Journal: Pharmaceutics

Article Title: Utilizing a Kidney-Targeting Peptide to Improve Renal Deposition of a Pro-Angiogenic Protein Biopolymer

doi: 10.3390/pharmaceutics11100542

Figure Lengend Snippet: In vitro cellular binding/uptake of all constructs in primary human renal cells. Primary human glomerular microvascular endothelial cells (HGME), proximal tubule epithelial cells (HRPTEpC), and podocytes were exposed to each fluorescently labeled ELP fusion protein at 10 µM for 24 h. Protein levels bound to or in each cell type were determined by flow cytometry ( A ). Expression of the VEGF receptors Flt-1 and Flk-1 were determined in each cell line by Western blot (( B ). Lane 1—HGME, Lane 2—HRPTEpC, and Lane 3—podocytes). * Statistically significant increase relative to ELP-treated cells (one-way ANOVA performed within each cell type with post-hoc Tukey’s multiple comparison).

Article Snippet: Human renal proximal tubular epithelial cells (HRPTEpC) were purchased from Cell Applications, Inc. (San Diego, CA, USA) and subcultured according to the manufacturer’s recommendations using RenaEpi Growth factor media.

Techniques: In Vitro, Binding Assay, Construct, Labeling, Flow Cytometry, Expressing, Western Blot, Comparison

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: Inhibition of BKPyV replication by IFNα HRPTECs were infected with BKPyV (1 × 10 6 copies/mL) for 2 h and then incubated in the presence or absence of human IFNα (0−20 ng/mL) for an additional 72 h. At the end of experiments, cell lysates were collected for Western blot analysis (A) to determine the protein expression of MxA and VP1, and the BKPyV viral titer was measured by qPCR (B) from the collected supernatant. (C) Eight-to ten-week Balb/c female mice received 30 min-ischemic reperfusion injury (IRI) of a unilateral kidney. Mice were then intraperitoneally inoculated with MuPyV wild-type strain (2 × 10 6 plaque-forming units). Two doses of IFNα (1 MIU/Kg) or PBS were subcutaneously injected on day 1 and day 4. After seven days of inoculation, the mice were sacrificed, and fold change of MuPyV VP1 mRNA relative expression using 18S rRNA (18S) as reference in the IRI and non-IRI kidneys was determined by RT-qPCR. The results of cell culture experiments represent three independent experiments. For animal study, each experimental group comprised 4 to 6 animals. The results are presented as mean ± SEM. Statistical significance: ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.005; ∗∗∗∗, p < 0.001.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Infection, Incubation, Western Blot, Expressing, Injection, Quantitative RT-PCR, Cell Culture

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: Inhibition of BKPyV replication by MxA HRPTECs were transfected with varying doses (0.1a2 μg) of FLAG-tagged MxA-expressing plasmids or an empty plasmid for 12 h. Subsequently, they were infected with BKPyV (1 × 10 6 copies/mL) for 2 h. After replacing the medium, the cells were cultured for an additional 72 h. BKPyV-infected cells transfected with the empty vector served as the negative control, while BKPyV-infected cells treated with 10 ng/mL of IFNα served as the positive control. At the end of the experiments, viral titer analysis (A) was performed on the collected supernatant, and Western blot analysis (B) was conducted on cell lysates to determine the expressions of TAg, VP1, and MxA proteins. Bar graphs depict the normalized levels of BKPyV TAg (C) and VP1 (D) relative to GAPDH. The data represent three independent experiments. The results are presented as mean ± SEM. Statistical significance: ns, not significant; ∗∗, p < 0.01; ∗∗∗, p < 0.005; ∗∗∗∗, p < 0.001.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Infection, Cell Culture, Negative Control, Positive Control, Western Blot

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: MxA silencing diminished IFNα′s anti-BKPyV effect HRPTECs were transfected with varying doses of MxA siRNA or scrambled RNA for 12 h. Afterward, they were treated with IFNα (10 ng/mL) and infected with BKPyV (1 × 10 6 copies/mL) for an additional 72 h. Western blot analysis was performed to assess the expression levels of TAg, VP1, and MxA proteins (A). Bar graphs depict the normalized levels of BKPyV MxA (B) and VP1 (C) relative to GAPDH. The data represent three independent experiments. The results are presented as mean ± SEM. Statistical significance: ns, not significant; ∗, p < 0.05; ∗∗∗∗, p < 0.001.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Infection, Western Blot, Expressing

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: Mutation of MxA residue 103 decreases antiviral activity against BK polyomavirus . HRPTECs were transfected with plasmids encoding FLAG-tagged wild-type MxA, GTP binding-defective MxA mutants (K83A and D250 N), or the GTPase-deficient MxA mutant (T103A) for 12 h. Cells transfected with the empty vector served as the negative control. Subsequently, the cells were infected with BKPyV (1 × 10 6 copies/mL) for an additional 72 h. Western blot analysis was conducted to determine the expression levels of wild-type MxA, MxA mutants, BKPyV TAg, and VP1 proteins. Bar graphs represent the normalized levels of BKPyV TAg (B) and VP1 (C) relative to GAPDH. The results are presented as mean ± SEM. Statistical significance: ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Residue, Activity Assay, Transfection, Binding Assay, Plasmid Preparation, Negative Control, Infection, Western Blot, Expressing

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: BKPyV TAg is co-immunoprecipitated with wild-type MxA but not with the MxA T103A mutant . A. HRPTECs were co-transfected with BKPyV TAg-expressing plasmid and FLAG-tagged wild-type MxA-expressing plasmid. Control transfections were the TAg-expressing or wild-type MxA-expressing plasmid alone. Immunoprecipitation assay was performed by immunoprecipitation with anti-SV40 TAg antibody. The TAg-binding MxA in the TAg-associated immunocomplexes was assessed by subsequent immunoblotting with an anti-FLAG antibody. The protein levels of inputted MxA, TAg, and GAPDH were determined through immunoblotting using their respective specific antibodies. B. HRPTECs were co-transfected with either FLAG-tagged MxA or FLAG-tagged MxA mutants (K83A and T103A)-expressing plasmids, along with the TAg-expressing plasmid. The immunoprecipitation assay was conducted using an anti-SV40 TAg antibody, followed by immunoblotting with an anti-FLAG antibody.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Immunoprecipitation, Mutagenesis, Transfection, Expressing, Plasmid Preparation, Control, Binding Assay, Western Blot

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: MxA and BKPyV TAg are colocalized in the cytoplasm . A. HRPTECs were infected with BKPyV (1 × 10 6 copies/mL). After 72 h, cells were fixed and stained for MxA and TAg. B&C. HRPTECs were initially infected with BKPyV (1 × 10 6 copies/mL) for 2 h, followed by incubation in the absence (B) or presence (C) of human IFNα (10 ng/mL) for an additional 72 h. D. HRPTECs were transfected with wild-type MxA-expressing plasmids for 12 h and infected with BKPyV (1 × 10 6 copies/mL). After 72 h, cells were fixed and stained for MxA and TAg. MxA was labeled with anti-MxA and a green fluorescent (Alexa Fluor 488) secondary antibody, and BKV TAg was labeled with anti-SV40 TAg and a red fluorescent (Alexa Fluor 647) secondary antibody. In MxA-expressing cells, MxA exhibited colocalization with BKPyV TAg in the cytoplasm, displaying a diffuse granulate pattern (indicated by white arrowheads) (C&D). In BKPyV infected non-MxA expressing cells, TAg could be seen exclusively in the nucleus (indicated by grey arrows) (A, C&D).

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Infection, Staining, Incubation, Transfection, Expressing, Labeling

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