Journal: Cell Death & Disease

Article Title: Cellular senescence promotes macrophage-to-myofibroblast transition in chronic ischemic renal disease

doi: 10.1038/s41419-025-07666-1

Figure Lengend Snippet: SA-β-gal staining ( A ) and the mRNA expression of senescence markers (PAI-1, p16 INK-4a , p21 Cip1/Waf1 , p53, IL-6, MCP-1, TNF-α) ( B – H ) were significantly increased in human renal proximal tubular epithelial cells (HRPTEpiC) following TNF-α and TGF-β treatment. Similarly, the protein levels of IFITM3 were elevated under the same conditions ( I , J ). Data are mean ± SD ( n = 3/group). * P < 0.05 vs. Normal. PAI-1: plasminogen activator inhibitor-1. Scale bar: 200 µm ( A ).

Article Snippet: Firstly, human renal proximal tubular epithelial cells (HRPTEpiC) (ScienCell, Carlsbad, CA) [ , ] were grown in an epithelial cell medium (ScienCell) containing epithelial cell growth supplement (ScienCell), in a humidified atmosphere of 95% air and 5% CO 2 at 37 °C.

Techniques: Staining, Expressing

Journal: Cell Death & Disease

Article Title: Cellular senescence promotes macrophage-to-myofibroblast transition in chronic ischemic renal disease

doi: 10.1038/s41419-025-07666-1

Figure Lengend Snippet: A Schematic of the in vitro experimental protocol. B , C : Immunofluorescence staining of Ki67 and quantitative analysis of Ki67 + cells. The number of Ki67 + cells decreased after co-culture with senescent human renal proximal tubular epithelial cells (HRPTEpiC) (senescent cells, SC). Scale bar: 100 µm ( B ). The data are mean ± SD (n = 3/group). D – H : The effects of IFITM-3 and ITGB-3 on macrophage senescence. IFITM3 and ITGB3 were manipulated in SC HRPTEpiC and macrophages, respectively. Macrophages were subsequently collected for Western blot analysis of senescence markers p21 Cip1/Waf1 , p53, p-p53.S15, and γ-H2AX. Silencing ITGB3 or IFITM3 individually using siRNA reduced the expression of these senescence markers. However, this blunting effect was mitigated when IFITM3 was overexpressed in HRPTEpiC, indicating that IFITM3 plays a critical role in maintaining senescence signaling despite ITGB3 knockdown. Data are mean ± SD ( n = 3/group). * P < 0.05 vs. Normal control (NC) and Non-SC groups; # P < 0.05 vs. SC group; and P < 0.05 vs. SC + IFITM3 over-expressing groups. I – K : Relative mRNA expression of the senescence markers p16 INK-4a , p21 Cip1/Waf1 , and p53 increased in macrophages co-incubated with senescent cells. β-actin was used as loading control. Data are mean ± SD ( n = 3/group). * P < 0.05 vs. Normal.

Article Snippet: Firstly, human renal proximal tubular epithelial cells (HRPTEpiC) (ScienCell, Carlsbad, CA) [ , ] were grown in an epithelial cell medium (ScienCell) containing epithelial cell growth supplement (ScienCell), in a humidified atmosphere of 95% air and 5% CO 2 at 37 °C.

Techniques: In Vitro, Immunofluorescence, Staining, Co-Culture Assay, Western Blot, Expressing, Knockdown, Control, Incubation

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: α-Klotho isoforms, sequence, and Western blot. A, Structure of the two isoforms of α-Klotho. Isoform 1 represents the full-length protein and contains a signal sequence domain (SS), two homologous domains (KL1, KL2), a short transmembrane domain (TM), and a short cytoplasmic tail. Shown is the site of the epitope for the antibody used in our experiments: AA 800 to 900, KL2. This epitope is absent from Isoform 2, a soluble, secreted protein that arises from alternative RNA splicing and contains only AA 1–549, and where the terminal 15 residues are replaced by the sequence shown. B, The full-length α-Klotho protein sequence of 1012 AA is shown, with KL1 and KL2 shown in red and green respectively, and TM highlighted (black). The peptides giving rise to the PRM signature are also shown (bold typeface; common to isoforms 1 and 2, red; exclusive to full-length α-Klotho, isoform 1, blue). C and D, Western blot analysis of cell lysates (C) and tissues (D) supports the presence of the full-length α-Klotho. Full-length rh α-Klotho protein (rh-α-Klotho). EC, epithelial cells.

