicates that H‐Ras was located on cell membrane of T24 sh‐Sc(+120) cells. However, the cell membrane remained red in the merged image of T24 sh‐Gal‐1(+120) cells, indicating that H‐Ras did not appear on the cell membrane because of Gal‐1 knockdown. (d) Interaction of Gal‐1 with H‐Ras in plasma membranes of shGal‐1(+120) cells. Protein lysate from purified plasma membrane immunoprecipitated ( IP ) with antibodies against H‐Ras and Gal‐1 proteins present in the immunocomplex was detected by western immunoblotting and vice versa. (e) Rac1 protein level in shGal‐1(+120) T24 cells. Protein lysates were collected for Western immunoblotting ( WB ). GTP or GDP ‐incorporated protein lysates were used as positive or negative controls. (f) MEKK 4 protein amount in shGal‐1(+120) T24 cells. (g) Interaction of Rac1 with H‐Ras or Gal‐1 in plasma membranes of shGal‐1(+120) cells. Protein lysate from purified plasma membrane was immunoprecipitated with antibody against H‐Ras or Gal‐1 protein and Rac1 protein present in the immunocomplex was detected by Western immunoblotting. Flotillin‐2 was the loading control. (h) JNK (left panel) and phosphorylated (p‐) JNK ) (right panel) protein levels in shGal‐1(+120) T24 cells. (i) p‐ JNK protein amount in shGal‐1(+120) J82 cells. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: Human urinary bladder urothelial carcinoma T24 cell, which is recognized as high grade and invasive, was purchased in 2010 from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and cultured at 37°C in McCoy's 5A (Gibco, Life Technologies, Grand Island, NY, USA), supplemented with 10% FBS (Gibco).

Techniques: Clone Assay, Knockdown, Western Blot, Reverse Transcription Polymerase Chain Reaction, Zymography, Expressing, Transfection, Plasmid Preparation, Membrane, Staining, Clinical Proteomics, Purification, Immunoprecipitation, Control

Journal: Cancer Science

Article Title: Role of galectin‐1 in urinary bladder urothelial carcinoma cell invasion through the JNK pathway

doi: 10.1111/cas.13016

Figure Lengend Snippet: Influences of galectin‐1 (Gal‐1) knockdown on c‐Jun and activator protein 1 ( AP 1) activities in urinary bladder urothelial carcinoma cells. (a) c‐Jun protein levels in nucleus (left panel) and cytoplasm (right panel) of cell clones with knockdown at +120 nt (shGal‐1(+120)). shGal‐1(+120) or scrambled (sh‐Sc(+120)) cells were treated with 2 μg/mL PMA at 37°C for 24 h, then the nuclear and cytoplasmic extracts were extracted from cells. (b) AP 1 activity was observed by Ch IP assay. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: Human urinary bladder urothelial carcinoma T24 cell, which is recognized as high grade and invasive, was purchased in 2010 from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and cultured at 37°C in McCoy's 5A (Gibco, Life Technologies, Grand Island, NY, USA), supplemented with 10% FBS (Gibco).

Techniques: Knockdown, Clone Assay, Activity Assay

Journal: Cancer Science

Article Title: Role of galectin‐1 in urinary bladder urothelial carcinoma cell invasion through the JNK pathway

doi: 10.1111/cas.13016

Figure Lengend Snippet: Effects of basic fibroblast growth factor (b FGF ), Rac1 activator, and SP 600125 on T24 cell clones with knockdown at +120 nt (sh‐Gal‐1(+120)). (a) Impacts of b FGF on sh‐Gal‐1(+120) T24 cell migration. Cells were treated with 1 μg/mL b FGF for 12 h then cells were evaluated for migration. (b) Influence of bFGF on sh‐Gal‐1(+120) T24 cell invasion. (c) Expression of phosphorylated (p‐) JNK in b FGF ‐treated sh‐Gal‐1(+120) T24 cells. (d) MMP 9 protein level in b FGF ‐treated sh‐Gal‐1(+120) T24 cells. (e) Rac1 activator‐incubated sh‐Gal‐1(+120) T24 cell invasion. (f) Rac1 activator‐treated sh‐Gal‐1(+120) T24 cell migration. (g) p‐ JNK protein level in Rac1 activator‐treated sh‐Gal‐1(+120) T24 cells. (h) MMP 9 protein amount in Rac1 activator‐treated sh‐Gal‐1(+120) T24 cells. (i) p‐Erk protein level in Rac1 activator‐treated sh‐Gal‐1(+120) T24 cells. (j) SP 600125‐incubated sh‐Gal‐1(+120) T24 cell migration. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: Human urinary bladder urothelial carcinoma T24 cell, which is recognized as high grade and invasive, was purchased in 2010 from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and cultured at 37°C in McCoy's 5A (Gibco, Life Technologies, Grand Island, NY, USA), supplemented with 10% FBS (Gibco).

Techniques: Clone Assay, Knockdown, Migration, Expressing, Incubation

Journal: Cancer Science

Article Title: Role of galectin‐1 in urinary bladder urothelial carcinoma cell invasion through the JNK pathway

doi: 10.1111/cas.13016

Figure Lengend Snippet: Impacts of galectin‐1 (Gal‐1) overexpression on TSGH 8301 low‐grade urinary bladder urothelial carcinoma cells. (a) Migration of TSGH 8301 cells overexpressing Gal‐1, constructed by transfection of PBK ‐ CMV ‐Gal‐1 vector inserted with Gal‐1 c DNA . (b) Invasion of TSGH 8301 cells overexpressing Gal‐1. (c) Phosphorylated (p‐) JNK and MMP 9 protein levels in TSGH 8301 cells overexpressing Gal‐1. Blot shows the typical result of three independent experiments. Right panel shows densitometer intensity data. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: Human urinary bladder urothelial carcinoma T24 cell, which is recognized as high grade and invasive, was purchased in 2010 from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and cultured at 37°C in McCoy's 5A (Gibco, Life Technologies, Grand Island, NY, USA), supplemented with 10% FBS (Gibco).

Techniques: Over Expression, Migration, Construct, Transfection, Plasmid Preparation