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: Cells

Article Title: Role of Aberrantly Activated Lysophosphatidic Acid Receptor 1 Signaling Mediated Inflammation in Renal Aging

doi: 10.3390/cells10102580

Figure Lengend Snippet: SA-β-gal stain and double immunofluorescence stain in senescence-induced cells: ( A ) representative image of SA-β-gal staining in senescence-induced HRPTEpiC; original magnification, ×200. ( B ) The HRPTEpiC treated with 100 nM DOXO and 100μM H 2 O 2 showed increases in the number of SA-β-gal-positive cells. ( C ) Representative image of double immunofluorescence staining of LPAR1 and NF-κB in senescent HRPTEpiC; original magnification, ×200. ( D ) LPAR1- and ( E ) NF-κB-positive area increased in senescence-induced HRPTEpiC. ‡ p < 0.001.

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

Techniques: Staining, Immunofluorescence, Double Immunofluorescence Staining

Journal: Cells

Article Title: Role of Aberrantly Activated Lysophosphatidic Acid Receptor 1 Signaling Mediated Inflammation in Renal Aging

doi: 10.3390/cells10102580

Figure Lengend Snippet: The ATX, LPAR1, PI3K, NF-κB, and inflammatory cytokines expression in senescent HRPTEpiC: ( A – C ) Representative Western blots demonstrating ATX, LPAR1, PI3K, NF-κB, and inflammatory cytokines expression in senescent cells. ( D , E ) LPAR1 and ( F , G ) NF-κB expression increased with DOXO and H 2 O 2 treatment. ( H – L ) The ATX, PI3K, and inflammatory cytokines expression up-regulated in senescence-induced HRPTEpiC. * p < 0.05, † p < 0.01, ‡ p < 0.001.

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

Techniques: Expressing, Western Blot

Journal: Cells

Article Title: Role of Aberrantly Activated Lysophosphatidic Acid Receptor 1 Signaling Mediated Inflammation in Renal Aging

doi: 10.3390/cells10102580

Figure Lengend Snippet: The expression of LPAR1, PI3K, NF-κB, and inflammatory cytokines in senescence-induced HRPTEpiC before and after si- LPAR1 and si- N F -κB transfection: ( A ) representative Western blots of each protein involved. ( B – G ) LPAR1, PI3K, NF-κB, and inflammatory cytokine expression highly increased with senescence-induction of DOXO and H 2 O 2 treatment, while si- LPAR1 treatment reduced the increases. (a: si-c vs. si-L, si-N; b: si-c+D vs. si-L+D, si-N+H; c: si-c+H vs. si-L+H, si-N+H; si-c = si-control, si-L = si- LPAR1 , si-N = si- NF- κ B , D = doxorubicin, H = H 2 O 2 ). ‡ p < 0.001.

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

Techniques: Expressing, Transfection, Western Blot, Control

Journal: Nephrology Dialysis Transplantation

Article Title: CRIM1 is localized to the podocyte filtration slit diaphragm of the adult human kidney

doi: 10.1093/ndt/gfn743

Figure Lengend Snippet: Results from real-time quantitative RT-PCR assessment of CRIM1 mRNA levels in two separate cultures of conditionally immortalized podocytes (Podocytes 1 and 2), normal human kidney samples (Normal 1 and 2), primary human renal proximal tubule cells (RPTEC 1 and 2) and primary human pulmonary artery smooth muscle cells (HPASMC 1 and 2). Results are in arbitrary units ± SD. The levels were normalized to three separate housekeeping genes and the identity of the PCR product was confirmed by sequencing.

Article Snippet: Primary human renal proximal tubular cells (RPTEC) were obtained from Cambrex Bio Science (Walkersville MD, USA).

Techniques: Quantitative RT-PCR, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis

doi: 10.3390/ijms21010192

Figure Lengend Snippet: Analysis of proteins involved in fission process of mitochondrial dynamics in untreated and cysteamine (MEA)-treated CTNS −/−compared to CTNS +/+. Cell cultures were treated with 100 μM cysteamine (MEA) or DMSO (vehicle) for 24 h as specified in the figure. ( a ) Representative immunoblotting analysis in cellular lysate of conditionally immortalized proximal tubular epithelial cells (ciPTEC) from a healthy subject ( CTNS +/+) and cystinotic patient ( CTNS −/−). ( b – e ) The histograms (Drp1 pS637 , panel ( b ), n = 8; mitochondrial fission factor (Mff), panel ( c ), n = 3; mitochondrial fission 1 protein (Fis1), panel ( d ), n = 4; ubiquitinated Fis1 (Ub-Fis1), panel ( e ), n = 3) represent the means values ± SEM of the relative expression normalized on actin level. Densitometric analysis was performed by Versa-Doc imaging system BioRad, using Quantity One software. p -value less than 0.05 was considered as statistically significant, (Student’s t test, *** p < 0.001; ** p < 0.01; * p < 0.05). For further details see under “materials and methods” section.

Article Snippet: Conditionally immortalized proximal tubular epithelial cells (ciPTEC), from healthy donor and cystinotic patients were obtained from Radboud University Medical Center, Nijmegen, The Netherlands and cultured as described in [ ].

