Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Loss of CTRP5 improves insulin action and hepatic steatosis

doi: 10.1152/ajpendo.00010.2016

Figure Lengend Snippet: Primers used in real-time PCR

Article Snippet: A ready-made human cDNA tissue panel (OriGene) was used to survey the tissue expression patterns of human CTRP5 .

Techniques:

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Loss of CTRP5 improves insulin action and hepatic steatosis

doi: 10.1152/ajpendo.00010.2016

Figure Lengend Snippet: Evolutionary conservation of C1q/TNF-related protein 5 (CTRP5) in vertebrates and its tissue expression profile in humans. A: sequence alignment of human (NP_001265360), mouse (NP_001177248), chicken (XP_001232467), frog (Xenopus; XP_002935065), and zebrafish (NP_001025124) CTRP5 using a web-based Clustal W (version 2) tool (26). Identical amino acids are shaded black, and similar amino acids are shaded gray. Shading was done using the web-based BoxShade tool. The NH2-terminal signal peptide collagen domain (with G-X-Y repeats) and the COOH-terminal globular C1q domain are indicated. B: quantitative real-time PCR analysis of human CTRP5 mRNA expression across 47 tissue types. Expression levels of CTRP5 in each tissue were normalized to GAPDH.

Article Snippet: A ready-made human cDNA tissue panel (OriGene) was used to survey the tissue expression patterns of human CTRP5 .

Techniques: Expressing, Sequencing, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Loss of CTRP5 improves insulin action and hepatic steatosis

doi: 10.1152/ajpendo.00010.2016

Figure Lengend Snippet: Ctrp5 expression in different metabolic states. A: quantitative real-time PCR analysis of Ctrp5 expression in epididymal white adipose tissue (eWAT), skeletal muscle, liver, and hypothalamus of mice subjected to overnight fast (fasted group; n = 7) or overnight fast followed by 3 h of refeeding (refed group; n = 8). Expression levels were normalized to β-actin. B and C: quantitative real-time PCR analysis of Ctrp5 expression in eWAT from leptin-deficient ob/ob (n = 10) and wild-type (WT) lean controls (n = 9) or in eWAT and inguinal white adipose tissue (iWAT) from mice fed a control low-fat diet (LFD; n = 8) vs. a high-fat diet (HFD; n = 8). D: expression levels of Ctrp5 in the brain and peripheral tissues of a separate cohort of LFD-fed (n = 11) and HFD-fed (n = 11) mice. Expression levels were normalized to β-actin. E: quantitative real-time PCR analysis of Ctrp5 expression in brain, heart, liver, kidney, and eWAT from mice fed a ketogenic diet (n = 8) or matched control diet (n = 8). Expression levels were normalized to the average of 18s rRNA, Gapdh, β-actin, and Rpl-22. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: A ready-made human cDNA tissue panel (OriGene) was used to survey the tissue expression patterns of human CTRP5 .

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Loss of CTRP5 improves insulin action and hepatic steatosis

doi: 10.1152/ajpendo.00010.2016

Figure Lengend Snippet: Expression of CTRP5 in lean and obese humans. Quantitative real-time PCR analysis of CTRP5 in omental (A and C) or subcutaneous (B and D) adipose tissue of human abdominal surgery subjects. Expression of CTRP5 in the subcutaneous fat depot is positively correlated with body mass index (BMI; B). Expression levels of CTRP5 are higher in obese individuals with or without type 2 diabetes relative to lean individuals (n = 7–8; D). Expression levels were normalized to β-actin levels in each sample. **P < 0.01.

Article Snippet: A ready-made human cDNA tissue panel (OriGene) was used to survey the tissue expression patterns of human CTRP5 .

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Loss of CTRP5 improves insulin action and hepatic steatosis

doi: 10.1152/ajpendo.00010.2016

Figure Lengend Snippet: Generation of Ctrp5-null mice. A: schematic showing the strategy for generating Ctrp5 knockout (KO) mice. The entire Ctrp5 gene, comprising two exons, was replaced by a neomycin resistance gene and lacZ reporter cassette. B: PCR genotyping results show the successful generation of wild-type (WT; +/+), heterozygous (+/−), and homozygous KO (−/−) alleles using the indicated primer pairs (TUF and TUR for WT allele, lacInF, and lacInR for KO allele) shown in A. C: the absence of Ctrp5 mRNA in eWAT from the KO mice was confirmed by RT-PCR with primers specific for Ctrp5 (mCtrp5F and mCtrp5R).

Article Snippet: A ready-made human cDNA tissue panel (OriGene) was used to survey the tissue expression patterns of human CTRP5 .

Techniques: Knock-Out, Reverse Transcription Polymerase Chain Reaction

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Loss of CTRP5 improves insulin action and hepatic steatosis

doi: 10.1152/ajpendo.00010.2016

Figure Lengend Snippet: Metabolic phenotypes of Ctrp5-null mice fed a standard laboratory chow diet. A: body weight of wild-type (WT) and knockout (KO) male mice over time. B: fat and lean mass in WT and KO mice quantified by Echo-MRI. C: WT and KO blood glucose levels were measured at the indicated time points during glucose tolerance test (GTT). D: WT and KO blood glucose levels were measured at the indicated time points during insulin tolerance test (ITT). E and F: fasting blood glucose and insulin levels. G: calculated insulin resistance (HOMA-IR) index for WT and KO mice at 20 wk of age. WT, n = 7; KO, n = 8. *P < 0.05; **P < 0.01.

Article Snippet: A ready-made human cDNA tissue panel (OriGene) was used to survey the tissue expression patterns of human CTRP5 .

Techniques: Knock-Out

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Loss of CTRP5 improves insulin action and hepatic steatosis

doi: 10.1152/ajpendo.00010.2016

Figure Lengend Snippet: Improved insulin sensitivity in Ctrp5-null mice fed a high-fat diet. A: body weight of wild-type (WT) and knockout (KO) male mice over time. B: fat and lean mass in WT and KO mice quantified by Echo-MRI at 21 wk of age. C–F: fasting blood glucose, insulin, and C-peptide levels as well as the calculated insulin resistance (HOMA-IR) index for WT and KO mice at 20 wk of age. G: real-time PCR for gluconeogenic gene (G6Pc and Pck1) expression in liver of WT and KO mice. H: blood glucose levels for WT and KO mice were measured at the indicated time points during glucose tolerance test (GTT). I: WT and KO serum insulin levels at 0 and 30 min after glucose injection. J: WT and KO blood glucose levels were measured at the indicated time point during insulin tolerance test (ITT). K: the decay constant (KITT) for WT and KO mice based on the ITT data. WT, n = 8; KO, n = 7. *P < 0.05; **P < 0.01.

Article Snippet: A ready-made human cDNA tissue panel (OriGene) was used to survey the tissue expression patterns of human CTRP5 .

Techniques: Knock-Out, Real-time Polymerase Chain Reaction, Expressing, Injection

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Loss of CTRP5 improves insulin action and hepatic steatosis

doi: 10.1152/ajpendo.00010.2016

Figure Lengend Snippet: Insulin-stimulated Akt phosphorylation in Ctrp5-null adipose tissue, skeletal muscle, and liver. Quantitative Western blot analysis of insulin-stimulated Akt (Ser473) phosphorylation in adipose tissue (A), skeletal muscle (B), and liver (C) of WT and KO mice injected with insulin (1 U/kg body wt). Tissues were harvested at 15 min post-insulin injection. A total of 10 μg protein lysate from each sample was loaded onto Western blot gels. *P < 0.05.

Article Snippet: A ready-made human cDNA tissue panel (OriGene) was used to survey the tissue expression patterns of human CTRP5 .

Techniques: Western Blot, Injection

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Loss of CTRP5 improves insulin action and hepatic steatosis

doi: 10.1152/ajpendo.00010.2016

Figure Lengend Snippet: Lipid and adipokine profiles of Ctrp5-null mice fed a high-fat diet. A: representative histological sections of liver from WT and KO mice stained with hematoxylin and eosin. B and C: liver triglyceride and cholesterol levels of WT and KO mice. D–G: serum concentrations of triglycerides, cholesterol, and nonesterified free fatty acids (NEFA) and β-hydroxybutyrate (ketone) in WT and KO mice. H: skeletal muscle triglyceride levels of WT and KO mice. I: quantitative PCR analysis of genes involved in de novo lipid synthesis (Scd1, Fasn, Srebp1c, and Acc1) and fat oxidation (Lcad and Mcad) in WT and KO mouse liver. J: expression of genes (Gpat, Agpat, and Dgat) involved in triglyceride synthesis in WT and KO mouse liver. All expression levels were normalized to 18s rRNA. WT, n = 8; KO, n = 7. *P < 0.05.

Article Snippet: A ready-made human cDNA tissue panel (OriGene) was used to survey the tissue expression patterns of human CTRP5 .

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Loss of CTRP5 improves insulin action and hepatic steatosis

doi: 10.1152/ajpendo.00010.2016

Figure Lengend Snippet: Inflammatory and fibrotic states of adipose tissue in Ctrp5-null mice. A: representative histological sections of eWAT from WT and KO mice stained with hematoxylin and eosin. B and C: quantitative PCR analysis of macrophage marker genes (F4/80 and Cd11) in visceral (epididymal; eWAT) and subcutaneous (inguinal; iWAT) white adipose tissue. D and E: expression levels of fibrotic collagen genes (Col3 and Col6) in the visceral (eWAT) and subcutaneous (iWAT) fat depots of WT and KO mice. F–I: ELISA quantification of serum leptin, adiponectin, IL-6, and TNFα levels in WT and KO mice. J and K: expression levels of adiponectin and CTRPs in the eWAT and iWAT of WT and KO mice. All expression levels were normalized to 18s rRNA levels. WT, n = 8; KO, n = 7.

Article Snippet: A ready-made human cDNA tissue panel (OriGene) was used to survey the tissue expression patterns of human CTRP5 .

Techniques: Staining, Real-time Polymerase Chain Reaction, Marker, Expressing, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Loss of CTRP5 improves insulin action and hepatic steatosis

doi: 10.1152/ajpendo.00010.2016

Figure Lengend Snippet: Indirect calorimetry analysis of Ctrp5-null mice fed a high-fat diet. A–D: oxygen consumption (V̇o2), CO2 production (V̇co2), respiratory exchange ratio (RER), and energy expenditure (EE) for male WT and KO mice at 22 wk of age. E: total physical activity levels for WT and KO mice during the dark and light phases of the photocycle. F: real-time food intake measurements for WT and KO mice during the dark and light phases of the photocycle. G: cumulative food intake (over a 12-h period) for WT and KO mice in the dark and light phases of the photocycle. (WT, n = 6; KO, n = 8).

Article Snippet: A ready-made human cDNA tissue panel (OriGene) was used to survey the tissue expression patterns of human CTRP5 .

Techniques: Activity Assay

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Loss of CTRP5 improves insulin action and hepatic steatosis

doi: 10.1152/ajpendo.00010.2016

Figure Lengend Snippet: Reduced insulin resistance and hepatic triglyceride synthesis gene expression in aged Ctrp5-null mice fed a high-fat diet later in life. Weaned WT and KO male mice were fed a chow diet for 21 wk and then a HFD for 16 wk. A: body weights of male WT (n = 9) and KO (n = 7) mice after being switched to a HFD. B–E: fasting blood glucose, serum insulin, and C-peptide levels as well as the calculated insulin resistance (HOMA-IR) index of aged WT and KO mice after high-fat feeding for 16 wk. F: blood glucose levels of WT (n = 7) and KO (n = 5) mice at the indicated time points during glucose tolerance test (GTT). G: serum insulin levels were measured in the same group of mice during GTT. H: blood glucose levels of WT (n = 7) and KO (n = 5) mice at the indicated time points during insulin tolerance test (ITT). I: the decay constant (KITT) for WT and KO mice based on the ITT data. J: quantitative PCR analysis of genes involved in de novo lipid synthesis (Scd1, Fasn, Srebp1c, and Acc1) and fat oxidation (Lcad and Mcad) in WT and KO mouse liver. K: expression of genes (Gpat, Agpat, and Dgat) involved in triglyceride synthesis in WT and KO mouse liver. Food was removed for 3 h before liver tissue was harvested from mice. Expression levels were normalized to 18s rRNA levels. WT, n = 7; KO, n = 6. *P < 0.05; **P < 0.01.

Article Snippet: A ready-made human cDNA tissue panel (OriGene) was used to survey the tissue expression patterns of human CTRP5 .

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Loss of CTRP5 improves insulin action and hepatic steatosis

doi: 10.1152/ajpendo.00010.2016

Figure Lengend Snippet: Recombinant mouse CTRP5 attenuates insulin-stimulated Akt phosphorylation. A and C: mouse 3T3-L1 adipocytes (A) and rat L6 myotubes (C) were treated overnight with control conditioned medium or conditioned medium containing recombinant mouse CTRP5. The following day, cells were washed once and then stimulated with vehicle control or 100 nM insulin for 5 min. Cell lysates were then subjected to Western blot analysis with total and phosphorylated Akt antibodies. B: quantification of immunoblot results for 3T3-L1 adipocytes based on 2 independent experiments (n = 4). C: quantification of immunoblot results for L6 myotubes based on 2 independent experiments (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: A ready-made human cDNA tissue panel (OriGene) was used to survey the tissue expression patterns of human CTRP5 .

Techniques: Recombinant, Western Blot

Journal: Cell & Bioscience

Article Title: Growth inhibition and apoptosis in colorectal cancer cells induced by Vitamin D-Nanoemulsion (NVD): involvement of Wnt/β-catenin and other signal transduction pathways

doi: 10.1186/s13578-019-0277-z

Figure Lengend Snippet: Inhibition of cologenecity in colorectal cancer cells by NVD. (Clonogenic assay; 7 days). a , b NVD administration (40 and 60 μM) of HCT116 and HT29 cells inhibiting colony formation. Each value represents a mean ± SD (n = 3)

Article Snippet: Two human colorectal cancer cell lines HCT116 and HT29 (obtained from College of Pharmacy, King Saud University, KSA) were cultured in a 5% CO 2 atmosphere at 37 °C in medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC ® 30–2002TM), 10% fetal bovine serum (FBS, Gibco) as well as 1% penicillin/streptomycin.

Techniques: Inhibition, Clonogenic Assay

Journal: Cell & Bioscience

Article Title: Growth inhibition and apoptosis in colorectal cancer cells induced by Vitamin D-Nanoemulsion (NVD): involvement of Wnt/β-catenin and other signal transduction pathways

doi: 10.1186/s13578-019-0277-z

Figure Lengend Snippet: a , b NVD treatment on HCT116 and HT29 cells resulted in accumulation of cells at the G2-phase and growth inhibition and apoptosis. After 24 h incubation of NVD treated cells and staining with propidium iodide, DNA content was analyzed by flow cytometry. c , d NVD treatment on HCT116 and HT29 cells resulted in accumulation of cells at the G2-phase. After 24 h incubation of NVD treated cells and staining with propidium iodide, DNA content was analyzed by flow cytometry. Percentage of cell population in G2-phase of the cell cycle is shown. Experiments were performed in triplicate

Article Snippet: Two human colorectal cancer cell lines HCT116 and HT29 (obtained from College of Pharmacy, King Saud University, KSA) were cultured in a 5% CO 2 atmosphere at 37 °C in medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC ® 30–2002TM), 10% fetal bovine serum (FBS, Gibco) as well as 1% penicillin/streptomycin.

Techniques: Inhibition, Incubation, Staining, Flow Cytometry

Journal: Cell & Bioscience

Article Title: Growth inhibition and apoptosis in colorectal cancer cells induced by Vitamin D-Nanoemulsion (NVD): involvement of Wnt/β-catenin and other signal transduction pathways

doi: 10.1186/s13578-019-0277-z

Figure Lengend Snippet: Percentage of cell population after administration of NVD

Article Snippet: Two human colorectal cancer cell lines HCT116 and HT29 (obtained from College of Pharmacy, King Saud University, KSA) were cultured in a 5% CO 2 atmosphere at 37 °C in medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC ® 30–2002TM), 10% fetal bovine serum (FBS, Gibco) as well as 1% penicillin/streptomycin.

Techniques:

Journal: Cell & Bioscience

Article Title: Growth inhibition and apoptosis in colorectal cancer cells induced by Vitamin D-Nanoemulsion (NVD): involvement of Wnt/β-catenin and other signal transduction pathways

doi: 10.1186/s13578-019-0277-z

Figure Lengend Snippet: Effect of NVD treatment of HCT116 and HT29 ( a , b ) cells on protein expression of cdc2, WAF1/p21, cdk 2, 4 cyclin A, B1, E2 and ( c , d ) showing active Caspase 3, 7, 9 and PARP. Cells were administrated with NVD (20, 40 and 60 μM) for 24 h and 48 h and harvested. Total cell lysates were prepared and 40 μM proteins was subjected to SDS page followed by immunoblot analysis and chemiluminescence detection. Equal loading of protein was verified by stripping the Immunoblot and again probing it for Actin. The immunoblots shown here are representative of three individual experiments with similar results

Article Snippet: Two human colorectal cancer cell lines HCT116 and HT29 (obtained from College of Pharmacy, King Saud University, KSA) were cultured in a 5% CO 2 atmosphere at 37 °C in medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC ® 30–2002TM), 10% fetal bovine serum (FBS, Gibco) as well as 1% penicillin/streptomycin.

Techniques: Expressing, SDS Page, Western Blot, Stripping Membranes

Journal: Cell & Bioscience

Article Title: Growth inhibition and apoptosis in colorectal cancer cells induced by Vitamin D-Nanoemulsion (NVD): involvement of Wnt/β-catenin and other signal transduction pathways

doi: 10.1186/s13578-019-0277-z

Figure Lengend Snippet: a Effect of treatment of NVD to HCT116 and HT29 cell lines on protein expression of Bax, Bcl2, Bak and Bcl-X L . b Immunoblot analysis of β-catenin expression of HCT116 and HT29 in NVD treated group as compared to control group. The cells were administrated with NVD for 48 h and harvested and cell lysates prepared. The data are representative of three independent experiments with similar results

Article Snippet: Two human colorectal cancer cell lines HCT116 and HT29 (obtained from College of Pharmacy, King Saud University, KSA) were cultured in a 5% CO 2 atmosphere at 37 °C in medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC ® 30–2002TM), 10% fetal bovine serum (FBS, Gibco) as well as 1% penicillin/streptomycin.

Techniques: Expressing, Western Blot, Control

Journal: Cell & Bioscience

Article Title: Growth inhibition and apoptosis in colorectal cancer cells induced by Vitamin D-Nanoemulsion (NVD): involvement of Wnt/β-catenin and other signal transduction pathways

doi: 10.1186/s13578-019-0277-z

Figure Lengend Snippet: a Modulation of β -catenin expression by NVD treatment in HCT116 ( a ) and HT29 ( b ) cells. mRNA expression of β-catenin in NVD treated HCT116 and HT29 cells (RT-PCR), experiment performed in triplicate (mean ± SD), **p < 0.01. b Effect of NVD on protein expression of β-catenin, Survivin and phosphorylation of Akt at Ser473 in HCT116 and HT29 cells., Total cell lysate were prepared and 40 μM proteins was subjected to SDS-page followed by Immunoblot analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-Actin

Article Snippet: Two human colorectal cancer cell lines HCT116 and HT29 (obtained from College of Pharmacy, King Saud University, KSA) were cultured in a 5% CO 2 atmosphere at 37 °C in medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC ® 30–2002TM), 10% fetal bovine serum (FBS, Gibco) as well as 1% penicillin/streptomycin.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Phospho-proteomics, SDS Page, Western Blot, Stripping Membranes

Journal: Cell & Bioscience

Article Title: Growth inhibition and apoptosis in colorectal cancer cells induced by Vitamin D-Nanoemulsion (NVD): involvement of Wnt/β-catenin and other signal transduction pathways

doi: 10.1186/s13578-019-0277-z

Figure Lengend Snippet: Imunofluorescence staining of HCT116 ( b ) and HT29 ( d ) demonstrating expression of β-catenin in both NVD treated (60 µM) as compared to control HCT116 ( a ) and HT29 ( c ) cells (untreated). Alexa fluor staining of β-catenin of both cell lines (green fluorescence) and counter stained with DAPI (blue fluorescence) were observed

Article Snippet: Two human colorectal cancer cell lines HCT116 and HT29 (obtained from College of Pharmacy, King Saud University, KSA) were cultured in a 5% CO 2 atmosphere at 37 °C in medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC ® 30–2002TM), 10% fetal bovine serum (FBS, Gibco) as well as 1% penicillin/streptomycin.

Techniques: Staining, Expressing, Control, Fluorescence

Journal: Cell & Bioscience

Article Title: Growth inhibition and apoptosis in colorectal cancer cells induced by Vitamin D-Nanoemulsion (NVD): involvement of Wnt/β-catenin and other signal transduction pathways

doi: 10.1186/s13578-019-0277-z

Figure Lengend Snippet: Western blot analysis of cellular lysates prepared from HCT116 and HT29 cells. Lysates prepared from HCT116 and HT29 cells, initially seeded at density of 1.5 × 10 4 cells were administrated with 20, 40 and 60 µM concentrations of NVD. The blots probed with CK2 αα’, PI3-K, STAT3, STAT P-Try705, NFκB p65 P-S529, IκBα P-S32/36 and xIAP antibodies were shown. Actin blots are shown as loading controls. Data based on three different experiments, each carried out in triplicate

Article Snippet: Two human colorectal cancer cell lines HCT116 and HT29 (obtained from College of Pharmacy, King Saud University, KSA) were cultured in a 5% CO 2 atmosphere at 37 °C in medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC ® 30–2002TM), 10% fetal bovine serum (FBS, Gibco) as well as 1% penicillin/streptomycin.

Techniques: Western Blot