Journal: International Journal of Medical Sciences

Article Title: Recombinant Klotho attenuates IFNγ receptor signaling and SAMHD1 expression through blocking NF-κB translocation in glomerular mesangial cells

doi: 10.7150/ijms.78279

Figure Lengend Snippet: Recombinant Klotho protein inhibited IFNγ-induced SAMHD1 protein expression in MES-13 cells. In (A), MES-13 cells were pretreated with recombinant Klotho protein (0.4 nM, 1 nM) for 24 h and then stimulated with 10 ng/mL IFNγ for 24 h. The SAMHD1 protein level in the IFNγ (10 ng/mL)-treated MES-13 cells was used as the control value and set to 100%. The relative protein levels were quantified through scanning densitometry and are expressed as a percentage of the maximal band intensity of the SAMHD1 protein from cultures treated with IFNγ (10 ng/mL). Data are presented as the mean ± SD of Klotho/GAPDH from at least three separate experiments. Asterisks indicate a significant difference from treatment without IFNγ. In (B), the effect of recombinant Klotho protein and PDTC on NFκB nuclear translocation in IFNγ-activated MES-13 cells was visualized through immunofluorescence microscopy. MES-13 cells were pretreated with recombinant Klotho protein (1 nM) for 24 h or with NFκB inhibitor PDTC (10 μM) for 30 min and then stimulated with 10 ng/mL IFNγ for 1.5 h. After fixation on slide glasses, nuclei of MES-13 cells were stained with DAPI to label nuclear DNA (top panels, blue signal). Samples were also stained with an NFκB antibody, and this was followed by signal amplification with a fluorescein isothiocyanate-conjugated secondary antibody (middle panels, green signal). The bottom panel is a merged image of the two upper panels. In (C), the levels of NFκB p65 in nuclear extracts were prepared by nuclear and cytoplasmic extraction as described in methods. Western blots were performed to detect the NFκB distribution in nuclear fractions. The protein amount was quantified by measuring the NFκB bands using densitometric analysis with AlphaEaseFC (Genetic Technologies, Miami, FL, USA) and normalized to histone H3 (H3). All experiments were performed at least in triplicate and reported as a percentage of the control. Data are presented as the mean ± SD of NFκB/H3 derived from at least three separate experiments. Asterisks indicate a significant difference from treatment with IFNγ (10 ng/mL) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The antibody for SAMHD1 (ab128107) and Klotho (ab203576) and recombinant human Klotho protein (ab84072) were purchased from Abcam (Cambridge, MA).

Techniques: Recombinant, Expressing, Translocation Assay, Immunofluorescence, Microscopy, Staining, Amplification, Western Blot, Derivative Assay

Journal: International Journal of Medical Sciences

Article Title: Recombinant Klotho attenuates IFNγ receptor signaling and SAMHD1 expression through blocking NF-κB translocation in glomerular mesangial cells

doi: 10.7150/ijms.78279

Figure Lengend Snippet: Recombinant Klotho protein inhibited IFNγ-induced SAMHD1 expression not through JAK-STAT1 signaling. MES-13 cells were pretreated with recombinant Klotho protein (0.4 nM, 1 nM) for 24 h and then stimulated with 10 ng/mL IFNγ for 1.5 h. The effects of recombinant Klotho protein on IFNγ-induced protein levels of STAT1 and phosphorylated STAT1 (Tyr 701) in MES-13 cells were then determined through Western blot analysis. Phosphorylation of Stat1 at Tyr701 induces transcription factor activation in response to IFNγ. The values of the STAT1 and phosphorylated STAT1 protein levels in IFNγ (10 ng/mL)-treated MES-13 cells were used as controls and set to 100%. Relative protein levels were quantified through scanning densitometry and are expressed as a percentage of the maximal band intensity of the STAT1 and phosphorylated STAT1 proteins from cultures treated with IFNγ (10 ng/mL). Data are presented as the mean ± SD of STAT1 and phosphorylated STAT1 protein/GAPDH derived from at least three separate experiments. Asterisks indicate a significant difference from treatment without IFNγ (∗∗∗∗ p < 0.0001).

