Journal: International Journal of Biological Sciences

Article Title: Triptolide Suppresses Glomerular Mesangial Cell Proliferation in Diabetic Nephropathy Is Associated with Inhibition of PDK1/Akt/mTOR Pathway

doi: 10.7150/ijbs.20485

Figure Lengend Snippet: The effect of TP on PDK1 expression in vivo and in vitro . (A) The expression of PDK1 in the kidney of HFD/STZ-induced diabetic rats. (B) Quantification of results in A. (C) The expression of PDK1 in HRMCs treated for 72 h. (D) Quantification of results in C. Data were reported as mean ± S.D.. * P < 0.05

Article Snippet: The primary antibodies and their dilutions used were as follows: PDK1 (1:1000, Cell Signaling Technology, USA), total-Akt (1:2000, Cell Signaling Technology, USA), phosphorylation-Akt (1:1000, Cell Signaling Technology, USA), total-mTOR (1:2000, Cell Signaling Technology, USA), phosphorylation-mTOR (1:1000, Cell Signaling Technology, USA), Ki-67 and PCNA (all diluted as 1:1000, Proteintech, USA).

Techniques: Expressing, In Vivo, In Vitro

Journal: International Journal of Biological Sciences

Article Title: Triptolide Suppresses Glomerular Mesangial Cell Proliferation in Diabetic Nephropathy Is Associated with Inhibition of PDK1/Akt/mTOR Pathway

doi: 10.7150/ijbs.20485

Figure Lengend Snippet: PDK1 activator reversed the effect of TP on mesangial cell proliferation. (A) MTT assay in the cells treated with HG for different times. * P < 0.05 vs. Control group, # P < 0.05 vs. HG+TP group. (B and C) Flow cytometry analysis of cell cycle in the HRMCs treated for 72 h. (D) Protein expression of PDK1/Akt/mTOR pathway in HRMCs treated for 72 h. (E) Protein expression of Ki-67 and PCNA in HRMCs treated for 72 h. (F) Immunofluorescence images of Ki-67 and PCNA. The scale bar represents 10 μm. Data were reported as mean ± S.D.. * P < 0.05; ns represents no significance

Article Snippet: The primary antibodies and their dilutions used were as follows: PDK1 (1:1000, Cell Signaling Technology, USA), total-Akt (1:2000, Cell Signaling Technology, USA), phosphorylation-Akt (1:1000, Cell Signaling Technology, USA), total-mTOR (1:2000, Cell Signaling Technology, USA), phosphorylation-mTOR (1:1000, Cell Signaling Technology, USA), Ki-67 and PCNA (all diluted as 1:1000, Proteintech, USA).

Techniques: MTT Assay, Control, Flow Cytometry, Expressing, Immunofluorescence

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) The landscape of PDK1-related clinicopathological features in osteosarcoma from the UCSCXena database. No statistically significant differences in PDK1 expression were observed among the following subgroups: (B) Age group (>18 vs. ≤ 18 years), (C) Gender group,(D) Metastasis status group, and (E) 3-year survival status group. Statistical significance was assessed using an unpaired t test.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Expressing

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) PDK1 was enriched in the deceased group of osteosarcoma patients in the UCSCXena databases. The significance of the difference was tested by unpaired t test. (B) The receiver-operating characteristic (ROC) curve showed the high-expression specificity of PDK1 in deceased group of osteosarcoma patients. AUC, area under the curve.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Expressing

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) AUC curve shows that the precision of this model is good. (B) Kaplan-Meier survival curves illustrate the increasing expression of PDK1 was negatively correlated with prognosis of patients with osteosarcoma. (C) The expression of PDK1 was negatively correlated with prognosis in non-metastic osteosarcoma group. (D)There was no significant correlation between PDK1 expression and prognosis in metastatic osteosarcoma group.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Expressing

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) Top 50 genes most negatively associated with PDK1 are shown in a heatmap. (B) Top 50 genes most positively associated with PDK1 are shown in a heatmap. (C) GO functional annotation analysis showed that PDK1 played a key role in biological processes such as metabolism and cell proliferation. (D) KEGG enrichment analysis showed that positive related genes with PDK1 were mostly enriched in cell metabolism and proliferation pathways, while negative related genes were enriched in immune-related signaling pathways. (E) Reactome analysis of significant gene pathways negatively correlated with PDK1. (F) Reactome analysis of significant gene pathways positively correlated with PDK1.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Functional Assay, Protein-Protein interactions