Techniques: Western Blot, Expressing, Imaging, Software

Journal: International Journal of Molecular Sciences

Article Title: Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis

doi: 10.3390/ijms21010192

Figure Lengend Snippet: Processing and oligomerization of optic atrophy 1 (OPA1) fusion protein in untreated and MEA-treated CTNS −/− compared to CTNS +/+. ( a ) Representative immunoblotting analysis of ciPTEC obtained from CTNS + / + and CTNS − / −. Where indicated, the cells were treated with MEA or DMSO (vehicle) for 24 h. The histograms of OPA1 ( b ) represent the percentage of relative expression of L and S forms of OPA1 in each lane ( n = 3). The histograms of OMA1 ( c ) represent the means values ± SEM of the relative expression normalized on actin level ( n = 3). ( d ) The fresh collected cells were treated with the cross-linker 1,6-bismaleimidohexane (BMH) 1 mM or with vehicle (DMSO) for 30 min at 37 °C, then centrifuged and resuspended in sodium dodecyl sulfate (SDS) lysis buffer for western blotting analysis with the antibody against OPA1. ( e ) The histograms represent the means values ± SEM of the relative expression of OPA1 oligomers ( n = 3). Densitometric analysis was performed by Versa-Doc imaging system BioRad, using Quantity One software. Student’s t test, *** p < 0.001; * p < 0.05. For further details see under “materials and methods” section.

Article Snippet: Conditionally immortalized proximal tubular epithelial cells (ciPTEC), from healthy donor and cystinotic patients were obtained from Radboud University Medical Center, Nijmegen, The Netherlands and cultured as described in [ ].

Techniques: Western Blot, Expressing, Lysis, Imaging, Software

Journal: International Journal of Molecular Sciences

Article Title: Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis

doi: 10.3390/ijms21010192

Figure Lengend Snippet: Expression and ubiquitination of mitofusin 2 (MFN2) in untreated and MEA-treated CTNS +/+ and CTNS −/−. ( a ) Representative immunoblotting analysis of untreated and MEA-treated ciPTEC CTNS +/+ and CTNS −/−. ( b ) The histogram of MFN2, n = 3, and ( c ) the histogram of ubiquitinated MFN2 Ub-MFN2, n = 3, represent the mean values ± SEM of the relative expression normalized on actin level. Densitometric analysis was performed by Versa-Doc imaging system BioRad, using Quantity One software. Student’s t test, *** p < 0.001.

Article Snippet: Conditionally immortalized proximal tubular epithelial cells (ciPTEC), from healthy donor and cystinotic patients were obtained from Radboud University Medical Center, Nijmegen, The Netherlands and cultured as described in [ ].

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Imaging, Software

Journal: International Journal of Molecular Sciences

Article Title: Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis

doi: 10.3390/ijms21010192

Figure Lengend Snippet: Parkin and ubiquitin carboxyl-terminal hydrolase 30 (USP30) proteins levels and MEA effect in CTNS +/+ and CTNS −/−. ( a ) Immunoblotting analysis of untreated and MEA-treated ciPTEC CTNS + / + and CTNS − / −. ( b ) The histogram represents the means values ± SEM of the relative expression of Parkin normalized on actin level ( n = 3). ( c ) The histogram represents the means values ± SEM of the relative expression of USP30 normalized on actin level ( n = 3). Densitometric analysis was performed by Versa-Doc imaging system BioRad, using Quantity One software. Student’s t test, *** p < 0.001; * p < 0.05.

Article Snippet: Conditionally immortalized proximal tubular epithelial cells (ciPTEC), from healthy donor and cystinotic patients were obtained from Radboud University Medical Center, Nijmegen, The Netherlands and cultured as described in [ ].

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Imaging, Software

Journal: International Journal of Molecular Sciences

Article Title: Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis

doi: 10.3390/ijms21010192

Figure Lengend Snippet: Comparative ultrastructural analysis of mitochondria in ciPTEC. ( a ) Representative images of TEM with magnification 16,000× of untreated and MEA-treated ciPTEC CTNS +/+ and CTNS −/−; scale bar = 1 µm. As shown in high magnification cropped micrographs and in ad hoc schematic reconstruction, mitochondria kept preserved ultrastructure in ciPTEC CTNS +/+ and in MEA-treated ciPTEC CTNS +/+ and CTNS −/−, whereas ciPTEC CTNS −/− showed disruption of mitochondrial cristae and the disarrangement of the internal structures; scale bar = 200 nm. ( b – e ) Quantitative analysis was performed with ImageJ v.1.52p in n ≥ 5 double-blind acquisitions for each experimental condition, red lines represent median with interquartile range. ( b ) Evaluation of relative mitochondrial size measured as area of n ≥ 27 mitochondrial sections. ( c ) Average number of cristae per mitochondrion in each cell ( n ≥ 27 mitochondrial sections). ( d ) The measure of distance of cristae junction near the inner membrane boundary and ( e ) the measure of cristae lumen, assessed on cristae membranes that outline the lumen boundary, were assessed in n ≥ 100 cristae. Non-parametric Mann-Whitney test was applied, *** p < 0.001; * p < 0.05.

Article Snippet: Conditionally immortalized proximal tubular epithelial cells (ciPTEC), from healthy donor and cystinotic patients were obtained from Radboud University Medical Center, Nijmegen, The Netherlands and cultured as described in [ ].

Techniques: Disruption, Membrane, MANN-WHITNEY