human bladder cancer cell lines rt4  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: The Reduction of PSMB4 in T24 and J82 Bladder Cancer Cells Inhibits the Angiogenesis and Migration of Endothelial Cells

doi: 10.3390/ijms25105559

Figure Lengend Snippet: PSMB4 expression in patients with bladder cancer and bladder cancer cell lines. ( A ) a–c: Nonneoplastic urinary bladder tissue; low-grade and high-grade urothelial carcinoma tissues (H&E, 400×). d–f: Immunohistochemical staining of PSMB4 in nonneoplastic urinary bladder tissue and in low-grade and high-grade urothelial carcinoma tissues (PSMB4, 400×). Scale bar = 50 μm. ( B ) PSMB4 protein expression after siPSMB4 transfection for 72 h in RT4, T24, and J82 cells ( n = 10). ( C ) The viability of RT4, T24, and J82 human bladder cancer cells was evaluated using an MTT assay after PSMB4 silencing for 72 and 96 h ( n = 6). ( D ) Colony formation assay of bladder cancer cells after PSMB4 downregulation ( n = 5). Data are presented as the mean ± SEM. *** p < 0.001 compared to the siRNA negative control group, determined by unpaired t -test with the Mann–Whitney test.

Article Snippet: The human bladder cancer cell lines RT4 (HTB-2) and T24 (HTB-4) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured with McCoy’s 5A medium.

Techniques: Expressing, Immunohistochemical staining, Staining, Transfection, MTT Assay, Colony Assay, Negative Control, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Reduction of PSMB4 in T24 and J82 Bladder Cancer Cells Inhibits the Angiogenesis and Migration of Endothelial Cells

doi: 10.3390/ijms25105559

Figure Lengend Snippet: The migration ability of bladder cancer cells after PSMB4 silencing. ( A ) The migration ability of RT4, T24, and J82 human bladder cancer cells was measured via a wound healing assay. Cells were incubated with siPSMB4 for 72 h; then, wounds were created by scratching the cell layer with a P200 pipette tip before observing them for 6 h. Scale bar = 200 μm. ( B ) Quantification of the relative migration rate. Data are presented as the mean ± SEM ( n = 5). *** p < 0.001 compared to the siRNA negative control group, determined by unpaired t-test with the Mann–Whitney test.

Article Snippet: The human bladder cancer cell lines RT4 (HTB-2) and T24 (HTB-4) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured with McCoy’s 5A medium.

Techniques: Migration, Wound Healing Assay, Incubation, Transferring, Negative Control, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Reduction of PSMB4 in T24 and J82 Bladder Cancer Cells Inhibits the Angiogenesis and Migration of Endothelial Cells

doi: 10.3390/ijms25105559

Figure Lengend Snippet: The levels of migration-related proteins in bladder cancer cells after PSMB4 knockdown. ( A ) The protein levels of FAK, p-FAK, integrin β1, integrin β3, MLC, and p-MLC in RT4, T24, and J82 cells after treatment with siPSMB4 for 72 h were measured by Western blotting. We used GAPDH as the loading control. ( B ) The relative quantification of the aforementioned proteins. Data are presented as the mean ± SEM ( n = 6). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the siRNA negative control group, determined by unpaired t-test with the Mann–Whitney test.

Article Snippet: The human bladder cancer cell lines RT4 (HTB-2) and T24 (HTB-4) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured with McCoy’s 5A medium.

Techniques: Migration, Knockdown, Western Blot, Control, Quantitative Proteomics, Negative Control, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Reduction of PSMB4 in T24 and J82 Bladder Cancer Cells Inhibits the Angiogenesis and Migration of Endothelial Cells

doi: 10.3390/ijms25105559

Figure Lengend Snippet: The effect of PSMB4 knockdown on HUVEC angiogenesis. ( A ) The conditioned medium of RT4, T24, and J82 cells transfected with siPSMB4 was collected. HUVECs were cultured with these conditioned media for 6 h. The tube formation ability was evaluated. Scale bar = 200 μm. ( B , C ) The length and branching of formed tubes were analyzed ( n = 5). ( D ) The VEGF protein content in the conditioned medium of RT4, T24, and J82 cells after treatment with siPSMB4 was measured with an ELISA kit ( n = 4). ( E ) The mRNA levels of VEGF-B in RT4, T24, and J82 cells after treatment with siPSMB4 for 72 h were measured via real-time PCR ( n = 4). ( F ) VEGF-B protein expression after siPSMB4 transfection for 72 h in RT4, T24, and J82 cells ( n = 7). Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the siRNA negative control group, determined by unpaired t-test with the Mann–Whitney test.

Article Snippet: The human bladder cancer cell lines RT4 (HTB-2) and T24 (HTB-4) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured with McCoy’s 5A medium.

