Journal: American Journal of Translational Research

Article Title: Klotho mitigates diquat-induced myocardial injury in rats by activating Nrf2/ARE-mediated suppression of oxidative stress

doi: 10.62347/SKBR3572

Figure Lengend Snippet: Klotho alleviated DQ-induced cardiomyocyte oxidative stress. (A, B) Cell viability was examined using Cell Counting Kit-8 (CCK-8) assay. (C) Klotho protein levels were examined using western blot analysis. (D) DQ-stimulated H9c2 cardiomyocytes were treated with recombinant Klotho protein, followed by viability assessment using CCK-8 assay. (E, F) Apoptosis was examined using flow cytometry. (G, H) ROS fluorescence signal, (I) MDA level, (J) GSH-ST, (K) GSH-PX, and (L) SOD activities in H9c2 cells were assessed using ELISA kits. (M, N) Nrf2, HO-1, and NQO1 protein levels were examined using western blot analysis. Data were presented as mean ± SD. * P <0.05, ** P <0.01, compared to control group. ## P <0.01, compared to DQ group.

Article Snippet: To induce oxidative injury, cells were treated with 50 μM DQ (45422, Sigma-Aldrich, USA) for 24 h. For Klotho intervention, cells were treated with 1 μg/mL recombinant Klotho protein (1819-KL, R&D Systems, USA) [ 19 , 20 ].

Techniques: Cell Counting, CCK-8 Assay, Western Blot, Recombinant, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: American Journal of Translational Research

Article Title: Klotho mitigates diquat-induced myocardial injury in rats by activating Nrf2/ARE-mediated suppression of oxidative stress

doi: 10.62347/SKBR3572

Figure Lengend Snippet: Nrf2 inhibition abrogated the protective effects of Klotho on DQ-induced oxidative stress. DQ-stimulated H9c2 cells received recombinant Klotho protein and Nrf2 inhibitor ML385. (A, B) Nrf2, HO-1, and NQO1 protein levels were examined using western blot analysis. (C) Cell viability was examined using CCK-8 assay. (D, E) Apoptosis was examined using flow cytometry. (F, G) ROS fluorescence signal, (H) MDA level, (I) GSH-ST, (J) GSH-PX, and (K) SOD activities in H9c2 cells were assessed using ELISA kits. Data were presented as mean ± SD. ** P <0.01, compared to control group. ## P <0.01, compared to DQ group. && P <0.01, compared to DQ+Klotho group.

Article Snippet: To induce oxidative injury, cells were treated with 50 μM DQ (45422, Sigma-Aldrich, USA) for 24 h. For Klotho intervention, cells were treated with 1 μg/mL recombinant Klotho protein (1819-KL, R&D Systems, USA) [ 19 , 20 ].

Techniques: Inhibition, Recombinant, Western Blot, CCK-8 Assay, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: American Journal of Translational Research

Article Title: Klotho mitigates diquat-induced myocardial injury in rats by activating Nrf2/ARE-mediated suppression of oxidative stress

doi: 10.62347/SKBR3572

Figure Lengend Snippet: Klotho alleviated DQ-induced acute myocardial injury in rats through Nrf2/ARE activation. Rats were intragastrically administered with DQ to induce acute myocardial injury and treated with recombinant Klotho protein for 5 days. (A, B) Klotho protein expression in myocardial tissues was assessed using immunohistochemistry. (C) H&E staining of myocardial tissue of rats in each group (D, E) ROS fluorescence signal, (F) MDA level, (G) GSH-ST, (H) GSH-PX, and (I) SOD activities in H9c2 cells were assessed using ELISA kits. (J, K) Nrf2, HO-1, and NQO1 protein levels in myocardial tissues was assessed using immunohistochemistry. ** P <0.01, compared to control group. ## P <0.01, compared to DQ group.

