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

Journal: International Journal of Molecular Sciences

Article Title: Pilot Proteomic Analysis of Urinary Extracellular Vesicles Supports the “Toxic Urine Hypothesis” as a Vicious Cycle in Refractory IC/BPS Pathogenesis

doi: 10.3390/ijms27010130

Figure Lengend Snippet: Urinary extracellular vesicles from IC/BPS patients enhance NF-κB activity in HEK293T reporter cells and human bladder epithelial cells. ( A ) Schematic representation of the dual-luciferase reporter assay workflow. HEK293T cells were seeded at 0 h, transfected with NF-κB reporter (Firefly luciferase) and internal control (Renilla luciferase) plasmids at 16 h, stimulated with uEVs (50 µg/mL) or TNFα (10 ng/mL) at 32 h, and assessed for luciferase activity at 56 h. pGL3-Basic vector lacking NF-κB binding sites served as a negative control. ( B ) NF-κB activity represented as normalized Firefly/Renilla luciferase ratios. Data are presented as the mean ± SEM. Statistical significance was determined between the indicated groups: *, p < 0.05. ( C ) Here, 50 µg/mL of uEVs from a healthy donor (H) or an IC patient (Pt) was used to treat human bladder epithelial cells (HBlEpC) for 48 h. The protein expressions of 14-3-3ζ, p-NF-κB p65 at serine536, and total NF-κB p65 were determined via Western blot. GAPDH was used as an internal protein loading control. The numbers below the blots indicate expression levels relative to H uEV–treated cells after normalization to GAPDH.

Article Snippet: For the detection of cellular proteins, human bladder epithelial cells (HBlEpC; Cell Applications, Inc., San Diego, CA, USA) were treated with uEVs for 48 h and lysed with RIPA buffer, followed by SDS-PAGE and immunoblot analysis using the indicated antibodies.

Techniques: Activity Assay, Luciferase, Reporter Assay, Transfection, Control, Plasmid Preparation, Binding Assay, Negative Control, Western Blot, Expressing

Journal: Cell reports

Article Title: Cancer Cells Resist Mechanical Destruction in Circulation via RhoA/Actomyosin-Dependent Mechano-Adaptation

doi: 10.1016/j.celrep.2020.02.080

Figure Lengend Snippet: (A) Effects of 2–10 FSS pulses on the viability of freshly isolated prostate cancer cells from tumor-bearing mice (Pten−/− and Pten−/−;Trp53−/−) and on wild-type prostate epithelial cells (****p < 0.0001, two-way ANOVA).

Article Snippet: Primary Bladder Epithelial Cells , ATCC , PCS-420-010.

Techniques: Isolation

Journal: Cell reports

Article Title: Cancer Cells Resist Mechanical Destruction in Circulation via RhoA/Actomyosin-Dependent Mechano-Adaptation

doi: 10.1016/j.celrep.2020.02.080

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary Bladder Epithelial Cells , ATCC , PCS-420-010.

Techniques: Virus, Recombinant, shRNA

Journal: Cell Genomics

Article Title: A cis -regulatory lexicon of DNA motif combinations mediating cell-type-specific gene regulation

doi: 10.1016/j.xgen.2022.100191

Figure Lengend Snippet:

Article Snippet: Primary Human Bladder Epithelial Cells (A/T/N) (Bladder) were obtained from ATCC (PCS-420-010) and grown in Bladder Epithelial Cell Basal Medium ((ATCC PCS-420-032) supplemented with Bladder Epithelial Cell Growth Kit (ATCC PCS-420-042).