Techniques: Knockdown, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Negative Control, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Reduction of PSMB4 in T24 and J82 Bladder Cancer Cells Inhibits the Angiogenesis and Migration of Endothelial Cells

doi: 10.3390/ijms25105559

Figure Lengend Snippet: The role of VEGF factors in PSMB4 knockdown in HUVEC angiogenesis. ( A ) The conditioned media from RT4 ( n = 7) and T24 ( n =11) cells transfected with siPSMB4 were collected. The angiogenesis factors of VEFG-121, VEGF-165, and VEGF-B (2 μg/mL) were added into the condition media. Tube formation was observed after 6 h. ( B , C ) The length and branching of formed tubes in RT4 and T24 cells were analyzed. Scale bar = 200 μm. Data are presented as the mean ± SEM. ** p < 0.01, and *** p < 0.001 compared to the siRNA negative control group. # p < 0.05 compared to the siPSMB4 control group, determined by unpaired t-test with the Mann–Whitney test.

Article Snippet: The human bladder cancer cell lines RT4 (HTB-2) and T24 (HTB-4) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured with McCoy’s 5A medium.

Techniques: Knockdown, Transfection, Negative Control, Control, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Reduction of PSMB4 in T24 and J82 Bladder Cancer Cells Inhibits the Angiogenesis and Migration of Endothelial Cells

doi: 10.3390/ijms25105559

Figure Lengend Snippet: The effect of PSMB4 knockdown on HUVEC migration. ( A ) The conditioned media of RT4, T24, and J82 cells transfected with siPSMB4 were collected. HUVECs were incubated with conditioned medium; then, wounds were created in the cell layer by scraping with a P200 pipette tip before observing them for 6 h. The migration ability of HUVECs was measured using a wound healing assay. Scale bar = 200 μm. ( B ) The quantitative relative migration rate is shown. Data are presented as the mean ± SEM ( n = 11). * p < 0.05 and ** p < 0.01 compared to the siRNA negative control group, determined by unpaired t-test with the Mann–Whitney test.

Article Snippet: The human bladder cancer cell lines RT4 (HTB-2) and T24 (HTB-4) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured with McCoy’s 5A medium.

Techniques: Knockdown, Migration, Transfection, Incubation, Transferring, Wound Healing Assay, Negative Control, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Reduction of PSMB4 in T24 and J82 Bladder Cancer Cells Inhibits the Angiogenesis and Migration of Endothelial Cells

doi: 10.3390/ijms25105559

Figure Lengend Snippet: The expression of angiogenesis-related proteins in HUVECs after the silencing of PSMB4. The conditioned media of RT4, T24, and J82 cells transfected with siPSMB4 were collected. HUVECs were cultured with these conditioned media for 6 h. The protein levels of VEGFR1 and VEGFR2 in HUVECs after treatment with conditioned medium were measured by Western blotting. GAPDH was used as the loading control. Data are presented as the mean ± SEM ( n = 5). ** p < 0.01 compared to the siRNA negative control group, determined by unpaired t-test with the Mann–Whitney test.

Article Snippet: The human bladder cancer cell lines RT4 (HTB-2) and T24 (HTB-4) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured with McCoy’s 5A medium.

Techniques: Expressing, Transfection, Cell Culture, Western Blot, Control, Negative Control, MANN-WHITNEY

Journal: Molecules

Article Title: Exploratory Metabolomics Underscores the Folate Enzyme ALDH1L1 as a Regulator of Glycine and Methylation Reactions

doi: 10.3390/molecules27238394

Figure Lengend Snippet: ( A ) shRNA targeting sequences of ALDH1L1 gene, ( B ) ALDH1L1 sequence in exon 3 targeted by CRISPR/Cas9, ( C ) genomic sequencing of the RT4 clone L1-CR and control RT4 cells confirms successful targeting ALDH1L1, ( D ) ALDH1L1 protein levels ( left panel) and bands quantification ( right panel), ( E ) Distribution of ALDH1L1 mRNA levels, ( F ) Immunofluorescence staining of ALDH1L1; plot shows quantification of green fluorescence (ALDH1L1) using Fiji-Image J (NIH). Apparent residual fluorescence in clones 572 and L1-CR represents background. Multigroup comparisons were performed by a one-way ANOVA with Dunnett’s multiple comparisons using GraphPad Prism 9. **** p < 0.0001; *** p < 0.001.