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Sequencing, Western Blot

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: α-Klotho protein expression and distribution in human epithelial and reproductive tissues. IHC, positive staining (brown) was found in all the cellular layers of the epidermis (A) and appendage tissue such as hair follicle and sebaceous gland (B). Intestinal expression was primarily found in epithelial cells as illustrated in jejunum (C) and colon (D). In reproductive tissues, positive staining was found in epithelial Sertoli cells (E), testosterone producing Leydig cells (illustrated with white arrows) of the testis (F), and epithelial cells of the prostate gland G). H–K, In mammary tissue (H), endometrium of uterus (I), and endometrium of salpinx (K), the epithelial cell layer was staining strongly for α-Klotho protein; insets are larger magnifications of the epithelial layer. n ≥ 5 for each tissue.

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Expressing, Staining

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: Mass spectrometry characterization of the transmembrane α-Klotho protein in human tissues and cells: extracellular α-Klotho peptide GLFYVDFLSQKD (exon 3). A–D, Representative mass spectrometry spectra (left) and Skyline data (right) confirmed the presence of full-length α-Klotho rh full-length α-Klotho protein (rh-α-Klotho) (A), renal proximal tubular epithelial cells (B), kidney tissue (C), and renal artery (D). E–G, The full-length specific (isoform 1) αKlotho peptide LWITMNEPYTR (exon 4). Representative mass spectrometry spectra (left) and Skyline data (right) confirmed the presence of full-length α-Klotho. E), rh full-length α-Klotho protein (rh-α-Klotho); F, kidney tissue; G, renal artery; and H, neuronal cells.

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Mass Spectrometry, Targeted Proteomics

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: Confirmation of Transmembrane α-Klotho Protein Expression in Human Tissues and Cells

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Expressing, Recombinant

Journal: Acta Pharmacologica Sinica

Article Title: Involvement of endoplasmic reticulum stress in angiotensin II-induced NLRP3 inflammasome activation in human renal proximal tubular cells in vitro

doi: 10.1038/aps.2015.21

Figure Lengend Snippet: Angiotensin II induced inflammasome activation in tubular epithelial cells. (A) Real-time PCR and (C, E, and G) western blot analysis show that the mRNA and protein expression of caspase-1, IL-1β, and IL-18 were increased after treatment with different amounts (0, 10, 100, or 1000 nmol/L) of angiotensin II for 12 h in serum-free medium. (B) Real-time PCR and (D, F, and H) western blot analysis indicate that the mRNA and protein expression of caspase-1, IL-1β, and IL-18 were increased after treatment with 100 nmol/L angiotensin II for various time periods (0, 6, 12, or 24 h) in serum-free medium. β-Actin served as an internal control gene. The results represent the mean±SD from four experiments. bP<0.05 vs control.

Article Snippet: Human kidney proximal epithelial cells (HK-2) were purchased from the China Center for Type Culture Collection (CCTCC).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Vericiguat attenuates cyclosporine A-induced nephropathy by targeting the NF-κB/TGF-β1 axis: an integrated network pharmacology, Mendelian randomization, and experimental study

doi: 10.3389/fimmu.2025.1756582

Figure Lengend Snippet: Proposed molecular mechanism of cyclosporine A-induced renal injury and the protective role of vericiguat via the ROS/NF-κB/TGF-β1/Smad signaling axis. Cyclosporine A (CsA) enters renal tubular epithelial cells and induces mitochondrial dysfunction, leading to excessive reactive oxygen species (ROS) production. ROS activates the IKK complex, resulting in IκBα phosphorylation and degradation, followed by nuclear translocation of NF-κB p65 and transcription of pro-inflammatory cytokines (e.g., TNF-α and IL-6). Concurrently, ROS promotes TGF-β1 signaling through ALK5-mediated phosphorylation of Smad2/3, complex formation with Smad4, and suppression of Smad7. This drives epithelial–mesenchymal transition (EMT), extracellular matrix deposition, and renal fibrosis. Vericiguat, as an sGC stimulator, interferes with this vicious cycle by inhibiting both NF-κB activation and TGF-β1/Smad signaling, thereby exerting anti-inflammatory, anti-fibrotic, and antioxidant effects in CsA-induced chronic kidney disease (CKD). ROS, reactive oxygen species; IKK, IκB Kinase; IκBα, Inhibitor of Nuclear Factor Kappa B Alpha; NF-κB, Nuclear Factor Kappa B; TNF-α, Tumor Necrosis Factor Alpha; IL-6, Interleukin-6; TGF-β1, Transforming Growth Factor Beta 1; ALK5, Activin Receptor-Like Kinase 5; Smad, Mothers Against Decapentaplegic Homolog (from Drosophila ); α-SMA, Alpha-Smooth Muscle Actin; EMT, epithelial–mesenchymal transition; ECM, extracellular matrix.

Article Snippet: The human renal proximal tubular epithelial cell line (HK-2) was obtained from Procell (Wuhan, China) and authenticated by Short Tandem Repeat (STR) profiling.

Techniques: Phospho-proteomics, Translocation Assay, Activation Assay