Article Snippet: The antibody for SAMHD1 (ab128107) and Klotho (ab203576) and recombinant human Klotho protein (ab84072) were purchased from Abcam (Cambridge, MA).

Techniques: Recombinant, Expressing, Western Blot, Activation Assay, Derivative Assay

Journal: Acta physiologica (Oxford, England)

Article Title: Klotho protein supplementation reduces blood pressure and renal hypertrophy in db/db mice, a model of type 2 diabetes

doi: 10.1111/apha.13190

Figure Lengend Snippet: The effects of exogenous klotho protein supplementation on kidney function

Article Snippet: 40 Exogenous supplementation of recombinant human klotho protein (rh-klotho, 10 μg/kg/d; PeproTech, Rocky Hill, NJ, USA) or vehicle was started on 8-week-old mice (n = 10 for each group) with daily subcutaneous injections.

Techniques: Control, Clinical Proteomics

Journal: Acta physiologica (Oxford, England)

Article Title: Klotho protein supplementation reduces blood pressure and renal hypertrophy in db/db mice, a model of type 2 diabetes

doi: 10.1111/apha.13190

Figure Lengend Snippet: Impact of exogenous klotho protein supplementation on renal expressions of TGF-β (A), collagen I (B), fibronectin (C), and E-cadherin (D), Smad3 distribution (E), and interstitial fibrosis (F) in db/db mice (db). The * indicates statistically significant differences between the two groups (n = 10 for each)

Article Snippet: 40 Exogenous supplementation of recombinant human klotho protein (rh-klotho, 10 μg/kg/d; PeproTech, Rocky Hill, NJ, USA) or vehicle was started on 8-week-old mice (n = 10 for each group) with daily subcutaneous injections.

Techniques:

Journal: Acta physiologica (Oxford, England)

Article Title: Klotho protein supplementation reduces blood pressure and renal hypertrophy in db/db mice, a model of type 2 diabetes

doi: 10.1111/apha.13190

Figure Lengend Snippet: Influences of exogenous klotho protein supplementation on phosphorylation of Akt (A, 56 kDa), mTOR (B, 289 kDa), and p70-S6k (C, 70 kDa), and phosphorylated mTOR staining (D) in db/db mice (db). The * indicates statistically significant differences between the two groups (n = 10 for each). db + k depicts db/db mice with klotho supplementation

Article Snippet: 40 Exogenous supplementation of recombinant human klotho protein (rh-klotho, 10 μg/kg/d; PeproTech, Rocky Hill, NJ, USA) or vehicle was started on 8-week-old mice (n = 10 for each group) with daily subcutaneous injections.

Techniques: Phospho-proteomics, Staining

Journal: Acta physiologica (Oxford, England)

Article Title: Klotho protein supplementation reduces blood pressure and renal hypertrophy in db/db mice, a model of type 2 diabetes

doi: 10.1111/apha.13190

Figure Lengend Snippet: Effects of exogenous klotho protein supplementation on aortic (A) and renal (B) expressions of superoxide dismutase (SOD), renal abundance of hypoxia-inducible factor-1α (C, 110 kDa, HIF-1α), renal expression of tumour necrosis factor-α (D, TNF-α), plasma concentration of TNF-α (E), and phosphorylation of Iκβ (F, 36 kDa) in db/db mice (db). β-actin was observed at 42 kDa. The * indicates statistically significant differences between the two groups (n = 10 for each). db + k depicts db/db mice with klotho supplementation

Article Snippet: 40 Exogenous supplementation of recombinant human klotho protein (rh-klotho, 10 μg/kg/d; PeproTech, Rocky Hill, NJ, USA) or vehicle was started on 8-week-old mice (n = 10 for each group) with daily subcutaneous injections.

