Journal: Scientific reports

Article Title: Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway.

doi: 10.1038/s41598-023-46091-2

Figure Lengend Snippet: Figure 1. Dose- and time-dependent inhibition of bladder cancer cell migration by hAM preparations. (A–E′) Representative images of wound healing of T24 cells. (F–J′) Representative images of wound healing of RT4 cells. (K–O′) Representative images of wound healing of NPU cells. (P-R) Percentage of wound healing of T24, RT4 and NPU cells, respectively. Data are presented as mean ± standard error of the mean (SEM) of at least three independent experiments. *P < 0.05 vs. control group. Scale bars 50 µm.

Article Snippet: Normal and cancer urothelial models Human muscle-invasive bladder cancer T24 cells and human non-invasive transitional cell papilloma RT4 cells were purchased from the American Type Culture Collection (ATCC) and grown in culture medium consisting of equal parts A-DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and F12 (Sigma-Aldrich, Merck, Darmstadt, Germany), 5% foetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 4 mM glutamax (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Inhibition, Migration, Control

Journal: Scientific reports

Article Title: Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway.

doi: 10.1038/s41598-023-46091-2

Figure Lengend Snippet: Figure 3. hAM homogenate and extract suppress the migration rate of non-invasive urothelial papilloma RT4 cells by decreasing the amount of lamellipodia and filopodia along the leading edge. RT4 cells treated with hAM homogenate, hAM extract or an appropriate culture medium (control) migrate collectively and maintain an epithelial morphology. However, migrating RT4 cells, treated with hAM preparations, contain less lamellipodia and filopodia (red arrows) along the leading edge than untreated control RT4 cells. Larger framed inset with black lines are the enlarged images of the corresponding smaller framed inserts. The presented images were acquired consecutively every 3 h and 15 min (shown in seconds) after the treatment. Shown is one representative experiment out of three independent experiments with different hAM preparations. Scale bars 50 µm.

Article Snippet: Normal and cancer urothelial models Human muscle-invasive bladder cancer T24 cells and human non-invasive transitional cell papilloma RT4 cells were purchased from the American Type Culture Collection (ATCC) and grown in culture medium consisting of equal parts A-DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and F12 (Sigma-Aldrich, Merck, Darmstadt, Germany), 5% foetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 4 mM glutamax (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Migration, Control

Journal: Scientific reports

Article Title: Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway.

doi: 10.1038/s41598-023-46091-2

Figure Lengend Snippet: Figure 6. hAM homogenate and extract down-regulate FAK expression in bladder cancer urothelial cells but not in normal urothelial cells. (A) Western blot analysis of FAK and p-FAK in T24, RT4 and NPU cells treated for 24 h with hAM homogenate, hAM extract or appropriate culture medium (control samples) without hAM preparations. (A′) The expression and phosphorylation of FAK were normalised to the level of α-tubulin. In addition, the expression and phosphorylation of FAK in the treated samples were normalised to the control samples and these values are presented as log2 fold change (log2FC). The results are expressed as median (solid black line) with data range (minimum and maximum). The dashed black lines represent the normalised value of the control sample (0). The results were obtained on the basis of 3–6 biological replicates. The most representative blot of the mean effect is presented. The original blots are presented in Supplementary Fig. 2. The absence of full-length membranes is due to the cropping of the membranes prior to hybridization with primary antibodies. * p ≤ 0.05. (B) qPCR analysis of PTK2 expression in T24, RT4 and NPU cells treated for 24 h with hAM homogenate, hAM extract or appropriate culture medium without hAM preparations (control samples). PTK2 expression was normalised to GAPDH levels. The qPCR results were presented as ΔCt (CtGAPDH − CtPTK2). Each dot in the graph represents the value of one biological sample. The solid red lines show the median and the solid black lines represent the interquartile range. *p ≤ 0.05.

