Journal: Journal of Cellular and Molecular Medicine

Article Title: Occludin facilitates tumour angiogenesis in bladder cancer by regulating IL8/STAT3 through STAT4

doi: 10.1111/jcmm.17257

Figure Lengend Snippet: Occludin (OCLN) promotes tumour angiogenesis in vitro and in vivo. (a and b) The knockdown efficiency was confirmed by performing (A) Western blotting and (B) RT‐qPCR assays using 5637 and T24 cells. (C), Tube formation by EA.hy926 cells incubated with CM derived from 5637 and T24 OCLN‐silenced bladder cancer (BLCA) cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. (d and e) The OCLN plasmid was transfected into 5637 and T24 cells; the efficiency of overexpression was analysed using (D) Western blotting and (E) RT‐qPCR. (F) Tube formation by EA.hy926 cells incubated with CM derived from 5637 and T24 OCLN overexpressing cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. (G) The segment lengths were analysed, and the meshes were quantified using ImageJ software. (H) Matrigel plugs containing 5637 and T24 OCLN stable knockdown cells were removed from BALB/c nude mice. (I) CD31 staining in the indicated cells embedded in Matrigel plugs after growth in BALB/c nude mice. Scale bar = 100 µm. (J) The density of microvessels in Matrigel plugs from BALB/c nude mice injected with the indicated cells. (K) IHC staining showing CD31 levels in clinical patients with high‐/low‐grade BLCA. Scale bar = 100 µm. The results are shown as the mean ± SD. *.01 < p < .05; **.001 < p < .01

Article Snippet: BLCA cell lines, namely T24 (ATCC HTB‐4), 5637 (ATCC HTB‐9) were derived from laboratory retained cells.

Techniques: In Vitro, In Vivo, Knockdown, Western Blot, Quantitative RT-PCR, Incubation, Derivative Assay, Staining, Imaging, Fluorescence, Microscopy, Plasmid Preparation, Transfection, Over Expression, Software, Injection, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Occludin facilitates tumour angiogenesis in bladder cancer by regulating IL8/STAT3 through STAT4

doi: 10.1111/jcmm.17257

Figure Lengend Snippet: Occludin (OCLN) mediates bladder cancer (BLCA) angiogenesis by regulating IL8 expression. (A) Venn diagram showing the differentially expressed genes (DEGs) in the two T24 OCLN knockdown groups compared with the control groups (fold change ≥1, FDR < 0.1, p < .05). (B) Heatmap of the RNA sequencing analysis showing the relative levels of proangiogenic factors. Columns represent probe sets, and rows represent samples receiving the indicated treatments. (C) The relative mRNA levels of proangiogenic factors were detected in control and OCLN shRNA transfected 5637 and T24 cells. (D) The relative IL8 levels in control and OCLN shRNA‐transfected 5637 and T24 cells were measured using an ELISA (pg/ml). (E) The relative IL8 mRNA levels were detected in 5637 and T24 cells following transfection with the vector or OCLN plasmid. (F) Tube formation by EA.hy926 cells cultured with CM derived from 5637 and T24 OCLN‐silenced BLCA cells. IL8 was added, and the cells were stained with Calcein AM and then imaged with a fluorescence microscope. Scale bar = 100 µm. (G) The segment lengths were analysed, and the meshes were quantified using ImageJ software. The results are shown as the mean ± SD. *.01 < p < .05; **.001 < p < .01; *** p < .001

Article Snippet: BLCA cell lines, namely T24 (ATCC HTB‐4), 5637 (ATCC HTB‐9) were derived from laboratory retained cells.

Techniques: Expressing, Knockdown, Control, RNA Sequencing, shRNA, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Cell Culture, Derivative Assay, Staining, Fluorescence, Microscopy, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Occludin facilitates tumour angiogenesis in bladder cancer by regulating IL8/STAT3 through STAT4

doi: 10.1111/jcmm.17257

Figure Lengend Snippet: IL8/ STAT3 is involved in the process of Occludin (OCLN)‐mediated angiogenesis in bladder cancer (BLCA). (A) p‐STAT3 and STAT3 protein levels were detected in OCLN knockdown 5637 and T24 cells. (B) 5637 and T24 cells were transfected with the OCLN plasmid and treated with or without the STAT3 inhibitor Stattic, and tube formation by EA.hy926 cells incubated with CM derived from the indicated cells was assessed by performing staining with Calcein AM and imagining using a fluorescence microscope. Scale bar = 100 µm. (C) The segment lengths in these images were analysed, and the meshes were quantified using ImageJ software. D, p‐STAT3 and STAT3 protein levels were detected in OCLN knockdown 5637 and T24 cells after IL8 supplementation. (E) p‐STAT3 and STAT3 protein levels were detected in OCLN‐overexpressing 5637 and T24 cells after blocking IL8. (F) 5637 and T24 cells were transfected with the OCLN plasmid or cultured with the IL8‐neutralizing antibody; tube formation by EA.hy926 cells incubated with CM derived from the indicated cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. G, The segment lengths in these images were analysed, and the meshes were quantified using ImageJ software. The results are shown as the mean ± SD. *.01< p < .05; **.001< p < .01

Article Snippet: BLCA cell lines, namely T24 (ATCC HTB‐4), 5637 (ATCC HTB‐9) were derived from laboratory retained cells.

Techniques: Knockdown, Transfection, Plasmid Preparation, Incubation, Derivative Assay, Staining, Fluorescence, Microscopy, Software, Blocking Assay, Cell Culture, Imaging

Journal: Frontiers in Oncology

Article Title: Identify and validate RUNX2 and LAMA2 as novel prognostic signatures and correlate with immune infiltrates in bladder cancer

doi: 10.3389/fonc.2023.1191398

Figure Lengend Snippet: LAMA2 and RUNX2 promote the proliferation, migration, and invasiveness, and inhibit apoptosis of BLCA. (A) High expression of LAMA2 and RUNX2 are associated with advanced clinical stages through analyzing GEPIA database. (B) The silencing of LAMA2 and RUNX2 assessed using qRT-PCR. (C) CCK8 assay showing the effects of LAMA2 or RUNX2 knockdown on proliferation of UMUC3 cells. (D) Effects of LAMA2 and RUNX2 on apoptosis of BLCA as determined by flow cytometry. (E) Effects of LAMA2 and RUNX2 knockdown on migration and invasiveness of UMUC3 and 5637 cells, assessed using Transwell assay (** p <0.01, *** p <0.001, ### p <0.001, n = 3).

Article Snippet: In this study, human BLCA cells (UMUC3 and 5637) were obtained from Procell company (Wuhan, Hubei, China).

Techniques: Migration, Expressing, Quantitative RT-PCR, CCK-8 Assay, Knockdown, Flow Cytometry, Transwell Assay

Journal: Frontiers in Immunology

Article Title: Deciphering the immunological and prognostic features of bladder cancer through platinum-resistance-related genes analysis and identifying potential therapeutic target P4HB

doi: 10.3389/fimmu.2023.1253586

Figure Lengend Snippet: Analysis of the PRR gene expression and prognosis in BLCA. (A) The intersection genes between the TCGA, GEO databases and the PRR genes from the HGSOC-Platinum database. (B) The network of the interaction and regulatory relationships of the PRR genes with significant prognostic relevance. (C) Differential expression analysis of the prognosis-related PRR genes between normal and tumor tissues. (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Human BLCA cell lines 5637 and EJ were obtained from the cell bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China) and T24 were obtained from Procell Life Science&Technology Co., Ltd (Wuhan, China).

Techniques: Gene Expression, Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: Deciphering the immunological and prognostic features of bladder cancer through platinum-resistance-related genes analysis and identifying potential therapeutic target P4HB

doi: 10.3389/fimmu.2023.1253586

Figure Lengend Snippet: Identification of the PRR molecular subtypes in BLCA. (A-C) The consensus matrix heatmap demonstrated that the optimal clustering solution for consensus clustering was K=2 (D) The heatmap visualizing differential expression of the 264 PRR genes between three different molecular subtypes (E) Principal component analysis of the PRR genes identified three different molecular subtypes. (F) K-M curves for overall survival of the three PRR clusters in BLCA patients (G) Immune cell infiltration abundance of the three PRR molecular subtypes. (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Human BLCA cell lines 5637 and EJ were obtained from the cell bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China) and T24 were obtained from Procell Life Science&Technology Co., Ltd (Wuhan, China).

Techniques: Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: Deciphering the immunological and prognostic features of bladder cancer through platinum-resistance-related genes analysis and identifying potential therapeutic target P4HB

doi: 10.3389/fimmu.2023.1253586

Figure Lengend Snippet: Prognostic analysis of the PRR scoring system. (A) The relationship between the PRR scoring system with survival time and status. (B) The K-M curve revealed that BLCA patients with a high PRR score had significantly worse survival than those with a low PRR score. (C) The ROCs demonstrated that the PRR score as a single predictor for patients 1, 3, and 5-year survival rates had good predictive performance. (D) Univariate and (E) Multivariate Cox regression analysis of BMR scores and clinicopathological characteristics. (F) The prognostic nomogram for predicting the 1-, 3- and 5-year overall survival of BLCA patients. (G) The calibration curves and ROCs are used for evaluating the predictive performance of the nomogram.

