Journal: The FASEB Journal

Article Title: Intravesical Delivery of P21 mRNA –Loaded Lipid Nanoparticles as a Tumor Suppressor Replacement Therapy for Bladder Cancer

doi: 10.1096/fj.202600049R

Figure Lengend Snippet: Rationale for selecting CDKN1A /p21 as a therapeutic target in bladder cancer and validation of p21 mRNA expression. (A) Pan‐cancer analysis of CDKN1A alteration frequencies based on TCGA datasets. The analysis was performed using the TIMER3 web server. (B) CDKN1A mRNA expression in bladder cancer and normal bladder tissues, shown as log2(TPM + 1), where TPM denotes transcripts per million. Data were analyzed using the TIMER3 web server. (C) Representative p21 immunohistochemical staining images from a bladder cancer tissue microarray stratified by pathological stage (Tis, T1, T2, T3, and T4). Scale bars, 200 μm. (D) Quantification of p21 H‐scores in Tis, T1, T2, T3, and T4 lesions. Compared with Tis lesions, T1, T2, T3, and T4 tumors showed significantly lower p21 H‐scores. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01 (one‐way ANOVA). (E) Western blot analysis of basal and p21 mRNA‐induced p21 protein expression in T24 and J82 bladder cancer cells. Basal p21 protein levels were very low in both cell lines, whereas transfection of p21 mRNA led to robust p21 expression. For p21 blots, both short‐exposure (se) and long‐exposure (le) images are shown. β‐Actin serves as a loading control. (F) Immunofluorescence analysis of EGFP‐HA and p21‐HA expression in T24 and J82 cells using an anti‐HA antibody. Nuclei were stained with DAPI. The p21‐HA signal was predominantly localized to the nucleus, whereas EGFP showed diffuse cytoplasmic distribution. Scale bars, 20 μm.

Article Snippet: The human bladder carcinoma cell line J82 and the human embryonic kidney cell line HEK293T were purchased from the American Type Culture Collection (ATCC).

Techniques: Biomarker Discovery, Expressing, Immunohistochemical staining, Staining, Microarray, Western Blot, Transfection, Control, Immunofluorescence

Journal: Open Medicine

Article Title: ARAF regulates malignant progression of bladder cancer through the p38MAPK pathway

doi: 10.1515/med-2026-1422

Figure Lengend Snippet: Effect of ARAF on the proliferation of T24 cells. (A) RT-qPCR analysis of si-ARAF transfection efficiency. (B and C) Western blot analysis of si-ARAF transfection efficiency. (D) EdU fluorescence assay showing proliferating cells. Data are presented as mean ± SD from three independent biological replicates. Statistical analysis was performed using one-way ANOVA. ***p<0.001, ****p<0.0001.

Article Snippet: The human ureter epithelial immortalized cell line SV-HUC-1 (CL-0222) and human bladder cancer cell lines T24 (CL-0227) and J82 (CL-0125) were purchased from Wuhan Procell Biological Company.

Techniques: Quantitative RT-PCR, Transfection, Western Blot, Fluorescence

Journal: Open Medicine

Article Title: ARAF regulates malignant progression of bladder cancer through the p38MAPK pathway

doi: 10.1515/med-2026-1422

Figure Lengend Snippet: Effect of ARAF on apoptosis of T24 cells. (A and B) Flow cytometry analysis of apoptosis levels. (C and D) Protein expression of cleaved-caspase 3 and total caspase 3. Data are presented as mean ± SD from three independent biological replicates. Statistical analysis was performed using one-way ANOVA. **p<0.01, ****p<0.0001.

Article Snippet: The human ureter epithelial immortalized cell line SV-HUC-1 (CL-0222) and human bladder cancer cell lines T24 (CL-0227) and J82 (CL-0125) were purchased from Wuhan Procell Biological Company.

Techniques: Flow Cytometry, Expressing

Journal: Open Medicine

Article Title: ARAF regulates malignant progression of bladder cancer through the p38MAPK pathway

doi: 10.1515/med-2026-1422

Figure Lengend Snippet: Effect of ARAF on the metastatic capacity of T24 cells. (A and B) Quantification of migrating cells. (A and C) Quantification of invading cells. (D and E) Expression of EMT-related proteins in T24 cells. Data are presented as mean ± SD from three independent biological replicates. Statistical analysis was performed using one-way ANOVA. ****p<0.0001.

Article Snippet: The human ureter epithelial immortalized cell line SV-HUC-1 (CL-0222) and human bladder cancer cell lines T24 (CL-0227) and J82 (CL-0125) were purchased from Wuhan Procell Biological Company.

Techniques: Expressing

Journal: Open Medicine

Article Title: ARAF regulates malignant progression of bladder cancer through the p38MAPK pathway

doi: 10.1515/med-2026-1422

Figure Lengend Snippet: Effect of ARAF on the p38 MAPK pathway in T24 cells. (A) The level of p-p38, p38, p-ERK, ERK, p-MEK, MEK in T24 cells was determined by western blot assay. (B) p-p38/p38 ratio. (C) p-ERK/ERK ratio. (D) p-MEK/MEK ratio. Data are presented as mean ± SD from three independent biological replicates. Statistical analysis was performed using one-way ANOVA. ***p<0.001, ****p<0.0001.

Article Snippet: The human ureter epithelial immortalized cell line SV-HUC-1 (CL-0222) and human bladder cancer cell lines T24 (CL-0227) and J82 (CL-0125) were purchased from Wuhan Procell Biological Company.

Techniques: Western Blot

Journal: Open Medicine

Article Title: ARAF regulates malignant progression of bladder cancer through the p38MAPK pathway

doi: 10.1515/med-2026-1422

Figure Lengend Snippet: Anisomycin activated p38 MAPK pathway in si-ARAF transfected T24 cells. (A) The level of p-p38, p38, p-ERK, ERK, p-MEK, MEK in T24 cells was determined by western blot assay. (B) p-p38/p38 ratio. (C) p-ERK/ERK ratio. (D) p-MEK/MEK ratio. Data are presented as mean ± SD from three independent biological replicates. Statistical analysis was performed using Student’s t-test. ***p<0.001, ****p<0.0001.

Article Snippet: The human ureter epithelial immortalized cell line SV-HUC-1 (CL-0222) and human bladder cancer cell lines T24 (CL-0227) and J82 (CL-0125) were purchased from Wuhan Procell Biological Company.

Techniques: Transfection, Western Blot

Journal: Open Medicine

Article Title: ARAF regulates malignant progression of bladder cancer through the p38MAPK pathway

doi: 10.1515/med-2026-1422

Figure Lengend Snippet: Anisomycin reversed the effect of ARAF on T24 cell proliferation and apoptosis. (A) EdU fluorescence for cell proliferation capacity. (B) The apoptotic rate of T24 cells. (C and D) The protein level of Cleaved-caspase3 and Caspase3. Data are presented as mean ± SD from three independent biological replicates. Statistical analysis was performed using Student’s t-test. **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The human ureter epithelial immortalized cell line SV-HUC-1 (CL-0222) and human bladder cancer cell lines T24 (CL-0227) and J82 (CL-0125) were purchased from Wuhan Procell Biological Company.

Techniques: Fluorescence

Journal: Open Medicine

Article Title: ARAF regulates malignant progression of bladder cancer through the p38MAPK pathway

doi: 10.1515/med-2026-1422

Figure Lengend Snippet: Anisomycin reversed the effect of ARAF on T24 cell metastasis and EMT. (A–C) The migratory and invasive ability of T24 cells. (D–F) The protein level of E-cadherin and N-cadherin. Data are presented as mean ± SD from three independent biological replicates. Statistical analysis was performed using Student’s t-test. **p<0.01, ****p<0.0001.

Article Snippet: The human ureter epithelial immortalized cell line SV-HUC-1 (CL-0222) and human bladder cancer cell lines T24 (CL-0227) and J82 (CL-0125) were purchased from Wuhan Procell Biological Company.

Techniques:

Journal: Renal Failure

Article Title: PCSK6 is a novel regulator of venous smooth muscle cell function in arteriovenous fistula remodeling

doi: 10.1080/0886022X.2026.2663246

Figure Lengend Snippet: PCSK6 is increased in smooth muscle cells of stenotic arteriovenous fistula. (A-B) Human stenotic and non-stenotic arteriovenous fistula (AVF) tissues were obtained as described in the Materials and Methods. (A) Representative images of hematoxylin and eosin (HE) staining and immunofluorescence staining for PCSK6, COL1A1, and MYH11 in tissue sections. Immunofluorescence intensity of PCSK6 in the two groups, as well as correlations between PCSK6 and COL1A1 immunofluorescence intensity, neointimal thickness, degree of luminal stenosis, and AVF blood flow were plotted. (B) Total protein and RNA were extracted from tissues. Protein expression of PCSK6 was analyzed by Western blot, and mRNA expression was determined by real-time PCR. (C-D) Primary cultured smooth muscle cells (SMCs) were derived from human stenotic and non-stenotic AVF tissues. (C) Immunofluorescence staining for PCSK6 and the SMC marker MYH11 in primary cultured SMCs. (D) Total protein and RNA were extracted from primary cultured SMCs. Protein expression of PCSK6 was analyzed by Western blot, and mRNA expression was determined by real-time PCR.

Article Snippet: Human venous smooth muscle cells were purchased from ATCC with the Vascular Smooth Muscle Cell Growth Kit (ATCC PCS-100-04).

Techniques: Staining, Immunofluorescence, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Cell Culture, Derivative Assay, Marker

Journal: Renal Failure

Article Title: PCSK6 is a novel regulator of venous smooth muscle cell function in arteriovenous fistula remodeling

doi: 10.1080/0886022X.2026.2663246

Figure Lengend Snippet: PCSK6 is increased in smooth muscle cells during venous remodeling after arteriovenous creation. (A-B) Mouse AVF models were generated as described in the Material and Methods. (A) Tissues from the AVF anastomosis were collected at the indicated time points. Representative images of HE staining and immunofluorescence staining for PCSK6 and MYH11 are shown. Neointimal thickness and PCSK6 immunofluorescence intensity across different time points, as well as the correlation between PCSK6 intensity and neointimal thickness were plotted. (B) Total protein and RNA were extracted from the tissues. Protein expression of PCSK6 at different time points was analyzed by Western blot, and mRNA expression was determined by real-time PCR. (C-D) Primary cultured SMCs were derived from the AVF at the indicated time points. (C) Immunofluorescence staining for PCSK6 and the SMC marker MYH11 in primary cultured SMCs. (D) Total protein and RNA were extracted from primary cultured SMCs. Protein expression of PCSK6 was analyzed by Western blot, and mRNA expression was determined by real-time PCR.

Article Snippet: Human venous smooth muscle cells were purchased from ATCC with the Vascular Smooth Muscle Cell Growth Kit (ATCC PCS-100-04).

Techniques: Generated, Staining, Immunofluorescence, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Cell Culture, Derivative Assay, Marker

Journal: Renal Failure

Article Title: PCSK6 is a novel regulator of venous smooth muscle cell function in arteriovenous fistula remodeling

doi: 10.1080/0886022X.2026.2663246

Figure Lengend Snippet: PCSK6 promotes smooth muscle cells phenotypic switch and ECM production. (A–G) Venous SMCs were transfected with control or PCSK6 expression vectors. (A) Total protein and RNA were extracted. Protein expression of COL1A1, fibronectin, VIM, and MMP2 was analyzed by Western blot, and mRNA expression was determined by real-time PCR. (B) Cell viability was assessed using CCK-8 assay. (C) Cell proliferation was measured by BrdU assay. (D) Cell migration was evaluated by wound healing assay. (E) Cell contractility was determined by collagen gel contraction assay. (F). Hydroxyproline levels were quantified. (G) MMPs activity was measured using MMPs activity kit as described in the Material and Methods section. (H–N) PrimaryM cultured SMCs were transfected with siRNA targeting either control or PCSK6. (H) Total protein and RNA were extracted. Protein expression of COL1A1, fibronectin, VIM, and MMP2 was analyzed by Western blot, and mRNA expression was determined by real-time PCR. (I) Cell viability was assessed using CCK-8 assay. (J) Cell proliferation was measured by BrdU assay. (K) Cell migration was evaluated by wound healing assay. (L) Cell contractility was determined by collagen gel contraction assay. (M) Hydroxyproline levels were quantified. (N) MMPs activity was measured using MMPs activity kit as described in the Material and Methods section.

Article Snippet: Human venous smooth muscle cells were purchased from ATCC with the Vascular Smooth Muscle Cell Growth Kit (ATCC PCS-100-04).

Techniques: Transfection, Control, Expressing, Western Blot, Real-time Polymerase Chain Reaction, CCK-8 Assay, BrdU Staining, Migration, Wound Healing Assay, Collagen Gel Contraction Assay, Activity Assay, Cell Culture

Journal: Renal Failure

Article Title: PCSK6 is a novel regulator of venous smooth muscle cell function in arteriovenous fistula remodeling

doi: 10.1080/0886022X.2026.2663246

Figure Lengend Snippet: Silencing of PCSK6 in VSMCs alleviated venous remodeling and AVF stenosis. (A) Smooth muscle cell-specific PCSK6 knockout mice were generated as described in the Material and Methods. The schematic illustrates the experimental timeline after AVF creation in both knockout and control mice. (B) AVF diameter and blood flow were monitored by ultrasound. Quantitative data are presented. (C-D) Functional analysis of harvested IVC segments assessing (C) contraction responses to 40mM KCl and (D) Relaxation responses to the cumulative addition of acetylcholine. (E) Total protein and RNA were extracted. Protein expression of COL1A1, fibronectin, MMP2 and VIM, was analyzed by Western blot, and mRNA expression was determined by real-time PCR. (F) Histological evaluation of IVC sections through HE/EVG/Masson staining and immunofluorescence for PCSK6 and MYH11. Neointimal thickness was quantified in both experimental groups.

Article Snippet: Human venous smooth muscle cells were purchased from ATCC with the Vascular Smooth Muscle Cell Growth Kit (ATCC PCS-100-04).

Techniques: Knock-Out, Generated, Control, Functional Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Staining, Immunofluorescence