Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The inhibitory activities of Taiwanese local pomegranate fruit. The ethylacetate, butanol, and water layers extracted from pomegranate peel extract (PEP) were tested for the inhibitory efficacy to T24 ( A ) or J82 ( B ) cells using MTT assay as described in Materials and Methods. PEPE2 and PEPE3 ( C ) were also investigated for the inhibitory effectiveness to T24 or J82 cells. 0.1% ( v / v ) Dimethyl sulfoxide (DMSO)-treated urinary bladder urothelial carcinoma (UBUC) cells were regarded as the solvent control. Each MTT result was the typical data of at least three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: MTT Assay, Solvent, Control

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The propidium iodide (PI) and PI/annexin V analyses of UBUC cells treated with PEPE2. The results of PI analyses were represented in ( A ) the T24 cells and ( B ) the J82 cells. The data of the PI/annexin V measurement were shown in ( C ) T24 cells and ( D ) J82 cells. Each flow cytometry figure was the typical result of three independent experiments. The diagram under the PI/annexin V panel was the results of the three independent experiments. The cell cycles and apoptosis detection of the T24 or J82 cells treated with PEPE2 were measured with PI and PI/annexin V analyses, respectively, using flow cytometry as described in the . The 0.1% ( v / v ) DMSO-treated UBUC cells were implemented as the vehicle control. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: Flow Cytometry, Control

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The molecular mechanisms of apoptotic pathway evoked in PEPE2-incubated UBUC cells. T24 and J82 cells were treated with 50 and 20 μg/mL PEPE2 respectively. Then the protein levels of ( A ) pro-/cleaved caspase-3; ( B ) pro-/cleaved caspase-8, DR4 and DR5; ( C ) pro-/cleaved caspase-9, Bax and Bcl-2 and ( D ) Bip, VCP and pro- caspase-12 in PEPE2-treated T24 cells were measured using western immunoblotting as described in the supporting information. The 0.1% ( v / v ) DMSO-treated UBUC cells were used as the solvent control; ( E ) The proposed molecular apoptotic pathway provoked in the PEPE2-treated UBUC cells. The immunoblot in each figure was the representative result of at least three independent experiments. The diagram (ratio (mean ± standard deviation (S.D.)) under each immunoblot indicated the ratio of the normalized protein intensity (observed protein/actin) of PEPE2-treated cells at the indicated time interval, divided by that at the 0-h time point. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: Incubation, Western Blot, Solvent, Control, Standard Deviation

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The inhibition of the xenografted UBUC growth in nude mice by the treatment of the EtOAc layer. T24 cells were injected s.c. into nude mice and were o.g. administrated with water (control), 2, 5, 10, or 100 mg/kg EtOAc layer as described in Materials and Methods. ( A ) The body weights of the extract-treated mice. *** p < 0.001, ** p < 0.005; ( B ) The effects of the EtOAc layer on tumor growth in the xenografted nude mice. The volumes of tumors from the extract-fed mice were compared to those of the water-fed mice. *** p < 0.001; ( C ) The tumor weight. The tumor weight was measured at the 10th week. *** p < 0.001; ( D ) The typical H/E images (400×) of tumor specimens dissected from xenografted nude mice; ( E ) The representative terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) images (400×) of tumor lesions from xenografted nude mice. H&E and TUNEL assays were performed as described in Material and Methods.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: Inhibition, Injection, Control, End Labeling, TUNEL Assay

Journal: BMC Physiology

Article Title: Mandatory role of proteinase-activated receptor 1 in experimental bladder inflammation

doi: 10.1186/1472-6793-7-4

Figure Lengend Snippet: A-E . PAR immunohistochemistry in J82 human urothelial carcinoma cell line. Representative photomicrograph obtained in J82 cell line stained with PAR specific antibodies. J82 cells were fixed and incubated with primary polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies: A = PAR-1 (1:20), B = PAR-2 (1:100), C = PAR-3 (1:5), and D = PAR-4 (1:50). Slides were washed and incubated with biotinylated secondary antibodies (Vector Labs), goat anti-rabbit (polyclonal antibodies). Orange dotted circles highlight some cells considered positives for the particular receptor. Original magnification was ×200. Figure 1E represents the average and SEM of number of PAR-positive cells as percent of the total cells per field.

Article Snippet: Human bladder carcinoma cell line J82 (HTB-1) were obtained from the American Tissue Culture Collection.

Techniques: Immunohistochemistry, Staining, Incubation, Plasmid Preparation

Journal: BMC Physiology

Article Title: Mandatory role of proteinase-activated receptor 1 in experimental bladder inflammation

doi: 10.1186/1472-6793-7-4

Figure Lengend Snippet: A-B . Polymerase Chain Reaction for detection of PARs message in J82 human urothelial carcinoma cell line. Figure 2A is a photomicrograph of the gel and Figure 2B represents the area under the curve as quantified using Image J software [74]. Primers used in this experiment are described in additional file (Table 1).

Article Snippet: Human bladder carcinoma cell line J82 (HTB-1) were obtained from the American Tissue Culture Collection.

Techniques: Polymerase Chain Reaction, Software

Journal: PLoS pathogens

Article Title: Point mutations in FimH adhesin of Crohn's disease-associated adherent-invasive Escherichia coli enhance intestinal inflammatory response.

doi: 10.1371/journal.ppat.1003141

Figure Lengend Snippet: Figure 2. Adhesion ability of AIEC and non-AIEC strains with regard to fimH clade and CEACAM6 expression. (A) Cell-associated bacteria were quantified using non differentiated T84 cells after a 3 H infection period, and results were analyzed with regard to fimH clade. (B) Cell- associated AIEC bacteria belonging to the fimH S70/N78 clade were quantified using undifferentiated and differentiated T84 cells after a 3 H infection period. (C) Western blot analysis of whole protein extracts from non-differentiated and differentiated T84 cells using anti-CEACAM6 and anti-GAPDH antibodies. (D) Cell-associated bacteria (AIEC and non-AIEC bacteria belonging to the fimH S70/N78 clade) were quantified using differentiated T84 cells after a 3 H infection period. (E) Yeast agglutination titer of AIEC and non-AIEC strains belonging to the fimH S70/N78 clade. (F) Transmission electron micrograph of negatively stained AIEC and non-AIEC bacteria belonging to the fimH S70/N78 clade, magnification 625 000. doi:10.1371/journal.ppat.1003141.g002

Article Snippet: Adhesion assay of human intestinal and bladder epithelial cells T84 cells (derived from human colorectal carcinoma) and T24 cells (human bladder epithelial cells) were purchased from ATCC and maintained in an atmosphere containing 5% CO2 at 37uC in appropriate medium.

Techniques: Expressing, Bacteria, Infection, Western Blot, Agglutination, Transmission Assay, Staining

Journal: PLoS pathogens

Article Title: Point mutations in FimH adhesin of Crohn's disease-associated adherent-invasive Escherichia coli enhance intestinal inflammatory response.

doi: 10.1371/journal.ppat.1003141

Figure Lengend Snippet: Figure 4. Impact of FimH amino acid substitutions on AIEC adhesion ability. Cell-associated bacteria were quantified using undifferentiated (A) or differentiated (B) T84 cells after a 3 H infection period. WT represents the original AIEC LF82 reference strain and DfimH represents the LF82- DfimH isogenic mutant. The other constructs were made in the LF82-DfimH isogenic mutant, in which various fimH variants were reintroduced at the fimH locus. Each value is the mean 6 SEM of at least four separate experiments (* P,0.05; ** P,0.001; *** P,0.001). (C) Relative ability to bind Man1 (1 M/3 M binding ratio) with regard to undifferentiated T84 index adhesion of the various fimH chromosomal mutants constructed. (D) Bacterial binding to 1M-BSA under various shear stresses (pN/m cm2) was evaluated by measuring bacterial accumulation over 3 min. doi:10.1371/journal.ppat.1003141.g004

Article Snippet: Adhesion assay of human intestinal and bladder epithelial cells T84 cells (derived from human colorectal carcinoma) and T24 cells (human bladder epithelial cells) were purchased from ATCC and maintained in an atmosphere containing 5% CO2 at 37uC in appropriate medium.

Techniques: Bacteria, Infection, Mutagenesis, Construct, Binding Assay, Shear

Journal: Cell death & disease

Article Title: The APC/C E3 ligase subunit ANAPC11 mediates FOXO3 protein degradation to promote cell proliferation and lymph node metastasis in urothelial bladder cancer.

doi: 10.1038/s41419-023-06000-x

Figure Lengend Snippet: Fig. 6 FOXO3 promotes the transcription of the CDK inhibitor p21 and GULP1. A The mRNA expression of 12 potential FOXO3 downstream effectors when FOXO3 was silenced in UBC cells, measured by RT–PCR. B The mRNA expression of GULP1 and p21 in T24 and UM-UC-3 cells overexpressing FOXO3, measured by RT-PCR. C, D Correlation analysis of CDKN1A and FOXO3 mRNA (C) and correlation analysis of GULP1 and FOXO3 mRNA (D) in TCGA-BLCA. E Enrichment of the CDKN1A promoter with anti-FOXO3 or IgG measured by ChIP followed by RT-PCR. F Luciferase reporter assays in 293 T to evaluate the regulation of FOXO3 expression on CDKN1A transcriptional activity. Firefly luciferase activity was normalized to Rluc intensity. G The prediction of FOXO3 binding motif via JASPAR. H Illustration of two potential binding sites of FOXO3 in GULP1 promoter. I Enrichment of two binding sites in GULP1 promoter with anti-FOXO3 or IgG measured by ChIP followed by RT- PCR. J The effect of FOXO3 expression on GULP1 transcriptional activity, evaluated by dual luciferase reporter assays. K, L Western blot analysis showed the expression of FOXO3, p21, and GULP1 when the FOXO3 level was downregulated (K) or upregulated (L) in UBC cells. Data are shown as mean ± SD. Unpaired, two-tailed student’s t-test, *P < 0.05, **P < 0.01.

Article Snippet: The human bladder urothelial carcinoma cell lines HT-1376, UM-UC-3, T24, J82, 5637, and RT4, the human immortalized uroepithelial cell line SV-HUC1 and the human embryonic kidney cell line 293 T were obtained from the American Type Culture Collection.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, Binding Assay, Western Blot, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Hispolon Methyl Ether, a Hispolon Analog, Suppresses the SRC/STAT3/Survivin Signaling Axis to Induce Cytotoxicity in Human Urinary Bladder Transitional Carcinoma Cell Lines

doi: 10.3390/ijms24010138

Figure Lengend Snippet: HME’s cytotoxic effect on human bladder TCC cell lines. ( A ) HME induces stronger cytotoxicity than cisplatin against human bladder TCC cells. Human bladder TCC cell lines J82, T24, and TCCSUP were treated with HME or cisplatin (0~200 μM) for 48 h, followed by cell viability determination using MTS assay. ( B ) HME impairs the colony-forming capacity of human bladder TCC cells. The levels of clonogenicity of HME-treated J82, T24, and TCCSUP cells were determined as described in . ***: p < 0.001. ( C ) Higher cytotoxic effect of HME than hispolon on TCCSUP cells while comparable cytotoxicity of these two drugs against J82 and T24 cells. J82, T24, and TCCSUP were treated with HME or hispolon (0~200 μM) for 48 h, followed by cell viability determination using MTS assay.

Article Snippet: Human urinary bladder transitional cell carcinoma cell lines J82 (ATCC HTB-1TM) and TCCSUP (ATCC HTB-5TM) were both cultured in Eagle’s Minimum Essential Medium, while the T24 cell line (ATCC HTB-4TM) in McCoy’s 5a medium in accordance with the recommendation of the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: MTS Assay

Journal: International Journal of Molecular Sciences

Article Title: Hispolon Methyl Ether, a Hispolon Analog, Suppresses the SRC/STAT3/Survivin Signaling Axis to Induce Cytotoxicity in Human Urinary Bladder Transitional Carcinoma Cell Lines

doi: 10.3390/ijms24010138

Figure Lengend Snippet: HME’s proapoptotic effect on human bladder TCC cell lines. ( A ) Induction of PARP cleavage by HME. J82, T24, and TCCSUP cells were treated with HME (0, 30, 60 μM) for 24 h, followed by immunoblotting to evaluate the levels of PARP cleavage. The levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as a control for equal loading. c-PARP: cleaved PARP. ( B ) Increase of annexin V-positive (apoptotic) cell population by HME. J82, T24, and TCCSUP cells were treated with HME (0, 30, 60 μM) for 24 h, followed by flow cytometry-based annexin V/propidium iodide (PI) dual staining as described in . ***: p < 0.001.

Article Snippet: Human urinary bladder transitional cell carcinoma cell lines J82 (ATCC HTB-1TM) and TCCSUP (ATCC HTB-5TM) were both cultured in Eagle’s Minimum Essential Medium, while the T24 cell line (ATCC HTB-4TM) in McCoy’s 5a medium in accordance with the recommendation of the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Western Blot, Control, Flow Cytometry, Staining

Journal: International Journal of Molecular Sciences

Article Title: Hispolon Methyl Ether, a Hispolon Analog, Suppresses the SRC/STAT3/Survivin Signaling Axis to Induce Cytotoxicity in Human Urinary Bladder Transitional Carcinoma Cell Lines

doi: 10.3390/ijms24010138

Figure Lengend Snippet: Blockade of STAT3 activation is a vital mechanism of HME’s anti-bladder cancer action. ( A ) HME inhibits constitutive STAT3 activation in human bladder TCC cell lines. J82, T24, and TCCSUP cells were treated with HME (0, 30, 60 μM) for 24 h, followed by immunoblotting for the levels of tyrosine 705-phosphorylated STAT3 (p-STAT3), total STAT3, and survivin, a canonical STAT3 target gene. GAPDH levels were used for equal loading control. ( B ) HME inhibits IL-6-induced STAT3 activation in human bladder TCC cell lines. J82, T24, and TCCSUP cells were treated without or with HME (0, 30, 60 μM) for 24 h in the absence or presence of IL-6 (100 ng/mL) stimulation for 30 min. The lysates from each treatment were subjected to immunoblotting for the levels of p-STAT3 and total STAT3. GAPDH levels were used for controlling equal loading. ( C ) Ectopic expression of STAT3-C (a dominant-active STAT3 mutant) impairs HME’s anti-bladder cancer activity. TCCSUP cells with stable expression of HA-tagged STAT3-C (HA-STAT3-C) and the respective vector control were treated with HME (0, 30, 60 μM) for 24 h, followed by immunoblotting to determine the levels of HA, c-PARP, and survivin. GAPDH levels were used for equal loading control (Left). Moreover, the HA-STAT3 stable clones and the respective vector control clones after 24 h-treatment with HME (0, 30, 60 μM) were subjected to annexin V/PI dual staining assay (center) and clonogenicity (right) for the evaluation of HME-induced apoptosis and cytotoxicity, respectively. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Article Snippet: Human urinary bladder transitional cell carcinoma cell lines J82 (ATCC HTB-1TM) and TCCSUP (ATCC HTB-5TM) were both cultured in Eagle’s Minimum Essential Medium, while the T24 cell line (ATCC HTB-4TM) in McCoy’s 5a medium in accordance with the recommendation of the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Activation Assay, Western Blot, Control, Expressing, Mutagenesis, Activity Assay, Plasmid Preparation, Clone Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Hispolon Methyl Ether, a Hispolon Analog, Suppresses the SRC/STAT3/Survivin Signaling Axis to Induce Cytotoxicity in Human Urinary Bladder Transitional Carcinoma Cell Lines

doi: 10.3390/ijms24010138

Figure Lengend Snippet: HME inhibits SRC to suppress the STAT3/survivin signaling axis. ( A ) HME inhibits SRC activation in all human bladder TCC cell lines examined. J82, T24, and TCCSUP cells were treated with HME (0, 30, 60 μM) for 24 h, followed by immunoblotting for the levels of tyrosine 1007/1008-phosphorylated JAK2 (p-JAK2), total JAK2, tyrosine 416-phosphorylated SRC (p-SRC), total SRC, and GAPDH (loading control). ( B ) Ectopic expression of v-src, a dominant-active SRC, rescues both p-STAT3 and survivin levels from HME-mediated suppression but also attenuates HME-induced PARP cleavage. TCCSUP stable clones of HA-v-src and the respective vector control were treated with HME (0, 30, 60 μM) for 24 h, followed by immunoblotting to evaluate the levels of HA, p-STAT3, total STAT3, survivin, c-PARP, and GAPDH (loading control). ( C ) v-src overexpression antagonizes the proapoptotic and cytotoxic effect of HME. TCCSUP stable clones of HA-v-src and the respective vector control were treated with HME (0, 30, 60 μM) for 24 h, followed by annexin V/PI dual staining assay (left) and clonogenicity assay (right) to examine the HME-induced apoptosis and cytotoxicity, respectively. *: p < 0.05; ***: p < 0.001.

Article Snippet: Human urinary bladder transitional cell carcinoma cell lines J82 (ATCC HTB-1TM) and TCCSUP (ATCC HTB-5TM) were both cultured in Eagle’s Minimum Essential Medium, while the T24 cell line (ATCC HTB-4TM) in McCoy’s 5a medium in accordance with the recommendation of the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Activation Assay, Western Blot, Control, Expressing, Clone Assay, Plasmid Preparation, Over Expression, Staining

Journal: eLife

Article Title: Dynamic persistence of UPEC intracellular bacterial communities in a human bladder-chip model of urinary tract infection

doi: 10.7554/elife.66481

Figure Lengend Snippet: Figure 1. Human Bladder-chip model of UTI recapitulates the physiology of bladder filling and voiding. (A) Schematic of the human bladder-chip with co-culture of the 5637 human bladder epithelial cell line (epithelium, top) and primary human bladder microvascular endothelial cells (endothelial, bottom) on either side of the stretchable and porous membrane. Pooled human urine diluted in PBS and endothelial cell medium were perfused in the apical and vascular channels respectively to mimic bladder physiology. A negative pressure in the ‘vacuum’ channels (magenta) on either side of the main channel was applied to stretch the porous membrane to mimic stretching of the bladder. (B, C) Immunofluorescence staining of confluent epithelial and endothelial cell monolayers (anti-EpCAM (magenta) and anti-CK7 (yellow) for the epithelial cells and anti-PECAM-1 (green) for the endothelial cells) in an uninfected control chip. Some endothelial cells also stained positive for CK7. Cell nuclei were labeled with DAPI (azure). (D) Schematic of the reconstitution of the bladder filling and voiding cycle via stretching of the membrane with a duty cycle of 6 hr. The cycle consisted of a linear increase in strain through stretching of the membrane (filling bladder, 0–2 hr), maintenance of the membrane under stretch (filled bladder, 2–4 hr), a quick relaxation of applied strain over 2 min (voiding bladder, 4:02 hr) and maintenance without applied strain (voided bladder, 4:02 hr to 6 hr). (E) An overview of the timeline of the experimental protocol including infection, addition of neutrophils via the vascular channel, and two cycles of antibiotic treatment interspersed by two bacterial growth cycles. The consecutive bladder duty cycles are indicated. The online version of this article includes the following figure supplement(s) for figure 1:

Article Snippet: 5637 human bladder epithelial carcinoma cell line (procured from ATCC, HTB-9TM).

Techniques: Co-Culture Assay, Membrane, Immunofluorescence, Staining, Control, Labeling, Infection