Article Snippet: To induce oxidative injury, cells were treated with 50 μM DQ (45422, Sigma-Aldrich, USA) for 24 h. For Klotho intervention, cells were treated with 1 μg/mL recombinant Klotho protein (1819-KL, R&D Systems, USA) [ 19 , 20 ].

Techniques: Activation Assay, Recombinant, Expressing, Immunohistochemistry, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: Diabetology & Metabolic Syndrome

Article Title: Klotho and SIRT1 changes from pre-diabetes to diabetes and pre-hypertension to hypertension

doi: 10.1186/s13098-021-00736-2

Figure Lengend Snippet: Box and whisker plot of serum Klotho levels among different groups. The boxes represent the 25th and 75th percentiles, and the central lines in the boxes represent the median values. Whiskers are presented as median ± 95% CI. CTL control; Pre-DM pre-diabetes; DM diabetes mellitus; Pre-HTN pre-hypertension; HTN hypertension. **P < 0.01 vs. CTL, $$ P < 0.01 vs. Pre-DM, ## P < 0.01 vs. Pre-HTN

Article Snippet: Serum levels of Klotho and SIRT1 were measured by ELISA method using Human Klotho ELISA kit (R&D Systems with coefficient of variation < 10%) and Human SIRT1 ELISA kit (Elab Science with coefficient of variation < 10%).

Techniques: Whisker Assay, Control

Journal: Diabetology & Metabolic Syndrome

Article Title: Klotho and SIRT1 changes from pre-diabetes to diabetes and pre-hypertension to hypertension

doi: 10.1186/s13098-021-00736-2

Figure Lengend Snippet: Correlations of serum levels of Klotho ( A , C , E ) or SIRT1 ( B , D , F ) with FBS over the normal/Pre-DM ( A , B ) or normal to DM range ( C – F ). C , D linear, and E , F quadratic versions. CTL control; Pre-DM pre-diabetes; DM diabetes mellitus; FBS fasting blood sugar

Article Snippet: Serum levels of Klotho and SIRT1 were measured by ELISA method using Human Klotho ELISA kit (R&D Systems with coefficient of variation < 10%) and Human SIRT1 ELISA kit (Elab Science with coefficient of variation < 10%).

Techniques: Control

Journal: Diabetology & Metabolic Syndrome

Article Title: Klotho and SIRT1 changes from pre-diabetes to diabetes and pre-hypertension to hypertension

doi: 10.1186/s13098-021-00736-2

Figure Lengend Snippet: Correlations of serum levels of Klotho ( A , C , E ) or SIRT1 ( B , D , F ) with MAP over the normal/Pre-HTN ( A , B ) or normal to HTN ( C – F ). C , D linear, and E , F quadratic versions. CTL control; Pre-HTN pre-hypertension; HTN hypertension; MAP mean arterial pressure

Article Snippet: Serum levels of Klotho and SIRT1 were measured by ELISA method using Human Klotho ELISA kit (R&D Systems with coefficient of variation < 10%) and Human SIRT1 ELISA kit (Elab Science with coefficient of variation < 10%).

Techniques: Control

Journal: Diabetology & Metabolic Syndrome

Article Title: Klotho and SIRT1 changes from pre-diabetes to diabetes and pre-hypertension to hypertension

doi: 10.1186/s13098-021-00736-2

Figure Lengend Snippet: Linear regression of serum Klotho or SIRT1 level, as dependent variables, with age, BMI, FBS, MAP in studied groups

Article Snippet: Serum levels of Klotho and SIRT1 were measured by ELISA method using Human Klotho ELISA kit (R&D Systems with coefficient of variation < 10%) and Human SIRT1 ELISA kit (Elab Science with coefficient of variation < 10%).

Techniques:

Journal: Cell death & disease

Article Title: FGF23 ameliorates ischemia-reperfusion induced acute kidney injury via modulation of endothelial progenitor cells: targeting SDF-1/CXCR4 signaling.

doi: 10.1038/s41419-021-03693-w

Figure Lengend Snippet: Fig. 5 FGF23 attenuates SDF-1-induced CXCR4 expression via interaction of FGF receptor-1 (FGFR1), but not Klotho. A FGFR-1, -2, -3, or -4 mRNA expression levels in EPCs were detected by RT-PCR. HK-2 lysates were used as positive controls. B EPCs were pre-treated with FGF23 and/or FGFR inhibitor (PD173074) for 30 min followed by SDF-1 stimulation for 15 min or 6 h. C EPCs harboring control or Klotho shRNA were pre-treated with FGF23 for 30 min followed by SDF-1 stimulation for 10 min or 6 h. D EPCs were pre-treated with Klotho neutralizing antibody for 60 min and FGF23 for 30 min followed by SDF-1 stimulation for 10 min or 24 h. E EPCs were treated with different doses of Klotho. F EPCs were collected at the indicated time points after Klotho treatment. G EPCs harboring control or Klotho shRNA were pre-treated with FGF23 for 24 h. Protein lysates were analyzed by immunoblot using indicated antibodies. β-actin or GAPDH were used as protein loading controls. The numbers under the gel lanes represent the relative protein level.

Article Snippet: For the Klotho knockdown assay, pGFP-C-shLenti vectors containing shRNA targeting human Klotho (TL303679A) or non-targeting scrambled shRNA cassette (TR30023) were purchased from Origene.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, shRNA, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Klotho Regulated by Estrogen Plays a Key Role in Sex Differences in Stress Resilience in Rats

doi: 10.3390/ijms24021206

Figure Lengend Snippet: E2 increased Klotho expression in the hippocampus of ovariectomized (OVX) rats and cultured hippocampal neurons. Experimental design ( A ). Representative images of KL immunostaining (red) in the hippocampal CA1 area of sham, OVX, and E2-treated OVX (OVX + E2) rats (48 h after vehicle or E2 injection ( B , C ); daily vehicle or E2 injection for 7 days ( F – H )). Quantification of fluorescence intensity of KL staining in the hippocampal CA1 area (48 h ( D ); 7 d ( I )). Western blot showing the relative KL protein levels in the hippocampus of sham, OVX, and OVX + E2 rats (48 h ( E ); 7 d ( J ); n = 6). The cultures were treated with the vehicle (Con), 10 nM E2, and E2 + 1 μM ICI (ICI182,780, estrogen receptor inhibitor) at Div13 for 48 h. Western blot result of hippocampal lysate prepared from primary hippocampal culture ( K ). Immunostaining of cultured hippocampal neurons with antibodies specific to KL and MAP2 in Con, E2, and E2 + ICI neurons ( L ). KL staining (green, ( L1 , L4 , L7 )), MAP2 staining (blue, ( L2 , L5 , L8 )), and merge of KL and MAP2 ( L3 , L6 , L9 ). Quantification of KL-positive clusters in cultured hippocampal neurons ( N ). Immunostaining of cultured hippocampal neurons with antibodies specific to Vglut1 and MAP2 in Con, E2, and E2 + ICI neurons ( M ). Vglut1 staining (red, ( M1 , M4 , M7 )), MAP2 staining (blue, ( M2 , M5 , M8 )), and merge of Vglut1 and MAP2 ( M3 , M6 , M9 ). The number of Vglut1-positive clusters in cultured hippocampal neurons ( O ). SO, stratum oriens of CA1 area; SP, stratum pyramidale of CA1 area; SR, stratum radiatum of CA1 area; Con, control. Data are shown as the mean ± SEM. D with the t -test, and others with one-way ANOVA followed by Tukey’s test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following reagents were used: β-estradiol 3-benzoate (E8515, Sigma-Aldrich, St. Louis, MO, USA), sesame oil (S3547, Sigma-Aldrich, St. Louis, MO, USA), β-estradiol suitable for cell culture (E2257, Sigma-Aldrich, St. Louis, MO, USA), ICI182,780 (estrogen receptor antagonist, 1047, TOCRIS, Bristol, UK), letrozole (aromatase inhibitor, 4382, TOCRIS, Bristol, UK), and primary antibodies (Klotho (AF1819, R&D Systems, Minneapolis, MN, USA, immunostaining and Western blot), Klotho (ab203576, Abcam, Cambridge, UK Western blot), vesicular glutamate transporter (Vglut1, AB5905, Millipore, Bredford, MA, USA), and MAP2 (sc-32791, Santa Cruz Biotechnology, Dallas, TX, USA)).

Techniques: Expressing, Cell Culture, Immunostaining, Injection, Fluorescence, Staining, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Klotho Regulated by Estrogen Plays a Key Role in Sex Differences in Stress Resilience in Rats

doi: 10.3390/ijms24021206

Figure Lengend Snippet: Klotho played an essential role in the E2-mediated increase in the number of Vglut1-positive clusters in hippocampal neurons. Primary cultured hippocampal neurons were transfected with a vector encoding scrambled control shRNA (sh-Scr, ( A1 – A4 )) or Klotho shRNA (sh-KL, ( A5 – A8 )) at Div10, and the neurons were fixed for double-immunostaining with anti-KL (red) and anti-MAP2 (blue) antibodies at Div14. The dashed lines in ( A6 ) show the dendrites of the non-transfected neuron, which did not express Klotho shRNA-GFP ( A ). Quantification of KL expression in cultured hippocampal neurons ( B ). Two different Klotho shRNAs, sh-KL #1 and sh-KL# 2, were designed. Vectors encoding sh-Scr and sh-KL were introduced into the hippocampal neurons by electroporation at the time of plating, and the Western blot result ( C ) showed that the expression of shRNA #1 and #2 reduces klotho (130 kDa) expression effectively. We used sh-KL #1 in subsequent experiments. Cultured hippocampal neurons were transfected with a vector encoding sh-Scr ( D1 – D8 ) and sh-KL ( D9 – D16 ) at Div10, and the cultures at Div13 were treated with the vehicle ( D1 – D4 , D9 – D12 ) or 10 nM E2 ( D5 – D8 , D13 – D16 ) for 48 h before fixing for double-immunostaining with antibodies specific to Vglut1 (red) and MAP2 (blue; D ). Quantification of Vglut1-positive clusters ( E ). One-way and two-way ANOVA followed by Tukey’s test. Data are shown as the mean ± SEM. ** p < 0.01, *** p < 0.001.

Article Snippet: The following reagents were used: β-estradiol 3-benzoate (E8515, Sigma-Aldrich, St. Louis, MO, USA), sesame oil (S3547, Sigma-Aldrich, St. Louis, MO, USA), β-estradiol suitable for cell culture (E2257, Sigma-Aldrich, St. Louis, MO, USA), ICI182,780 (estrogen receptor antagonist, 1047, TOCRIS, Bristol, UK), letrozole (aromatase inhibitor, 4382, TOCRIS, Bristol, UK), and primary antibodies (Klotho (AF1819, R&D Systems, Minneapolis, MN, USA, immunostaining and Western blot), Klotho (ab203576, Abcam, Cambridge, UK Western blot), vesicular glutamate transporter (Vglut1, AB5905, Millipore, Bredford, MA, USA), and MAP2 (sc-32791, Santa Cruz Biotechnology, Dallas, TX, USA)).

Techniques: Cell Culture, Transfection, Plasmid Preparation, Control, shRNA, Double Immunostaining, Expressing, Electroporation, Western Blot

Journal: Scientific Reports

Article Title: Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats

doi: 10.1038/s41598-017-11260-7

Figure Lengend Snippet: Comparison of systemic factors using enzyme-linked immunosorbent assays (ELISA). Brain-derived nerve growth factor (BDNF) was upregulated after both AE and NMES, with the most dramatic change evident after a medium dose of NMES resulting in an almost 8-fold increase. Little change was evident in VEGF-A, with only the low AE condition showing a minor non-significant 6% increase. The low AE condition also increased IGF-1 by 33% and showed an AE dose-dependent decrease, with the medium condition being equivalent to control levels. NMES did not affect IGF-1 levels. In contrast, Klotho revealed a significant AE dose-dependent modulation of levels with a medium dose of AE doubling the amount of Klotho in the blood compared to controls. NMES also produced an increase of Klotho for the low and high condition, but not for the medium dose. (*p < 0.05; **p < 0.01).

Article Snippet: Brain-derived neurotrophic factor (BDNF, ERBDNF, Thermo scientific), Vascular endothelial growth factor-A (VEGF-A, RRV00, R&D), Insulin-like growth factor 1 (IGF-1, CSB-E04582r, CUSABIO) and Klotho (CSB-E14958r, CUSABIO) levels were determined by quantitative enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control, Produced

Journal: Scientific Reports

Article Title: Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats

doi: 10.1038/s41598-017-11260-7

Figure Lengend Snippet: Correlations between biological variables and MCT.

Article Snippet: Brain-derived neurotrophic factor (BDNF, ERBDNF, Thermo scientific), Vascular endothelial growth factor-A (VEGF-A, RRV00, R&D), Insulin-like growth factor 1 (IGF-1, CSB-E04582r, CUSABIO) and Klotho (CSB-E14958r, CUSABIO) levels were determined by quantitative enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats

doi: 10.1038/s41598-017-11260-7

Figure Lengend Snippet: Contour maps – systemic factors. Based on the factorial design that arrayed treatment interventions to a control (0 dose), low, medium and high dose, contour plots of the dependent variables can be drawn to illustrate how these variables interact across the experimental space. It is evident here that systemic BDNF levels were most dramatically increased using NMES, but due to the lack of increase in MCT performance there was little interaction between treatment dose and % change in speed. BDNF did also not reveal a dramatic dose-dependent interaction for AE. A similar lack of interaction was evident for VEGF-A, with some evidence of an increase in levels for a low AE condition that results in a 25–50% change in speed. A clearer interaction between dosing and MCT was evident for AE on IGF-1. A low to medium dose of therapy here produced higher levels of factor release, but this was associated with a poorer performance. Lower levels of IGF-1 were actually induced by the high AE condition, producing the most significant performance change (upper right corner). Minimal interaction was seen for NMES, considering that there was less of an effect on MCT, demonstrating that IGF-1 levels in this paradigm were almost homogenous throughout experimental space. In the AE condition, the contour plot for Klotho indicated that the medium dose produced the highest level of Klotho release, which was associated with the highest level of behavioral change. In contrast the plot for NMES did not reveal a clear interaction between these variables.

Article Snippet: Brain-derived neurotrophic factor (BDNF, ERBDNF, Thermo scientific), Vascular endothelial growth factor-A (VEGF-A, RRV00, R&D), Insulin-like growth factor 1 (IGF-1, CSB-E04582r, CUSABIO) and Klotho (CSB-E14958r, CUSABIO) levels were determined by quantitative enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Control, Produced

Journal: Scientific Reports

Article Title: Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats

doi: 10.1038/s41598-017-11260-7

Figure Lengend Snippet: Hippocampal gene expression. BDNF mRNA was upregulated after AE, but did not exhibit a dose-dependency. A significant 10% increase in hippocampal BDNF was evident after low NMES and showed a dose dependent decrease to the level of controls. VEGF-A was upregulated in all NMES conditions, but did not change after a medium and high dose of AE. A low dose actually decreased VEGF-A expression. IGF-1 was upregulated in all conditions by approx. 17%. Only the low AE condition produced a lower 5% upregulation. Klotho revealed an expression profile similar to IGF-1 (27% increase), but with a more marked increase in expression in the low AE condition (12%). (*p < 0.05; **p < 0.01).

Article Snippet: Brain-derived neurotrophic factor (BDNF, ERBDNF, Thermo scientific), Vascular endothelial growth factor-A (VEGF-A, RRV00, R&D), Insulin-like growth factor 1 (IGF-1, CSB-E04582r, CUSABIO) and Klotho (CSB-E14958r, CUSABIO) levels were determined by quantitative enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Gene Expression, Expressing, Produced

Journal: Scientific Reports

Article Title: Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats

doi: 10.1038/s41598-017-11260-7

Figure Lengend Snippet: Correlations ELISA and mRNA.

Article Snippet: Brain-derived neurotrophic factor (BDNF, ERBDNF, Thermo scientific), Vascular endothelial growth factor-A (VEGF-A, RRV00, R&D), Insulin-like growth factor 1 (IGF-1, CSB-E04582r, CUSABIO) and Klotho (CSB-E14958r, CUSABIO) levels were determined by quantitative enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats

doi: 10.1038/s41598-017-11260-7

Figure Lengend Snippet: Contour maps – hippocampal gene expression. By accounting for the different levels of dosing for AE and NMES, contour maps were generated to expose interactions between variables in the experimental space. It was evident here that BDNF expression in the hippocampus was highest for the high AE and low NMES conditions. As there was no significant improvement in MCT performance from NMES, this produced a rather flat banding of expression levels. The highest BDNF levels were evident between no change to a 25% performance decrease. For AE, the highest levels were achieved by a high dose that resulted in the highest level of performance increase. VEGF-A revealed the highest increase in the NMES group with a low to a medium dosing leading to peak gene expression. This was associated with improvements in MCT. AE yielded a dose-dependent increase in IGF-1 signaling that was linked to improved performance with a diagonal pattern across the experimental space. A similar effect was evident for Klotho with lower levels being associated with poorer performance and higher levels indicating better performance. A medium dosing of AE is sufficient to achieve peak performance and high levels of Klotho. It was also highly upregulated in the NMES condition with only very high decreases in performance showing a low level of Klotho.

Article Snippet: Brain-derived neurotrophic factor (BDNF, ERBDNF, Thermo scientific), Vascular endothelial growth factor-A (VEGF-A, RRV00, R&D), Insulin-like growth factor 1 (IGF-1, CSB-E04582r, CUSABIO) and Klotho (CSB-E14958r, CUSABIO) levels were determined by quantitative enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Gene Expression, Generated, Expressing, Produced

Journal: Genetics Research

Article Title: Upregulation of Klotho Aggravates Insulin Resistance in Gestational Diabetes Mellitus Trophoblast Cells

doi: 10.1155/2022/1500768

Figure Lengend Snippet: Klotho affected insulin sensitivity. (a). RT-qPCR of klotho levels. (b) CCK-8 detection was used to detect cell viability. (c) The cells were subjected to a glucose uptake assay. (d) Western blot of GLUT4, INSR- α , INSR- β , IRS1, and IRS2 protein levels. ∗ p < 0.05 compared with the NC group. # p < 0.05 compared with the HG group.

Article Snippet: Anti-klotho antibody (28100-1-AP, proteintech), anti-Insulin receptor-alpha (INSR- α ) antibody (ab203037, Abcam), Insulin receptor-beta (INSR- β ) antibody (ab69508, Abcam), anti-Glucose transporter type 4 (GLUT4) antibody (66846-1-Ig, proteintech), anti-IRS1 antibody (17509-1-AP, proteintech), anti-IRS2 antibody (20702-1-AP, proteintech), anti-IGF-1 antibody (28530-1-AP, proteintech), anti-p-IGF-1R antibody (ab5681, Abcam), anti-IGF-1R antibody (20254-1-AP, proteintech), anti-p-mTOR antibody (67778-1-Ig, proteintech) anti-mTOR (66888-1-Ig, proteintech), anti-PI3K (ab140307, Abcam), anti-p-PI3K antibody (ab182651, Abcam), anti-Akt antibody (10176-2-AP, proteintech), anti-p-Akt (66444-1-Ig, proteintech) antibody and anti- β -actin antibody (66009-1-Ig, proteintech) were a primary antibody.

Techniques: Quantitative RT-PCR, CCK-8 Assay, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Klotho overexpression suppresses apoptosis by regulating the Hsp70/Akt/Bad pathway in H9c2(2-1) cells

doi: 10.3892/etm.2021.9917

Figure Lengend Snippet: Klotho mitigates H/R injury by regulating the Hsp70/Akt/Bad pathway. (A) H9c2(2-1) cells were transfected with klotho plasmid for 24 h prior to H/R injury. Expression levels of klotho and Hsp70 were detected by immunofluorescence microscopy (magnification, x200). DAPI was used to stain the nuclei. (B) Expression of klotho, p-Akt, p-Bad, total Akt and total Bad was detected by western blotting. β-actin was used as the internal reference gene. (C) H9c2(2-1) cells were infected with klotho shRNA lentivirus or control shRNA lentivirus for 48 h. The levels of klotho, p-Akt and total Akt were detected by western blotting. H/R, hypoxia/reoxygenation; Ctrl, control; Hsp70, heat shock protein 70; p-, phosphorylated; sh, short hairpin.

Article Snippet: The klotho overexpression lentivirus and control plasmid lentivirus were constructed by and purchased from Kilton Biotechnology (Shanghai) Co., Ltd. Klotho shRNA (m) and control shRNA lentiviral particles (containing a scrambled shRNA sequence) were purchased from Santa Cruz Biotechnology, Inc.

Techniques: Transfection, Plasmid Preparation, Expressing, Immunofluorescence, Microscopy, Staining, Western Blot, Infection, shRNA, Control

Journal: Stem Cell Research & Therapy

Article Title: Human umbilical cord-derived mesenchymal stromal cells protect against premature renal senescence resulting from oxidative stress in rats with acute kidney injury

doi: 10.1186/s13287-017-0475-8

Figure Lengend Snippet: Characterisation of the huMSCs employed. a Spindle-shaped cells in cultures of MSCs from Wharton’s jelly. b Fluorescence-activated cell immunophenotyping analysis of huMSCs, showing positivity (for CD90, CD29, CD73, CD105 and CD44) and negativity (for human leukocyte antigen-D region (HLA-DR), CD45 and CD34). c Fluorescence-activated cell immunophenotyping analysis of aMSCs, showing positivity (for CD90, CD29, CD73, CD105 and CD44) and negativity (for HLA-DR, CD45 and CD34). d Analysis of the differentiation capacity of the huMSCs. e Analysis of the differentiation capacity of the aMSCs. f Klotho: immunoblots and densitometric analysis of samples from huMSCs ( n = 2) and aMSCs ( n = 4). g β-gal: immunoblots and densitometric analysis of samples from huMSCs ( n = 2) and aMSCs ( n = 4). a p < 0.05 vs huMSCs. aMSC adipose-derived mesenchymal stromal cell, huMSC human umbilical cord-derived mesenchymal stromal cell, β-gal β-galactosidase

Article Snippet: Blots were then incubated overnight with antibodies against β-galactosidase (β-gal) at 1:1000 (Sigma-Aldrich); against AQP2 at 1:10,000, Klotho at 1:500, p21 Waf1/Cip1 (hereafter p21) at 1:500, p16 INK4a (hereafter p16) at 1:1000, transforming growth factor beta 1 (TGF-β1) at 1:1000 and Actin at 1:2000 (all Santa Cruz Biotechnology, Dallas, TX, USA); against HO-1 at 1:1000 (Assay Designs, Ann Arbor, MI, USA); and against MnSOD at 1:4000 (Cayman Chemical, Ann Arbor, MI, USA).