Article Snippet: Human BLCA cell lines 5637 and EJ were obtained from the cell bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China) and T24 were obtained from Procell Life Science&Technology Co., Ltd (Wuhan, China).

Techniques:

Journal: Frontiers in Immunology

Article Title: Deciphering the immunological and prognostic features of bladder cancer through platinum-resistance-related genes analysis and identifying potential therapeutic target P4HB

doi: 10.3389/fimmu.2023.1253586

Figure Lengend Snippet: Assessment of the reliability and validity of the PRR scoring system. (A) The heatmap visualizes the expression of the seven key PRR genes in the training and testing cohorts. (B) The relationship between the PRR score, survival time, and status of BLCA patients in the training and testing cohorts. (C) The relationship between the PRR score and the survival status of BLCA patients in the training and testing cohorts. (D) The K-M cure for the different PRR score groups in the training and testing cohorts. (E) The ROCs of predicting 1, 3, and 5-year survival rates of BLCA patients in the training and testing cohorts.

Article Snippet: Human BLCA cell lines 5637 and EJ were obtained from the cell bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China) and T24 were obtained from Procell Life Science&Technology Co., Ltd (Wuhan, China).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Deciphering the immunological and prognostic features of bladder cancer through platinum-resistance-related genes analysis and identifying potential therapeutic target P4HB

doi: 10.3389/fimmu.2023.1253586

Figure Lengend Snippet: The relationship between the PRR scoring system and immunotherapy. (A) The expression of immune checkpoint-associated molecules in the low and high PRR score groups. (B) The efficiency of immunotherapy in BLCA patients with different expression of CLA-4 and PD1 by analysis of the TCIA dataset. (C) The IC and (D) TC levels of patients in the low and high PRR score groups. (E, F) The relationship between the PRR scoring system and treatment efficacy between the different score groups by using data from a clinical trial conducted by the IMvigor210 cohort. (G) The K-M curve analysis of the low and high PRR score groups in the IMvigor210 cohort. (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Human BLCA cell lines 5637 and EJ were obtained from the cell bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China) and T24 were obtained from Procell Life Science&Technology Co., Ltd (Wuhan, China).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Deciphering the immunological and prognostic features of bladder cancer through platinum-resistance-related genes analysis and identifying potential therapeutic target P4HB

doi: 10.3389/fimmu.2023.1253586

Figure Lengend Snippet: P4HB in BLCA Progression and Platinum Resistance. (A) Expression levels of P4HB in BLCA cell lines. qPCR analysis was performed to assess the expression levels of P4HB in various BLCA cell lines (5637, BIU, J82, EJ, T24 and UM-UC-3) and normal bladder epithelial cells (SV-HUC-1). (B) Knockdown efficiency of P4HB in 5637 and EJ cells. Representative images (C) and corresponding quantitative analysis (D) of the results were obtained from the wound healing assay, demonstrating the effects of P4HB knockdown in cell migration. Similarly, representative images (E) and corresponding quantitative analysis (F) of the results were acquired from the transwell assay, illustrating the impact of P4HB knockdown on cell migration. (G) CCK8 assay demonstrated the impact of P4HB knockdown on drug-sensitivity of platinum in BLCA cell. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Article Snippet: Human BLCA cell lines 5637 and EJ were obtained from the cell bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China) and T24 were obtained from Procell Life Science&Technology Co., Ltd (Wuhan, China).

Techniques: Expressing, Knockdown, Wound Healing Assay, Migration, Transwell Assay, CCK-8 Assay

Journal: Frontiers in Immunology

Article Title: Exploration and validation of a novel reactive oxygen species–related signature for predicting the prognosis and chemotherapy response of patients with bladder cancer

doi: 10.3389/fimmu.2024.1493528

Figure Lengend Snippet: (A) IHC representation chart and western blot (WB) showed AKR1B1 expression in normal bladder tissue and BLCA tissue. Scale bar: 100μm. (B) WB detection of AKR1B1 relative expression in control, NC, and siAKR1B1 groups. (C) Colony formation experiment results with AKR1B1 expression. (D) Results of silencing AKR1B1 expression at different time points of CCK-8:24, 48, 72, 96h. (E) Edu assay showing proliferating cells (T24 and 5637); Edu (red) and DAPI (blue) staining. Scale bar: 50μm. (F) Transwell assay results in control, NC, and siAKR1B1 groups. Scale bar: 100μm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns p > 0.05.

Article Snippet: Human BLCA cells (T24 and 5637) were procured from Procell Life Science & Technology Company (Hubei, China).

Techniques: Western Blot, Expressing, Control, CCK-8 Assay, EdU Assay, Staining, Transwell Assay