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

Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet: REIMS Analysis Predicts Breast Cancer Molecular Markers Including Oncogenic Mutations in PIK3CA (A) Schematic overview of sample preparation for REIMS analysis. (B) Area under the curve (AUC) classification accuracies for ER, PR, HER2 receptor, and triple negative status of 43 breast cancer (BC) cell lines (median intensity of n = 3 biological replicates) following feature selection for phospholipids in the m/z range 600–900, and leave-one-out cross validation. (C) Immunoblot analysis of estrogen inducible protein pS2 and predicted ESR1 expression in ER +ve MCF7 cells following treatment with 0.1% DMSO or indicated concentrations of 4-OHT for 72 h. (D) Unsupervised hierarchical clustering of 872 lipid species detected by REIMS across 43 BC cell lines. (E) Dendrogram of BC cell lines and isogenic MCF10A cells harboring either WT or MUT (E545K or H1047R) PIK3CA. (F) Immunoblot analysis of mature SREBP1 transcription factor expression in nuclear extracts of the MCF10A PIK3CA isogenic panel. (G) Relative exogenous fatty acid uptake in MCF10A PIK3CA WT and MUT cells following serum starvation for 1 h and supplementation with fluorescently labeled dodecanoic acid (n = 5 replicates). (H and I) Unsupervised hierarchical clustering of 9 PIK3CA WT and 9 MUT breast PDX tumors (H) and (I) 5 WT and 7 MUT primary breast tumors. Individual rows in the heatmaps in (D), (H) and (I) correspond to scaled Z score phospholipid intensities (n = 3 biological replicates). Error bars represent ± SEM. n.s., not significant; ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001. p values in (C, bottom panel) and (G) were calculated with one-way ANOVA, followed by unpaired, two-tailed Student’s t test with Bonferroni correction.

Article Snippet: MCF7 (human breast carcinoma) , ATCC , Cat# HTB-22; RRID: CVCL_0031.

Techniques: Sample Prep, Selection, Biomarker Discovery, Western Blot, Expressing, Labeling, Two Tailed Test

Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet: Related to Figure 2 (A) Cell viability of MCF10A PIK3CA MUT cells following treatment with increasing concentrations of rapamycin, torin 1, BYL-719, BKM120, MK2206 or GSK690693 for 72 hours. (B) Unsupervised hierarchical clustering of the median phospholipid intensities of 5 PIK3CA MUT breast cancer cell lines (MCF7, T47D, MDAMB361, MDAMB453 and BT474) treated with 20 nM rapamycin, 100 nM BYL-719 and 150 nM MK2006 for 72 hours. (C) Immunoblot analysis of mTORC1 and mTORC2 signaling in the PIK3CA MUT isogenic panel. Cells were serum and growth-factor starved for 16 hours and subsequently stimulated with 5% horse serum, 20 ng/ml EGF, 0.5 mg/ml hydrocortisone and 10 μg/ml insulin for 30 min. Data in (A) are presented as the mean ± SEM of n = 4 biological replicates and are representative of at least two independent experiments. n.s., not significant; ∗ p ≤ 0.05; ∗∗ p ≤ 0.01. P value s in (A) were calculated with unpaired, two-tailed Student’s t test.

Article Snippet: MCF7 (human breast carcinoma) , ATCC , Cat# HTB-22; RRID: CVCL_0031.

Techniques: Western Blot, Two Tailed Test

Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet:

Article Snippet: MCF7 (human breast carcinoma) , ATCC , Cat# HTB-22; RRID: CVCL_0031.

Techniques: Produced, Virus, Recombinant, Transfection, Protease Inhibitor, Lysis, Mutagenesis, Proliferation Assay, In Situ, Calcium Assay, Reverse Transcription, Kinase Assay, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Proximity Ligation Assay, CRISPR, Control, shRNA, Amplification, Plasmid Preparation, Positive Control, Software, Modification, Targeted Proteomics, Mass Spectrometry, Western Blot

Journal: PLoS ONE

Article Title: Detection of Hepatocyte Growth Factor (HGF) Ligand-c-MET Receptor Activation in Formalin-Fixed Paraffin Embedded Specimens by a Novel Proximity Assay

doi: 10.1371/journal.pone.0015932

Figure Lengend Snippet: A. VeraTag FFPE quantification of HGF in unstimulated and HGF stimulated A549 cells. Isotype IgG control signals (inset) were subtracted from the HGF signals. Bi. VeraTag quantification of HGF in Ln18, Ln229, U138, U118, and U87MG glioma cell lines. Isotype IgG control signals (inset) were subtracted from HGF assay signals. ND: Not detectable. Bii. ELISA determinations of HGF in A549, Ln18, Ln229, U138, U118, and U87MG cell lysates. ND: Not detectable. Ci. VeraTag FFPE quantification of HGF in HEK293/HGF (clone 1) and U138 glioma cell lines. Cii. IHC detection of HGF in HEK293/HGF stable clone (clone 1) and U138 glioma cells.

Article Snippet: H441, H226, A549, H2170, MCF7, H661, Ln229, U87MG, Ln18, U138, U118, and H596 cell lines were obtained from ATCC (American Type culture collection, Manassas, VA) and maintained in DMEM/F12 (50∶50) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 1% penicillin-streptomycin, 2 mM L-Glutamine (CellGro, Manassas, VA).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Stable Transfection

Journal: PLoS ONE

Article Title: Detection of Hepatocyte Growth Factor (HGF) Ligand-c-MET Receptor Activation in Formalin-Fixed Paraffin Embedded Specimens by a Novel Proximity Assay

doi: 10.1371/journal.pone.0015932

Figure Lengend Snippet: Ai. Schematic of the HGF/c-MET VeraTag FFPE proximity assay. Aii. Quantification of HGF/c-MET levels in unstimulated and HGF stimulated A549 cells. Isotype IgG control signals (inset) were subtracted from the HGF/c-MET assay signals. B. VeraTag quantification of HGF/c-MET ligand receptor complexes in H661, MCF7, HGF stimulated MCF7, Ln18, Ln229, U138, U118, U87MG cell lines. C. Immunoprecipitation/Western blot analysis of c-MET (Tyr1003) phosphorylation and c-MET in Ln18, Ln229, U138, U118, and U87 MG glioma cells. The H596 cell line with exon 14 (L964-D1010) deletion was included as a negative control for c-MET (Tyr1003) phosphorylation.

Article Snippet: H441, H226, A549, H2170, MCF7, H661, Ln229, U87MG, Ln18, U138, U118, and H596 cell lines were obtained from ATCC (American Type culture collection, Manassas, VA) and maintained in DMEM/F12 (50∶50) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 1% penicillin-streptomycin, 2 mM L-Glutamine (CellGro, Manassas, VA).

Techniques: Proximity Assay, Control, Immunoprecipitation, Western Blot, Phospho-proteomics, Negative Control