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) Connected dot plot comparing TP53 expression with PDK1 expression between the PDK1-high and PDK1-low groups. (B) Connected dot plot comparing CDKN2A expression with PDK1 expression between the PDK1-high and PDK1-low groups. (C) Connected dot plot comparing MDM2 expression with PDK1 expression between the PDK1-high and PDK1-low groups. (D) Connected dot plot comparing RB1 expression with PDK1 expression between the PDK1-high and PDK1-low groups.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Expressing

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) Distributions of PDK1 expression levels are displayed using box plots. The statistical significance computed by the Wilcoxon test is annotated by the number of stars. (B) Overall survival analysis the prognostic relationship of PDK1 expression in different types of cancer by Cox proportional hazards regression model. (C) DFI analysis the prognostic relationship of PDK1 expression in different types of cancer by Cox proportional hazards regression model.(D-I) Pan-cancer Kaplan-Meier overall survival of PDK1 in indicated tumor types from TCGA database. The median value of PDK1 in each tumor was taken as the percentile (50%) of PDK1 expression.(J-L) Pan-cancer differential expression of PDK1 in WHO stages in indicatedumor types from TCGA database. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Expressing, Quantitative Proteomics

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) PDK1 expression and clinical features. (B)The calibration of the nomogram at 1, 3, and 5 years in the training cohort.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Expressing

Journal: PLOS One

Article Title: PDK1, associated with glycolytic metabolism, is a potential prognostic biomarker in osteosarcoma

doi: 10.1371/journal.pone.0332494

Figure Lengend Snippet: (A) Comparison of PDK1 mRNA expression between U-2 OS, 143B, MG63 and hFOB1.19 by qRT-PCR(n = 3). (B) Comparison of PDK1 protein expression between U-2 OS, 143B, MG63 and hFOB1.19 by western blotting(n = 3). (C) Analysis of PDK siRNA transfection efficiency in 143B cells by qRT-PCR(n = 3). (D) Detection of 143B viability at 24 hrs, 48 hrs and 72 hrs after silencing PDK1 by CCK-8 analysis(n = 3). (E) The effect of PDK1 siRNA on colony forming ability of 143B was detected by clone formation assay(n = 1). (F) Flow cytometry was used to detect the effect of PDK1 siRNA group and control group on apoptosis of 143B cells(n = 3). (G) Flow cytometry was used to detect the effect of PDK1 siRNA group and control group on cell cycle of 143B cells(n = 3). (H) Analysis of lactate content: The lactate produced during the glycolytic metabolism of 143B cells in the silenced PDK1 group and the control group(n = 3). (I) Analysis of ATP content: The ATP produced during the glycolytic metabolism of 143B cells in the silenced PDK1 group and the control group(n = 3). (J) Analysis of C6P content: The C6P produced during the glycolytic metabolism of 143B cells in the silenced PDK1 group and the control group(n = 3). (K) Analysis of glucose content: The glucose content during the glycolytic metabolism of 143B cells in the silenced PDK1 group and the control group(n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Membranes were blocked with 5% skim milk and incubated with PDK1 primary antibody (1:2000; Proteintech, cat. 18262–1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000).

Techniques: Comparison, Expressing, Quantitative RT-PCR, Western Blot, Transfection, CCK-8 Assay, Tube Formation Assay, Flow Cytometry, Control, Produced

Journal: PLoS ONE

Article Title: PDK1-Foxo1 in Agouti-Related Peptide Neurons Regulates Energy Homeostasis by Modulating Food Intake and Energy Expenditure

doi: 10.1371/journal.pone.0018324

Figure Lengend Snippet: ( A ) Representative immunofluorescence images of hypothalamic regions doubly labeled for AGRP and PDK1 in 24-week-old AGRPPdk1 +/+ (top panel) and AGRPPdk1 −/− (bottom panel) mice fed ad libitum . Green, red and blue indicate AGRP, PDK1, and DAPI staining, respectively. White arrows indicate AGRP neurons. Scale bars indicate 50 µm. ( B ) Quantification of PDK1-positive AGRP neurons from AGRPPdk1 +/+ (gray bar) and AGRPPdk1 −/− (blue bar) mice. The percentage of PDK1-positive AGRP neurons among AGRP neurons was determined as described in Experimental Procedures. Values are means ± SEM of three mice of each genotype. An asterisk indicates a statistically significant difference between AGRPPdk1 +/+ and AGRPPdk1 −/− mice with p<0.001 (one-factor ANOVA). ( C ) Body weight of male (left panel) and female (right panel) control (open circles) and AGRPPdk1 −/− (closed circles) mice fed normal chow. Values represent mean ± SEM of 35–40 mice in each genotype. An asterisk indicates a statistically significant difference between control and AGRPPdk1 −/− mice with p<0.05 (one-factor ANOVA). ( D ) Naso-anal length (NAL) of male (left panel) and female (right panel) control mice (gray bar) and AGRPPdk1 −/− mice (blue bar) at 24 weeks of age. Values represent mean ± SEM of 30 mice in each genotype. An asterisk indicates a statistically significant difference between control and AGRPPdk1 −/− mice with p<0.05 (one-factor ANOVA). ( E ) The calculated weights of muscle, adipose tissue, visceral fat, and subcutaneous fat in male control (gray bar) and AGRPPdk1 −/− (blue bar) mice at 24 weeks of age. Skeletal muscle weight, adipose tissue weight (sum of visceral and subcutaneous fats), visceral fat weight, and subcutaneous fat weight were calculated as described in Experimental Procedures. The data represent the mean weight ± SEM of 10 mice per genotype. Asterisks indicate statistically significant differences between control and AGRPPdk1 −/− mice with *p<0.001, **p<0.005, and ***p<0.05 (one-factor ANOVA). ( F ) Four-day food intake of male and female control mice (gray bar) and AGRPPdk1 −/− mice (blue bar) at 24 weeks of age. Values represent mean ± SEM of 40 mice in each genotype. An asterisk indicates a statistically significant difference between control and AGRPPdk1 −/− mice with p<0.05 (one-factor ANOVA). ( G ) Oxygen consumption in male control (gray bar) and AGRPPdk1 −/− (blue bar) mice at 24 weeks of age. The values represent the mean ± SEM of 10 male mice per genotype. ( H ) Respiratory quotient of male control (gray bar) and AGRPPdk1 −/− (blue bar) mice at 24 weeks of age. The values represent the mean ± SEM of 10 mice per genotype. ( I ) Basal locomotor activity for male control (gray bar) and AGRPPdk1 −/− (blue bar) mice at 24 weeks of age during the light and dark phases. The values represent the mean ± SEM of 5 mice per genotype. ( J–K ) Effects of intraperitoneal leptin injections on body weight and food intake. Leptin was injected into control (open circle, n = 8) or AGRPPdk1 −/− mice (closed circle, n = 5) for three consecutive days. Body weight (J) and food intake (K) were monitored for seven days. Body weight and food intake are presented as percentages of values from saline-injected mice for each genotype. An asterisk in (J) indicates a statistically significant difference between control and AGRPPdk1 −/− mice with p<0.05 (one-factor ANOVA). An asterisk in (K) indicates a statistically significant difference between saline- and leptin-injected mice in AGRPPdk1 −/− mice with p<0.05 (one-factor ANOVA).

Article Snippet: The primary antibodies were Ab-241 (Signalway Antibody, Pearland, TX) for PDK1 and GT15023 (Neuromics, Edina, MN) for AGRP; the secondary antibodies were Alexa Fluor R 594 chicken anti-rabbit IgG and Alexa Fluor R 488 donkey anti-goat IgG (Molecular Probes, Eugene, OR).

Techniques: Immunofluorescence, Labeling, Staining, Activity Assay, Injection

Journal: PLoS ONE

Article Title: PDK1-Foxo1 in Agouti-Related Peptide Neurons Regulates Energy Homeostasis by Modulating Food Intake and Energy Expenditure

doi: 10.1371/journal.pone.0018324

Figure Lengend Snippet: ( A ) Subcellular localization of Foxo1 in AGRP neurons from AGRPPdk1 +/+ (top panel) and AGRPPdk1 −/− (bottom panel) mice. Representative double immunofluorescence images show hypothalamic regions doubly-labeled for AGRP and Foxo1. Green, AGRP; red, Foxo1; blue, DAPI staining. White arrows indicate AGRP neurons. ( B ) Quantification of nuclear Foxo1 in AGRP neurons from AGRPPdk1 +/+ and AGRPPdk1 −/− mice. The percentage of AGRP neurons with nuclear Foxo1 among all AGRP neurons was determined as described in Experimental Procedures. Values are means ± SEM of three mice in each genotype. An asterisk indicates a statistically significant difference between AGRPPdk1 +/+ and AGRPPdk1 −/− mice with p<0.001 (one-factor ANOVA). ( C ) Body weights of male control (open circles), Δ256Foxo1 AGRP (open triangles), AGRPPdk1 −/− (closed circles), and Δ256Foxo1 AGRP Pdk1 −/− (closed triangles) mice. Values represent the mean ± SEM of 20 mice in each genotype. Asterisks indicate statistically significant differences between AGRPPdk1 −/− and Δ256Foxo1 AGRP Pdk1 −/− with *p<0.001, **p<0.01, and ***p<0.05 (one-factor ANOVA). ( D ) The calculated weights of muscle, adipose tissue, visceral fat, and subcutaneous fat in male control (gray bar), Δ256Foxo1 AGRP (white bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice at 24 weeks of age. Skeletal muscle weight, adipose tissue weight (sum of visceral and subcutaneous fats), visceral fat weight, and subcutaneous fat weight were calculated as described in Experimental Procedures. The data represent the mean weight ± SEM of 10 mice per genotype. Asterisks indicate statistically significant differences as indicated with *p<0.001, **p<0.005, and ***p<0.05 (one-factor ANOVA). ( E ) Four-day food intake for male control (gray bar), Δ256Foxo1 AGRP (white bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice at 24 weeks of age. The data represent the mean ± SEM of 10 mice per genotype. An asterisk indicates a statistically significant difference as indicated with *p<0.005 (one-factor ANOVA). ( F ) Expression in the fed-state of hypothalamic neuropeptide genes in control (gray bar), Δ256Foxo1 AGRP (white bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice. Data were normalized to β-actin expression and represent the mean ± SEM of six mice per genotype. An asterisk indicates a statistically significant difference between control and Δ256Foxo1 AGRP Pdk1 −/− mice with p<0.05 (one-factor ANOVA). ( G ) Oxygen consumption in male control (gray bar), Δ256Foxo1 AGRP (white bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice at 24 weeks of age. The values represent the mean ± SEM of 10 male mice per genotype. ( H ) Respiratory quotient of male control (gray bar), Δ256Foxo1 AGRP (white bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice at 24 weeks of age. The values represent the mean ± SEM of 10 mice per genotype. An asterisk indicates a statistically significant difference between AGRPPdk1 −/− and Δ256Foxo1 AGRP Pdk1 −/− mice with p<0.05 (one-factor ANOVA). ( I ) Basal locomotor activity for male control (gray bar), Δ256Foxo1 AGRP (white bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice at 24 weeks of age during the light and dark phases. The values represent the mean ± SEM of 5 mice per genotype. An asterisk indicates a statistically significant difference as indicated with p<0.001 (one-factor ANOVA).

Article Snippet: The primary antibodies were Ab-241 (Signalway Antibody, Pearland, TX) for PDK1 and GT15023 (Neuromics, Edina, MN) for AGRP; the secondary antibodies were Alexa Fluor R 594 chicken anti-rabbit IgG and Alexa Fluor R 488 donkey anti-goat IgG (Molecular Probes, Eugene, OR).

Techniques: Immunofluorescence, Labeling, Staining, Expressing, Activity Assay

Journal: PLoS ONE

Article Title: PDK1-Foxo1 in Agouti-Related Peptide Neurons Regulates Energy Homeostasis by Modulating Food Intake and Energy Expenditure

doi: 10.1371/journal.pone.0018324

Figure Lengend Snippet: ( A ) Effects of 4 weeks of calorie restriction on body weight of 20-week-old male control (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice. Values represent means ± SEM of the percentages of reduction of body weight at the end (4 week) of pair feeding compared to at the beginning of pair feeding. An asterisk indicates a statistically significant difference between bodyweight at the beginning and at the end of calorie restriction with p<0.05 (one-factor ANOVA; n = 10 male mice per genotype). ( B ) Oxygen consumption of male calorie-restricted control (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) during the light and dark phases. Values represent the mean ± SEM of 10 mice per genotype. An asterisk indicates a statistically significant difference between AGRPPdk1 −/− and control or Δ256Foxo1 AGRP Pdk1 −/− mice with p<0.05 (one-factor ANOVA). ( C ) Respiratory quotients of male pair-fed control (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice during the light and dark phases. Values represent the mean ± SEM of 10 mice per genotype. An asterisk indicates a statistically significant difference between AGRPPdk1 −/− and control or Δ256Foxo1 AGRP Pdk1 −/− mice with p<0.001 (one-factor ANOVA). ( D ) Basal locomotor activity of male pair-fed control (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) during the light and dark phases. Values represent the mean ± SEM of 10 mice per genotype. An asterisk indicates a statistically significant difference between AGRPPdk1 −/− and Δ256Foxo1 AGRP Pdk1 −/− mice with p<0.05 (one-factor ANOVA). ( E–F ) Effects of intraperitoneal leptin injections on body weight and food intake of pair-fed control (open squares, n = 10), AGRPPdk1 −/− mice (blue closed circles, n = 10), and Δ256Foxo1 AGRP Pdk1 −/− (red squares, n = 10) mice. Leptin was injected on 3 consecutive days. Body weight (E) and food intake (F) were monitored for eight days. Body weight and food intake data are presented as the percentage of that for saline-injected mice for each genotype. An asterisk in (F) indicates statistically significant differences between control and AGRPPdk1 −/− mice with *p<0.001, **p<0.01, and ***p<0.05 by one-factor ANOVA. ( G ) Cumulative food intake for male pair-fed control (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice injected with saline or ghrelin after a 24-h fast. Cumulative food intake was monitored for 6 h. Values represent the ratio of the mean cumulative food intakes of ghrelin:vehicle injected mice for each genotype. An asterisk indicates statistically significant differences between control and AGRPPdk1 −/− mice with *p<0.02 (one-factor ANOVA; n = 10 per genotype for each condition). ( H ) Cumulative food intake of male pair-fed control (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice injected with saline or MTII after a 24-h fast. Cumulative food intake was monitored for 24 h. Values represent the ratio of the mean cumulative food intakes of MTII:vehicle injected mice for each genotype. Asterisks indicate statistically significant differences between control and AGRPPdk1 −/− mice with p<0.05 (one-factor ANOVA; n = 10 per genotype).

Article Snippet: The primary antibodies were Ab-241 (Signalway Antibody, Pearland, TX) for PDK1 and GT15023 (Neuromics, Edina, MN) for AGRP; the secondary antibodies were Alexa Fluor R 594 chicken anti-rabbit IgG and Alexa Fluor R 488 donkey anti-goat IgG (Molecular Probes, Eugene, OR).

Techniques: Activity Assay, Injection

Journal: PLoS ONE

Article Title: PDK1-Foxo1 in Agouti-Related Peptide Neurons Regulates Energy Homeostasis by Modulating Food Intake and Energy Expenditure

doi: 10.1371/journal.pone.0018324

Figure Lengend Snippet: ( A )–( C ) Administration of 10 −10 M of ghrelin increased cytosolic calcium ([Ca 2+ ] i ) in AGRP neurons from AGRPPdk1 +/+ (A), AGRPPdk1 −/− (B), and Δ256Foxo1 AGRP Pdk1 −/− (C) mice. Administration of 10 −12 M of leptin inhibited increases in [Ca 2+ ] i induced by ghrelin in AGRPPdk1 +/+ (A) and Δ256Foxo1 AGRP Pdk1 −/− (B) neurons, but not in AGRPPdk1 −/− neurons (C). ( D ) Amplitude of [Ca 2+ ] i responses to ghrelin in the absence or presence of leptin (10 −12 M) in AGRP neurons of AGRPPdk1 +/+ (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar) mice. Values are means ± SEM of the ratio of [Ca 2+ ] i in the presence and absence of leptin. An asterisk indicates a statistically significant difference between the absence and the presence of leptin with p<0.05 (one-factor ANOVA: n = 10 AGRP neurons per genotype). ( E ) Fura-2 detection of [Ca 2+ ] i in response to ghrelin. Values are means ± SEM of ghrelin-stimulated fura-2 ratios in AGRP neurons from AGRPPdk1 +/+ (gray bar), AGRPPdk1 −/− (blue bar), and Δ256Foxo1 AGRP Pdk1 −/− (magenta bar). An asterisk indicates a statistically significant difference between AGRPPdk1 −/− and control or Δ256Foxo1 AGRP Pdk1 −/− mice with p<0.05 (one-factor ANOVA: n = 10 AGRP neurons per genotype).

Article Snippet: The primary antibodies were Ab-241 (Signalway Antibody, Pearland, TX) for PDK1 and GT15023 (Neuromics, Edina, MN) for AGRP; the secondary antibodies were Alexa Fluor R 594 chicken anti-rabbit IgG and Alexa Fluor R 488 donkey anti-goat IgG (Molecular Probes, Eugene, OR).

Techniques:

Journal: BMC musculoskeletal disorders

Article Title: Pyruvate Dehydrogenase Kinase 1 inhibition mediated oxidative phosphorylation enhancement in cartilage promotes osteoarthritis progression.

doi: 10.1186/s12891-023-06585-6

Figure Lengend Snippet: Fig. 2 PDK1 is involved in OA progression based on GEO. A Differential expression of PDK1 in GSE55235. B Differential expression of PDK1 in GSE98918. C The PPI network for PDK1 was constructed in GeneMANIA. D KEGG analysis for genes that highly correlated with PDK1. E GO analysis for genes that highly correlated with PDK1

Article Snippet: The sections were stained with a primary antibody against PDK1 (A01268-1, Boster), followed by the secondary antibody and Dapi.

Techniques: Quantitative Proteomics, Construct

Journal: BMC musculoskeletal disorders

Article Title: Pyruvate Dehydrogenase Kinase 1 inhibition mediated oxidative phosphorylation enhancement in cartilage promotes osteoarthritis progression.

doi: 10.1186/s12891-023-06585-6

Figure Lengend Snippet: Fig. 3 PDK1 is downregulated in human and mice osteoarthritic cartilage. A Immunofluorescence staining of PDK1 in articular cartilage from healthy and OA human. PDK1 stains red. Dapi stains the nucleus. B Quantification of an absolute number of PDK1.+ cells in articular cartilage from human. C Quantification analysis of the fluorescence intensity of PDK1 expression in articular cartilage from human. D Immunofluorescence staining of PDK1 in articular cartilage at sham or 4 weeks post-surgery. PDK1 stains red. Dapi stains the nucleus. E Quantifying an absolute number of PDK1 + cells in upper articular cartilage in the knee at the sham or 4 weeks post-surgery. F Quantification analysis of the fluorescence intensity of PDK1 expression in articular cartilage in the knee at sham or 4 weeks post-surgery. G PDH activity in articular cartilage at sham or 4 weeks post-surgery. In B, C, and E–G, horizontal lines and error bars show the mean ± SD (n ≥ 3 mice per group). * = P < 0.05; ** = P < 0.01; *** = P < 0.001

Article Snippet: The sections were stained with a primary antibody against PDK1 (A01268-1, Boster), followed by the secondary antibody and Dapi.

Techniques: Immunofluorescence, Staining, Fluorescence, Expressing, Activity Assay

Journal: BMC musculoskeletal disorders

Article Title: Pyruvate Dehydrogenase Kinase 1 inhibition mediated oxidative phosphorylation enhancement in cartilage promotes osteoarthritis progression.

doi: 10.1186/s12891-023-06585-6

Figure Lengend Snippet: Fig. 5 Suppressing of PDK1 disrupted anabolism and catabolism. A-C Representative immunohistochemistry images of Col2, Acan, MMP13 staining for mice at sham,4 or 8 weeks after DMM with or without jx06. D-F The mean ratio of integrated optical density (IOD) to area (IOD/area) was used to semi-quantify Col2, Acan, MMP13 amount, horizontal lines and error bars show the mean ± SD (n ≥ 3 mice per group). * = P < 0.05; ** = P < 0.01; *** = P < 0.001

Article Snippet: The sections were stained with a primary antibody against PDK1 (A01268-1, Boster), followed by the secondary antibody and Dapi.

Techniques: Immunohistochemistry, Staining

Journal: BMC musculoskeletal disorders

Article Title: Pyruvate Dehydrogenase Kinase 1 inhibition mediated oxidative phosphorylation enhancement in cartilage promotes osteoarthritis progression.

doi: 10.1186/s12891-023-06585-6

Figure Lengend Snippet: Fig. 6 Suppressing of PDK1 accelerated synovium inflammation. A Representative immunohistochemistry images of F4/80 staining for mice at sham,4 or 8 weeks after DMM with or without jx06. B Representative immunohistochemistry images of TNF-a staining for mice at sham,4 or 8 weeks after DMM with or without jx06. C Quantification of absolute number of F4/80.+ cells in synovium in the knee at sham or 4 weeks post-surgery. D The mean ratio of integrated optical density (IOD) to area (IOD/area) was used to semi-quantify TNF-a amount. E Model of PDK1 inhibitor modulates ECM degradation and synovium inflammation in mechanical stress-induced OA. Horizontal lines and error bars show the mean ± SD (n ≥ 3 mice per group). * = P < 0.05; ** = P < 0.01; *** = P < 0.001

Article Snippet: The sections were stained with a primary antibody against PDK1 (A01268-1, Boster), followed by the secondary antibody and Dapi.

Techniques: Immunohistochemistry, Staining