Article Snippet: The human bladder cancer cell line RT4 (ATCC HTB-2) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in McCoy’s 5A medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Bio-Techne, Minneapolis, MN, USA) and 1% of antibiotic-antimycotic (Thermo Fisher Scientific) at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: shRNA, Sequencing, CRISPR, Genomic Sequencing, Control, Immunofluorescence, Staining, Fluorescence, Clone Assay

Journal: Molecules

Article Title: Exploratory Metabolomics Underscores the Folate Enzyme ALDH1L1 as a Regulator of Glycine and Methylation Reactions

doi: 10.3390/molecules27238394

Figure Lengend Snippet: Comparison of RT4 cells and ALDH1L1-deficient clones based on all peaks from untargeted metabolomics data. PCA ( A ) and OPLS-DA ( B ) between all groups with the CRISPR and 572 clones clustered closely (R2X: 0.920, R2Y: 0.981, Q2: 0.808). ( C ) A heat map (generated using MetaboAnalyst 5.0 ) of measured metabolites (13,339 total, ) demonstrates significant differences between groups’ metabotypes, with WT RT4 cells and clone 506 being most distant and CRISPR and 572 clones being the farthest apart. The heatmap is auto-scaled (mean-centered and divided by standard deviation) for each variable; n = 5 per group (RT4 cells) and 6 per groups for each clone. Orange colors represent higher auto-scaled values whereas blue colors represent lower auto-scaled values. Colors for experimental groups are as follows: 506, red; 572, green; L1-CR, dark blue; WT, cyan. Hierarchical clustering was performed on samples in MetaboAnalyst 5.0 using Euclidean distance measures. Each sample number (as in ) is indicated at the bottom of the heatmap.

Article Snippet: The human bladder cancer cell line RT4 (ATCC HTB-2) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in McCoy’s 5A medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Bio-Techne, Minneapolis, MN, USA) and 1% of antibiotic-antimycotic (Thermo Fisher Scientific) at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Comparison, Clone Assay, CRISPR, Generated, Standard Deviation

Journal: Molecules

Article Title: Exploratory Metabolomics Underscores the Folate Enzyme ALDH1L1 as a Regulator of Glycine and Methylation Reactions

doi: 10.3390/molecules27238394

Figure Lengend Snippet: Boxplots showing the distribution of normalized peak area counts from the MS analysis for top significant metabolites (OL1 and OL2a ontology levels) based on the volcano plot in that differentiate RT4 cells and three ALDH1L1 depleted clones. The values on the y axis represent normalized peak area counts. FDR-corrected p values are: **** p < 0.0001; *** p < 0.001; ** p < 0.01. Non-corrected and Bonferroni-corrected p values for the pairwise comparison of RT4 cells and each clone are shown in (metabolites from are highlighted in the table).

Article Snippet: The human bladder cancer cell line RT4 (ATCC HTB-2) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in McCoy’s 5A medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Bio-Techne, Minneapolis, MN, USA) and 1% of antibiotic-antimycotic (Thermo Fisher Scientific) at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Clone Assay, Comparison

Journal: Molecules

Article Title: Exploratory Metabolomics Underscores the Folate Enzyme ALDH1L1 as a Regulator of Glycine and Methylation Reactions

doi: 10.3390/molecules27238394

Figure Lengend Snippet: Analysis of metabolomics data (PCA and OPLS-DA) based on the ALDH1L1 expression levels in three-groups (WT RT4 cells with high levels of ALDH1L1, high group; clone 506 with intermediate levels of ALDH1L1, medium group; L1-CR and 572 clones, low/undetectable ALDH1L1, low group).

Article Snippet: The human bladder cancer cell line RT4 (ATCC HTB-2) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in McCoy’s 5A medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Bio-Techne, Minneapolis, MN, USA) and 1% of antibiotic-antimycotic (Thermo Fisher Scientific) at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Expressing, Clone Assay

Journal: Molecules

Article Title: Exploratory Metabolomics Underscores the Folate Enzyme ALDH1L1 as a Regulator of Glycine and Methylation Reactions

doi: 10.3390/molecules27238394

Figure Lengend Snippet: The heat map generated using OL1 and OL2a metabolites to visualize differences between groups in the three-group analysis. The heatmap is auto-scaled (mean-centered and divided by standard deviation) for each variable; n = 5 (high ALDH1L1, RT4 cells, cyan); n = 6 (medium ALDH1L1, clone 506, red); n = 12 (low/undetectable ALDH1L1, L1-CR and 572 clones, green). Orange colors represent higher auto-scaled values whereas blue colors represent lower auto-scaled values. Hierarchical clustering was performed on samples in MetaboAnalyst 5.0 using Euclidean distance measures.

Article Snippet: The human bladder cancer cell line RT4 (ATCC HTB-2) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in McCoy’s 5A medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Bio-Techne, Minneapolis, MN, USA) and 1% of antibiotic-antimycotic (Thermo Fisher Scientific) at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Generated, Standard Deviation, Clone Assay