Techniques: Virus, Recombinant, Reverse Transcription, SYBR Green Assay, Staining, Library Quantification, HiChIP, Software

Journal: PLoS Genetics

Article Title: Hypomethylation of a LINE-1 Promoter Activates an Alternate Transcript of the MET Oncogene in Bladders with Cancer

doi: 10.1371/journal.pgen.1000917

Figure Lengend Snippet: (A) Map of alternate transcript from L1- MET . Exons are represented by black boxes and a red box represents the specific L1. The bent arrows indicate transcriptional start sites and ATGs indicate translational start sites. Horizontal arrows indicate the primers for PCR of bisulfite converted DNA and RT–PCR. The bisulfite-specific primers Bi-L1-5′ and Bi-MET-3′ were used to amplify L1- MET for methylation analysis and Bi-L1-5′ and Bi-L1-3′ for global L1 methylation analysis. The RT–PCR primers, RT-L1-MET-5′ and RT-MET-3′ were used to amplify cDNA of the L1- MET transcript for expression analysis and RT-MET-3′ and RT-MET-5′ for the full length MET expression analysis. The lower tick marks represent each CpG site. Vertical arrows indicate the CpG sites analyzed by the Ms-SNuPE assay. (B) L1- MET methylation (red bars) and L1 methylation (black bars) was analyzed by Ms-SNuPE in 8 normal tissues, one normal bladder fibroblast cell line (LD419), two non-tumorigenic urothelial cell lines (UROtsa and NK2426), and 20 bladder carcinoma cell lines. Values are the average of one CpG site for L1 and an average of two CpG sites for L1- MET from technical triplicates. Error bars represent the standard deviation. (C) Expression of L1- MET was measured using real-time RT PCR in one normal bladder fibroblast cell line, two normal urothelial cell lines and 10 bladder carcinoma cell lines. There is clearly a strong correlation between DNA methylation and expression in all 13 cell lines examined. Values are the average from technical duplicates. Red bars indicate the methylation status of L1- MET , which is also represented in (B), and green bars represent the level of expression relative to GAPDH .

Article Snippet: Human bladder carcinoma cell lines were obtained commercially (T24, J82, HT1376, SCaBER, UM-UC-3, TCCSUP, and RT4; American Type Culture Collection, Manassas, VA) or derived in our laboratory (prefix LD).

Techniques: Reverse Transcription Polymerase Chain Reaction, Methylation, Expressing, Snupe Assay, Standard Deviation, Quantitative RT-PCR, DNA Methylation Assay

Journal: PLoS Genetics

Article Title: Hypomethylation of a LINE-1 Promoter Activates an Alternate Transcript of the MET Oncogene in Bladders with Cancer

doi: 10.1371/journal.pgen.1000917

Figure Lengend Snippet: (A) DNA methylation at L1- MET and global L1s was determined by pyrosequencing in the immortalized urothelial cell line UROtsa and bladder carcinoma cell line T24. Chromatin immunoprecipitation was performed using antibodies for H3K4me3, acetylated H3, and H2A.Z. The values of the ChIP assay are the average of three experiments with technical duplicates. Error bars represent the standard deviation, and p16 represents a single copy gene control. The presence of active histone marks was associated with absence of DNA methylation at L1- MET in the cancer cell line. Methylase dependent single promoter analysis (MSPA) with M. CviP I, a GpC methyltransferase, of the (B) endogenously methylated L1- MET promoter (ch7:116364020–116364664) in the UROtsa immortalized urothelial cell line and the (C) endogenously unmethylated L1- MET promoter in T24 bladder carcinoma cells. (D) DNA methylation at L1- MET and global L1s was determined by pyrosequencing in the colon cancer cell line HCT116 and HCT116 DKO cells (DNMT1 hypomorph/DNMT3B knockout) , . Chromatin immunoprecipitation was performed using antibodies for H2A.Z. The presence of active histone marks was associated with absence of DNA methylation at L1- MET in the DKO cell line. Methylase dependent single promoter analysis (MSPA) with M. CviP I, a GpC methyltransferase, of the (E) endogenously methylated L1- MET promoter in HCT116 colon cancer cells, and (F) endogenously unmethylated L1- MET promoter in HCT116 DKO cells. White circles indicate unmethylated sites and black circles indicate methylated sites. Orange bars indicate areas of protection consistent with the presence of a nucleosome.

Article Snippet: Human bladder carcinoma cell lines were obtained commercially (T24, J82, HT1376, SCaBER, UM-UC-3, TCCSUP, and RT4; American Type Culture Collection, Manassas, VA) or derived in our laboratory (prefix LD).

Techniques: DNA Methylation Assay, Chromatin Immunoprecipitation, Standard Deviation, Control, Methylation, Knock-Out