Article Snippet: Normal and cancer urothelial models Human muscle-invasive bladder cancer T24 cells and human non-invasive transitional cell papilloma RT4 cells were purchased from the American Type Culture Collection (ATCC) and grown in culture medium consisting of equal parts A-DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and F12 (Sigma-Aldrich, Merck, Darmstadt, Germany), 5% foetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 4 mM glutamax (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Western Blot, Control, Phospho-proteomics, Hybridization

Journal: Scientific reports

Article Title: Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway.

doi: 10.1038/s41598-023-46091-2

Figure Lengend Snippet: Figure 8. hAM homogenate inhibits the expression of proteins involved in actin cytoskeleton reorganisation. (A) Western blot analysis of cortactin, RhoA, RhoC, Cdc42, Rac1/2/3 and phosphorylation of cortactin in T24, RT4 and NPU cells treated for 24 h with hAM homogenate, hAM extract or appropriate culture medium (controls) without hAM preparations. (B–D) Quantification of relative protein expression and phosphorylation. The expression and phosphorylation of the target proteins were normalised to the α-tubulin values. In addition, the expression and phosphorylation of the target proteins in the treated samples were normalised to the control samples (culture medium without hAM preparation) and these values were presented as log2 fold change (log2FC). The results are as median (solid red line) with data range (minimum and maximum). The dashed black lines represent the normalised value of the control sample (0). Results were obtained on the basis of 3–6 biological replicates. The most representative blot of the mean effect is presented. The original blots are presented in Supplementary Fig. 2. The absence of full-length membranes is due to the cropping of the membranes prior to hybridization with primary antibodies. *p ≤ 0.05.

Article Snippet: Normal and cancer urothelial models Human muscle-invasive bladder cancer T24 cells and human non-invasive transitional cell papilloma RT4 cells were purchased from the American Type Culture Collection (ATCC) and grown in culture medium consisting of equal parts A-DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and F12 (Sigma-Aldrich, Merck, Darmstadt, Germany), 5% foetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 4 mM glutamax (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Western Blot, Phospho-proteomics, Control, Hybridization

Journal: The Journal of Pathology

Article Title: YWHAZ amplification/overexpression defines aggressive bladder cancer and contributes to chemo‐/radio‐resistance by suppressing caspase‐mediated apoptosis

doi: 10.1002/path.5274

Figure Lengend Snippet: YWHAZ overexpression enhances survival activity to overcome environmental stresses induced by chemo−/radio‐therapy by downregulating caspase‐mediated apoptosis. (A) Western blotting was performed to determine endogenous YWHAZ levels in UCUB cell lines. Levels of β‐actin served as internal controls. (B) RT‐qPCR was performed to determine the relative expression levels of YWHAZ in UCUB cell lines using RT4 cells as the control. (C) Western blotting was performed to determine levels of the indicated effectors involved in caspase‐mediated apoptosis in RT4 cells 48 h after YWHAZ gene transfection. (D) RT4 cells overexpressing YWHAZ were treated with the indicated concentrations of doxorubicin and cisplatin. Cell viability was measured using Alamar Blue assays 4 days after drug treatments. (E) RT4 cells overexpressing YWHAZ were treated with 6 Gy irradiation, after which cell viability was measured every 24 h for 4 days. (F) Incidence rate of preapoptosis among treated cells 48 h after chemo−/radio‐therapy was determined by annexin‐V staining. Cells treated with empty vector served as controls in panels C to F. Data are expressed as means ± SD from five replicates in each experimental group. Paired t ‐tests were performed to evaluate differences between vector‐ and YWHAZ‐treated cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The BFTC905, HT1376, T24, 5637, TSGH8301, and RT4 human bladder cancer cell lines were purchased from the Bioresource Collection and Research Center (BCRC), Taiwan.

Techniques: Over Expression, Activity Assay, Western Blot, Quantitative RT-PCR, Expressing, Control, Transfection, Irradiation, Staining, Plasmid Preparation

Journal: The Journal of Pathology

Article Title: YWHAZ amplification/overexpression defines aggressive bladder cancer and contributes to chemo‐/radio‐resistance by suppressing caspase‐mediated apoptosis

doi: 10.1002/path.5274

Figure Lengend Snippet: YWHAZ overexpression promotes chemo‐resistance. (A) Western blotting was performed to detect YWHAZ levels in RT4 cells showing drug resistance after exposure to various concentrations of doxorubicin or cisplatin. (B) Representative IHC images of paired UCUBs before (primary) and after (recurrent) chemotherapy (left). YWHAZ staining scores were shown in the bar chart using data from 12 paired UCUBs (right).

Article Snippet: The BFTC905, HT1376, T24, 5637, TSGH8301, and RT4 human bladder cancer cell lines were purchased from the Bioresource Collection and Research Center (BCRC), Taiwan.

Techniques: Over Expression, Western Blot, Staining