Techniques: Fluorescence, Western Blot, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: Human umbilical cord-derived mesenchymal stromal cells protect against premature renal senescence resulting from oxidative stress in rats with acute kidney injury

doi: 10.1186/s13287-017-0475-8

Figure Lengend Snippet: Densitometric analysis and immunoblotting of markers of stress-induced senescence in kidney tissue in control ( n = 2), IRI ( n = 5) and IRI + huMSC ( n = 7) rats, on D2. a Immunoblots and densitometric analysis of Klotho. b Immunoblots and densitometric analysis of β-gal. c Immunoblots and densitometric analysis of HO-1. d Immunoblots and densitometric analysis of MnSOD. e Bar graphs showing renal miR-29a expression. f Bar graphs showing renal miR-34a expression. g Bar graphs showing renal miR-29b expression. h Bar graphs showing renal miR-335 expression. i Immunoblots and densitometric analysis of p21. j Immunoblots and densitometric analysis of p16. a p < 0.05 vs control. b p < 0.05 vs IRI + huMSC. c p = 0.05 vs control. d p = 0.05 vs IRI + huMSC. huMSC human umbilical cord-derived mesenchymal stromal cell, IRI ischaemia/reperfusion injury, β-gal β-galactosidase, MnSOD manganese superoxide dismutase, HO-1 heme oxygenase-1

Article Snippet: Blots were then incubated overnight with antibodies against β-galactosidase (β-gal) at 1:1000 (Sigma-Aldrich); against AQP2 at 1:10,000, Klotho at 1:500, p21 Waf1/Cip1 (hereafter p21) at 1:500, p16 INK4a (hereafter p16) at 1:1000, transforming growth factor beta 1 (TGF-β1) at 1:1000 and Actin at 1:2000 (all Santa Cruz Biotechnology, Dallas, TX, USA); against HO-1 at 1:1000 (Assay Designs, Ann Arbor, MI, USA); and against MnSOD at 1:4000 (Cayman Chemical, Ann Arbor, MI, USA).

Techniques: Western Blot, Control, Expressing, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: Human umbilical cord-derived mesenchymal stromal cells protect against premature renal senescence resulting from oxidative stress in rats with acute kidney injury

doi: 10.1186/s13287-017-0475-8

Figure Lengend Snippet: Ischaemia/reperfusion-induced kidney damage on D49. a AQP2: immunoblots and densitometric analysis of samples from IRI ( n = 3) and IRI + huMSC ( n = 4) rats. b Urinary flow rate over the study period. c Representative light microscopy of Masson’s trichrome staining and chronic renal damage score in IRI and IRI + huMSC rats (magnification, ×4). d Klotho: immunoblots and densitometric analysis of samples from IRI ( n = 3) and IRI + huMSC ( n = 4) rats. e β-gal: immunoblots and densitometric analysis of samples from IRI ( n = 3) and IRI + huMSC ( n = 4) rats. a p ≤ 0.05 vs IRI + huMSC. huMSC human umbilical cord-derived mesenchymal stromal cell, IRI ischaemia/reperfusion injury, AQP2 aquaporin, β-gal β-galactosidase, BW body weight

Article Snippet: Blots were then incubated overnight with antibodies against β-galactosidase (β-gal) at 1:1000 (Sigma-Aldrich); against AQP2 at 1:10,000, Klotho at 1:500, p21 Waf1/Cip1 (hereafter p21) at 1:500, p16 INK4a (hereafter p16) at 1:1000, transforming growth factor beta 1 (TGF-β1) at 1:1000 and Actin at 1:2000 (all Santa Cruz Biotechnology, Dallas, TX, USA); against HO-1 at 1:1000 (Assay Designs, Ann Arbor, MI, USA); and against MnSOD at 1:4000 (Cayman Chemical, Ann Arbor, MI, USA).

Techniques: Western Blot, Light Microscopy, Staining, Derivative Assay