Techniques: Expressing, Clinical Proteomics, Concentration Assay, Phospho-proteomics

Journal: Acta physiologica (Oxford, England)

Article Title: Klotho protein supplementation reduces blood pressure and renal hypertrophy in db/db mice, a model of type 2 diabetes

doi: 10.1111/apha.13190

Figure Lengend Snippet: Summary of in vitro studies in HK-2 cells. Hydrogen peroxide induced angiotensinogen expression (A) and klotho suppressed this response (For time: F = 36, df = 1, P < 0.005; for klotho treatment: F = 14, df = 1, P < 0.001; for interaction: F = 5, df = 1, P < 0.05; for error: df = 20). An interaction between time and klotho treatment may relate to transcytosis of klotho protein by proximal tubular cells.17 Similarly, hydrogen peroxide induced the expression of tumour necrosis factor-α (B, TNF-alpha), and klotho inhibited this (For time: F = 85, df = 1, P < 0.001; for klotho treatment: F = 8, df = 1, P < 0.05; for interaction: F = 13, df = 1, P < 0.01; for error: df = 20). Insulin-like growth factor repressed expression of superoxide dismutase (C, SOD), and klotho opposed this response (For time: F = 96, df = 1, P < 0.001; for klotho treatment: F = 6, df = 1, P < 0.05; for interaction: F = 9, df = 1, P < 0.01; for error: df = 20). Blue and grey bars depict control and klotho-treated groups respectively. The * indicates statistically significant differences between the two groups

Article Snippet: 40 Exogenous supplementation of recombinant human klotho protein (rh-klotho, 10 μg/kg/d; PeproTech, Rocky Hill, NJ, USA) or vehicle was started on 8-week-old mice (n = 10 for each group) with daily subcutaneous injections.

Techniques: In Vitro, Expressing, Control

Journal: Pathogens

Article Title: Exogenous Klotho Extends Survival in COVID-19 Model Mice

doi: 10.3390/pathogens12121404

Figure Lengend Snippet: Correlation between Klotho levels or expression and risk factors for severity and lethality in COVID-19.

Article Snippet: Recombinant mouse Klotho protein was acquired from R&D Systems, Inc.

Techniques: Expressing

Journal: Pathogens

Article Title: Exogenous Klotho Extends Survival in COVID-19 Model Mice

doi: 10.3390/pathogens12121404

Figure Lengend Snippet: Correlation between Klotho levels or expression and clinical complications similar to those found in COVID-19 cases.

Article Snippet: Recombinant mouse Klotho protein was acquired from R&D Systems, Inc.

Techniques: Expressing, Infection, Over Expression, Recombinant

Journal: Pathogens

Article Title: Exogenous Klotho Extends Survival in COVID-19 Model Mice

doi: 10.3390/pathogens12121404

Figure Lengend Snippet: Baseline characteristics of female mice in the first animal study (n = 15) and b. non-parametric comparisons of weight and temperature data across cohort groups in the first experiment.

Article Snippet: Recombinant mouse Klotho protein was acquired from R&D Systems, Inc.

Techniques: Control

Journal: Pathogens

Article Title: Exogenous Klotho Extends Survival in COVID-19 Model Mice

doi: 10.3390/pathogens12121404

Figure Lengend Snippet: Kaplan–Meier survival curves for treatment with mouse Klotho versus human Klotho versus vehicle (all female mice). Log rank test p = 0.026; p trend = 0.00.

Article Snippet: Recombinant mouse Klotho protein was acquired from R&D Systems, Inc.

Techniques:

Journal: Pathogens

Article Title: Exogenous Klotho Extends Survival in COVID-19 Model Mice

doi: 10.3390/pathogens12121404

Figure Lengend Snippet: Kaplan–Meier survival curves for experiment with eight cohorts: ( a ) Klotho treatment versus vehicle, with sex and mode of treatment cohorts combined (log rank test p < 0.001); ( b ) Klotho treatment versus vehicle in male mice (mode of treatment combined, log rank test p = 0.019); ( c ) Klotho treatment versus vehicle in female mice (mode of treatment combined, log rank p = 0.01); ( d ) minipump versus Klotho in i.p. injections versus vehicle (sexes combined, log rank test p < 0.001, p trend < 0.001).

Article Snippet: Recombinant mouse Klotho protein was acquired from R&D Systems, Inc.

Techniques: