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Millipore materials metformin
Degradation of pSTAT3 ser727 in grade 1 endometrial cancer cells treated with meformin. After Ishikawa cells were treated with control, 10 mM, or 20 mM <t>metformin</t> in high glucose media for 48h, samples were subjected to ubiquitin assay. The ubiquitinated proteins were subjected to immunoblot for pSTAT3 Ser727 and blotted with ubiquitin antibody. A ubiquitination smear of pSTAT3 Ser727 is seen in the metformin treated ishikawa cells under proteasomal inhibition using MG-132.
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1) Product Images from "High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer"

Article Title: High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0170318

Degradation of pSTAT3 ser727 in grade 1 endometrial cancer cells treated with meformin. After Ishikawa cells were treated with control, 10 mM, or 20 mM metformin in high glucose media for 48h, samples were subjected to ubiquitin assay. The ubiquitinated proteins were subjected to immunoblot for pSTAT3 Ser727 and blotted with ubiquitin antibody. A ubiquitination smear of pSTAT3 Ser727 is seen in the metformin treated ishikawa cells under proteasomal inhibition using MG-132.
Figure Legend Snippet: Degradation of pSTAT3 ser727 in grade 1 endometrial cancer cells treated with meformin. After Ishikawa cells were treated with control, 10 mM, or 20 mM metformin in high glucose media for 48h, samples were subjected to ubiquitin assay. The ubiquitinated proteins were subjected to immunoblot for pSTAT3 Ser727 and blotted with ubiquitin antibody. A ubiquitination smear of pSTAT3 Ser727 is seen in the metformin treated ishikawa cells under proteasomal inhibition using MG-132.

Techniques Used: Ubiquitin Assay, Inhibition

Metformin inhibits grade 1 endometrial cancer cell proliferation, survival, migration, and induces apoptosis. A . Sulforhodamine B (SRB) assay measured Ishikawa cell proliferation with increasing concentrations of metformin after 24h or 48h, in high-glucose media. B . Proportion of Ishikawa cells surviving (compared to control) after treatment with 10 mM or 20 mM of metformin. C . Cell migration assay, with Ishikawa cells subjected to control, 10 mM, or 20 mM metformin for 24h. Control (0h) is also shown. D . Quantification of % wound closure; the 10 mM and 20 mM treatment groups were each significantly different than control (p
Figure Legend Snippet: Metformin inhibits grade 1 endometrial cancer cell proliferation, survival, migration, and induces apoptosis. A . Sulforhodamine B (SRB) assay measured Ishikawa cell proliferation with increasing concentrations of metformin after 24h or 48h, in high-glucose media. B . Proportion of Ishikawa cells surviving (compared to control) after treatment with 10 mM or 20 mM of metformin. C . Cell migration assay, with Ishikawa cells subjected to control, 10 mM, or 20 mM metformin for 24h. Control (0h) is also shown. D . Quantification of % wound closure; the 10 mM and 20 mM treatment groups were each significantly different than control (p

Techniques Used: Migration, Sulforhodamine B Assay, Cell Migration Assay

Xenograft endometrial tumor weight and expression of STAT3 in mice treated with metformin. A xenograft study was done in which nude mice were injected with 1 x 10 6 Ishikawa endometrial cancer cells subcutaneously in the right flank. After tumors were at least 3–5 mm in diameter, treatment with control, metformin 100 mg/kg, or metformin 200 mg/kg was started. Mice were sacrificed after 4 weeks of treatment. A . Tumor weight (in grams) from the mice with the 3 largest tumors in each group. B . Average body weight (g) of the mice in each group at conclusion of the study. C . Western blot results of STAT3 and associated proteins after treatment with control, 100 mg/kg, or 200 mg/kg of metformin.
Figure Legend Snippet: Xenograft endometrial tumor weight and expression of STAT3 in mice treated with metformin. A xenograft study was done in which nude mice were injected with 1 x 10 6 Ishikawa endometrial cancer cells subcutaneously in the right flank. After tumors were at least 3–5 mm in diameter, treatment with control, metformin 100 mg/kg, or metformin 200 mg/kg was started. Mice were sacrificed after 4 weeks of treatment. A . Tumor weight (in grams) from the mice with the 3 largest tumors in each group. B . Average body weight (g) of the mice in each group at conclusion of the study. C . Western blot results of STAT3 and associated proteins after treatment with control, 100 mg/kg, or 200 mg/kg of metformin.

Techniques Used: Expressing, Mouse Assay, Injection, Western Blot

Expression of apoptosis and cell proliferation-related proteins in metformin-treated grade 1 endometrial cancer cells overexpressing STAT3. Ishikawa endometrial cancer cells were transfected with a STAT3-overexpressing plasmid. A . Western blot confirming overexpression of pSTAT3 ser727 and total STAT3. B . Western blot of proteins involved in apoptosis or cell proliferation in control or STAT3-overexpressing Ishikawa cells treated with control or 20 mM metformin in high-glucose medium for 48h. (C: control, TR: transfection reagent only, OE: transfected with STAT3-overexpressing plasmid, Ctrl: control, Met: metformin).
Figure Legend Snippet: Expression of apoptosis and cell proliferation-related proteins in metformin-treated grade 1 endometrial cancer cells overexpressing STAT3. Ishikawa endometrial cancer cells were transfected with a STAT3-overexpressing plasmid. A . Western blot confirming overexpression of pSTAT3 ser727 and total STAT3. B . Western blot of proteins involved in apoptosis or cell proliferation in control or STAT3-overexpressing Ishikawa cells treated with control or 20 mM metformin in high-glucose medium for 48h. (C: control, TR: transfection reagent only, OE: transfected with STAT3-overexpressing plasmid, Ctrl: control, Met: metformin).

Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Over Expression

Metformin inhibits STAT3, its regulatory proteins and upregulated apoptosis-related proteins, in grade 1 endometrial cancer cells. A . Western blot of STAT3 and its regulatory proteins in Ishikawa cells after treatment with control, 10 mM, or 20 mM metformin for 48h in high-glucose conditions. B . qPCR of STAT3 and some of its regulatory genes in Ishikawa cells after treatment with control, 10 mM, or 20 mM metformin for 48h. Groups significantly different than control (p
Figure Legend Snippet: Metformin inhibits STAT3, its regulatory proteins and upregulated apoptosis-related proteins, in grade 1 endometrial cancer cells. A . Western blot of STAT3 and its regulatory proteins in Ishikawa cells after treatment with control, 10 mM, or 20 mM metformin for 48h in high-glucose conditions. B . qPCR of STAT3 and some of its regulatory genes in Ishikawa cells after treatment with control, 10 mM, or 20 mM metformin for 48h. Groups significantly different than control (p

Techniques Used: Western Blot, Real-time Polymerase Chain Reaction

2) Product Images from "Repurposing phenformin for the targeting of glioma stem cells and the treatment of glioblastoma"

Article Title: Repurposing phenformin for the targeting of glioma stem cells and the treatment of glioblastoma

Journal: Oncotarget

doi: 10.18632/oncotarget.10919

A combined treatment of phenformin and TMZ exerts an enhanced cytotoxic effect on GSCs in vitro and in vivo ( A ) MTT assay determined cytotoxicity of combined treatments of phenformin or metformin with TMZ (100 μM) for 24 hr. Statistical analysis compares TMZ treated (pink bars) vs control (blue bars). * p
Figure Legend Snippet: A combined treatment of phenformin and TMZ exerts an enhanced cytotoxic effect on GSCs in vitro and in vivo ( A ) MTT assay determined cytotoxicity of combined treatments of phenformin or metformin with TMZ (100 μM) for 24 hr. Statistical analysis compares TMZ treated (pink bars) vs control (blue bars). * p

Techniques Used: In Vitro, In Vivo, MTT Assay

DCA treatment increases the inhibitory effect of phenformin on the stemness and survival of GSCs in vitro and in vivo ( A ) A combined treatment of phenformin or metformin with DCA on the inhibition of GSC self-renewal in 10 days. * represents the statistical analysis based on the DCA concentration, i.e, comparing 5 mM or 10 mM DCA treated groups with control untreated group ; + represents the statistical analysis that compares biguanide treatment vs. control. * or + p
Figure Legend Snippet: DCA treatment increases the inhibitory effect of phenformin on the stemness and survival of GSCs in vitro and in vivo ( A ) A combined treatment of phenformin or metformin with DCA on the inhibition of GSC self-renewal in 10 days. * represents the statistical analysis based on the DCA concentration, i.e, comparing 5 mM or 10 mM DCA treated groups with control untreated group ; + represents the statistical analysis that compares biguanide treatment vs. control. * or + p

Techniques Used: In Vitro, In Vivo, Inhibition, Concentration Assay

3) Product Images from "Metformin Ameliorates Aβ Pathology by Insulin-Degrading Enzyme in a Transgenic Mouse Model of Alzheimer's Disease"

Article Title: Metformin Ameliorates Aβ Pathology by Insulin-Degrading Enzyme in a Transgenic Mouse Model of Alzheimer's Disease

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2020/2315106

Metformin ameliorates oxidative stress and neuroinflammation in APP/PS1 mice. The level of (a) MDA and the activity of (b) SOD in the brain of APP/PS1 mice. The levels of (a) IL-1 β and (b) IL-6 in the brain of APP/PS1 mice. Experimental values were expressed as the mean ± SEM ( n = 6 per group). ∗ p
Figure Legend Snippet: Metformin ameliorates oxidative stress and neuroinflammation in APP/PS1 mice. The level of (a) MDA and the activity of (b) SOD in the brain of APP/PS1 mice. The levels of (a) IL-1 β and (b) IL-6 in the brain of APP/PS1 mice. Experimental values were expressed as the mean ± SEM ( n = 6 per group). ∗ p

Techniques Used: Mouse Assay, Multiple Displacement Amplification, Activity Assay

Metformin decreases A β levels in APP/PS1 mice. The levels of (a) A β 1-40 and (b) A β 1-42 in the brain of APP/PS1 mice. ThT staining of the brain slides in APP/PS1 mice. Experimental values were expressed as the mean ± SEM ( n = 6 per group). ∗ p
Figure Legend Snippet: Metformin decreases A β levels in APP/PS1 mice. The levels of (a) A β 1-40 and (b) A β 1-42 in the brain of APP/PS1 mice. ThT staining of the brain slides in APP/PS1 mice. Experimental values were expressed as the mean ± SEM ( n = 6 per group). ∗ p

Techniques Used: Mouse Assay, Staining

Metformin improves glucose metabolism in APP/PS1 mice. (a) PET-CT images. (b) 18 F-FDG uptake of mice brains. Data represent the mean ± SEM ( n = 3 per group). ∗ p
Figure Legend Snippet: Metformin improves glucose metabolism in APP/PS1 mice. (a) PET-CT images. (b) 18 F-FDG uptake of mice brains. Data represent the mean ± SEM ( n = 3 per group). ∗ p

Techniques Used: Mouse Assay, Positron Emission Tomography

Metformin activates AMPK and increases IDE in the brain of APP/PS1 mice. (a) The representative bands of p-AMPK, AMPK, IDE, NEP, and ACTB. Western blot analysis: (b) p-AMPK/AMPK, (c) IDE/ACTB, and (d) NEP/ACTB. Experimental values were expressed as the mean ± SEM ( n = 3 per group). ∗ p
Figure Legend Snippet: Metformin activates AMPK and increases IDE in the brain of APP/PS1 mice. (a) The representative bands of p-AMPK, AMPK, IDE, NEP, and ACTB. Western blot analysis: (b) p-AMPK/AMPK, (c) IDE/ACTB, and (d) NEP/ACTB. Experimental values were expressed as the mean ± SEM ( n = 3 per group). ∗ p

Techniques Used: Mouse Assay, Western Blot

Metformin improves neurotrophic factors in APP/PS1 mice. The mRNA levels of (a) Syp , (b) Bdnf , and (c) Ngf in the APP/PS1 mice. Data represent the mean ± SEM ( n = 6 per group). ∗ p
Figure Legend Snippet: Metformin improves neurotrophic factors in APP/PS1 mice. The mRNA levels of (a) Syp , (b) Bdnf , and (c) Ngf in the APP/PS1 mice. Data represent the mean ± SEM ( n = 6 per group). ∗ p

Techniques Used: Mouse Assay

Metformin improves learning and memory impairment in APP/PS1 mice. (a) Escape latency of the five-day Morris water maze. (b) Time spent in the target quadrant in the Morris water maze. (c) Crossing times of the target platform in the Morris water maze. (d) Swimming speed in the Morris water maze. (e) Percentage of spontaneous alternation of Y-maze. Data represent the mean ± SEM ( n = 15 per group). ∗ p
Figure Legend Snippet: Metformin improves learning and memory impairment in APP/PS1 mice. (a) Escape latency of the five-day Morris water maze. (b) Time spent in the target quadrant in the Morris water maze. (c) Crossing times of the target platform in the Morris water maze. (d) Swimming speed in the Morris water maze. (e) Percentage of spontaneous alternation of Y-maze. Data represent the mean ± SEM ( n = 15 per group). ∗ p

Techniques Used: Mouse Assay

Metformin has no effect on A β production and transportation-related genes in APP/PS1 mice. The activities of (a) α -, (b) β -, and (c) γ -secretases. The mRNA expressions of (d) ADAM10 , (e) BACE1 , (f) PS1 , (g) LRP1 , and (h) RAGE . Experimental values were expressed as the mean ± SEM ( n = 6 per group). ∗ p
Figure Legend Snippet: Metformin has no effect on A β production and transportation-related genes in APP/PS1 mice. The activities of (a) α -, (b) β -, and (c) γ -secretases. The mRNA expressions of (d) ADAM10 , (e) BACE1 , (f) PS1 , (g) LRP1 , and (h) RAGE . Experimental values were expressed as the mean ± SEM ( n = 6 per group). ∗ p

Techniques Used: Mouse Assay

4) Product Images from "Dual Effects of Metformin on Adipogenic Differentiation of 3T3-L1 Preadipocyte in AMPK-Dependent and Independent Manners"

Article Title: Dual Effects of Metformin on Adipogenic Differentiation of 3T3-L1 Preadipocyte in AMPK-Dependent and Independent Manners

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19061547

Dose response of 1.25–10 mM of metformin on phosphorylation of AMPK, Akt, and MAPKs in 3T3-L1 preadipocytes. Cells were cultured in differentiation medium containing 0, 1.25, 2.5, 5, or 10 mM of metformin for 15 min to examine AMPK, p38, JNK, and Akt activation and for 30 min to examine ERK activation. Results were quantified using densitometry analysis and normalized to total MAPKs, Akt, or AMPK accordingly ( n = 3). Data represent the means ± SEM from three experiments. * p
Figure Legend Snippet: Dose response of 1.25–10 mM of metformin on phosphorylation of AMPK, Akt, and MAPKs in 3T3-L1 preadipocytes. Cells were cultured in differentiation medium containing 0, 1.25, 2.5, 5, or 10 mM of metformin for 15 min to examine AMPK, p38, JNK, and Akt activation and for 30 min to examine ERK activation. Results were quantified using densitometry analysis and normalized to total MAPKs, Akt, or AMPK accordingly ( n = 3). Data represent the means ± SEM from three experiments. * p

Techniques Used: Cell Culture, Activation Assay

Effects of metformin at various concentrations on 3T3-L1 preadipocytes differentiation. ( A ) Effects of metformin (Met) on adipogenesis in a dose-dependent manner. The 3T3-L1 preadipocytes were treated with a differentiation cocktail and various doses of metformin for 9 days as observed by a microscope, 200×; ( B ) Time course of low or high concentrations of metformin (1.25 or 5 mM) on adipogenesis at day 1 (D1), 3 (D3), 5 (D5), 7 (D7), and 9 (D9) as observed by a microscope, 200×. Control cells were treated with differentiation medium I (DMI) only. Rosiglitazone (Rosi, 2.5 µM) was used as positive control.
Figure Legend Snippet: Effects of metformin at various concentrations on 3T3-L1 preadipocytes differentiation. ( A ) Effects of metformin (Met) on adipogenesis in a dose-dependent manner. The 3T3-L1 preadipocytes were treated with a differentiation cocktail and various doses of metformin for 9 days as observed by a microscope, 200×; ( B ) Time course of low or high concentrations of metformin (1.25 or 5 mM) on adipogenesis at day 1 (D1), 3 (D3), 5 (D5), 7 (D7), and 9 (D9) as observed by a microscope, 200×. Control cells were treated with differentiation medium I (DMI) only. Rosiglitazone (Rosi, 2.5 µM) was used as positive control.

Techniques Used: Microscopy, Positive Control

Effects of 1.25 or 5 mM of metformin on expression of adipogenic and lipogenic related genes. ( A ) Time course of gene expression upon treatment for marker genes during early stage of adipocyte differentiation ( n = 6); ( B ) Time course of gene expression upon treatment for marker genes during late stage of adipocyte differentiation ( n = 6). Post-confluent 3T3-L1 cells were treated with DMI mixture alone (Con) or DMI with metformin (Met: 1.25 or 5mM) for different times (0, 9, 12, 18, 24 h, 3, 5, 7, 9 day) and gene expression was determined by qRT-PCR; ( C ) Effects of metformin on expression of UCP-1 and aP2 during the differentiation of 3T3-L1 cells ( n = 6, duplicate for each time and repeated 3 times); Data represents the means ± SEM from three independent experiments. Two-way ANOVA with post hoc Bonferroni’s correction for multiple comparison was used to determine statistical significance. “*” indicates treatment effects, and “ # ” indicates time effects. * p
Figure Legend Snippet: Effects of 1.25 or 5 mM of metformin on expression of adipogenic and lipogenic related genes. ( A ) Time course of gene expression upon treatment for marker genes during early stage of adipocyte differentiation ( n = 6); ( B ) Time course of gene expression upon treatment for marker genes during late stage of adipocyte differentiation ( n = 6). Post-confluent 3T3-L1 cells were treated with DMI mixture alone (Con) or DMI with metformin (Met: 1.25 or 5mM) for different times (0, 9, 12, 18, 24 h, 3, 5, 7, 9 day) and gene expression was determined by qRT-PCR; ( C ) Effects of metformin on expression of UCP-1 and aP2 during the differentiation of 3T3-L1 cells ( n = 6, duplicate for each time and repeated 3 times); Data represents the means ± SEM from three independent experiments. Two-way ANOVA with post hoc Bonferroni’s correction for multiple comparison was used to determine statistical significance. “*” indicates treatment effects, and “ # ” indicates time effects. * p

Techniques Used: Expressing, Marker, Quantitative RT-PCR

Effects of different concentrations of metformin (1.25–10 mM) on lipid accumulation in 3T3-L1 cells. ( A ) Effects of metformin on adipocyte formation at day 9 as determined by Oil Red O staining, 100×. Microscopic observation ( left ); 6-well plate ( right ); ( B ) Optical density (OD) of quantified Oil Red O staining in 3T3-L1 cells ( n = 3); ( C ) Effects of metformin on intracellular and extracellular TG contents at day 9 as determined by triglyceride GPO-POD (glycerophosphate oxidase-phenol aminophenazone) enzymatic assay. Results are normalized by protein concentration in cell lysates ( n = 3). The data represents the means ± SEM (standard error of mean) from three independent experiments. * p
Figure Legend Snippet: Effects of different concentrations of metformin (1.25–10 mM) on lipid accumulation in 3T3-L1 cells. ( A ) Effects of metformin on adipocyte formation at day 9 as determined by Oil Red O staining, 100×. Microscopic observation ( left ); 6-well plate ( right ); ( B ) Optical density (OD) of quantified Oil Red O staining in 3T3-L1 cells ( n = 3); ( C ) Effects of metformin on intracellular and extracellular TG contents at day 9 as determined by triglyceride GPO-POD (glycerophosphate oxidase-phenol aminophenazone) enzymatic assay. Results are normalized by protein concentration in cell lysates ( n = 3). The data represents the means ± SEM (standard error of mean) from three independent experiments. * p

Techniques Used: Staining, Enzymatic Assay, Protein Concentration

Effects of different doses of metformin (1.25–10 mM) on the expression of adipogenesis markers FASN, PPARγ, and C/EBPα in 3T3-L1 cells. ( A ) Expression levels at day 5 (DAY 5) after differentiation initiation as determined by Western blot analysis ( n = 3); ( B ) Expression levels at day 9 (DAY 9) ( n = 3). Rosiglitazone (Rosi, 2.5 µM) was used as a positive control. Results were quantified using densitometry analysis and normalized to β-Actin. The data represent the means ± SEM from three independent experiments. * p
Figure Legend Snippet: Effects of different doses of metformin (1.25–10 mM) on the expression of adipogenesis markers FASN, PPARγ, and C/EBPα in 3T3-L1 cells. ( A ) Expression levels at day 5 (DAY 5) after differentiation initiation as determined by Western blot analysis ( n = 3); ( B ) Expression levels at day 9 (DAY 9) ( n = 3). Rosiglitazone (Rosi, 2.5 µM) was used as a positive control. Results were quantified using densitometry analysis and normalized to β-Actin. The data represent the means ± SEM from three independent experiments. * p

Techniques Used: Expressing, Western Blot, Positive Control

Effects of compound C on metformin-induced adipogenesis inhibition in 3T3-L1 cells. ( A ) Effects of compound C at 10 or 20 μM on AMPK phosphorylation in 3T3-L1 cells treated with or without 5 mM of metformin ( n = 3); ( B ) Compound C at 10 μM inhibited adipogenesis, but rescued metformin-induced inhibition of adipogenesis. After pretreated with or without 10 μM compound C for 1 h, the 3T3-L1 preadipocytes were treated with DMI and metformin for the first two days, and the differentiation was continued for another 3 (Day 5) or 7 (Day 9) days, adipogenic differentiation was then determined by microscopic observation (200×) or Oil Red O staining (100×); ( C ) OD of quantified Oil Red O staining in 3T3-L1 cells ( n = 3); ( D ) Effects of compound C on 5 mM of metformin-induced inhibition of C/EBPα, FASN, and PPARγ expression at day 9 ( n = 3). Cells were treated in the same way as above and the protein expressions were analyzed by Western blot. Blots were quantified using densitometry analysis and results were expressed relatively after normalization to β-Actin. Data represent the means ± SEM from three experiments. * p
Figure Legend Snippet: Effects of compound C on metformin-induced adipogenesis inhibition in 3T3-L1 cells. ( A ) Effects of compound C at 10 or 20 μM on AMPK phosphorylation in 3T3-L1 cells treated with or without 5 mM of metformin ( n = 3); ( B ) Compound C at 10 μM inhibited adipogenesis, but rescued metformin-induced inhibition of adipogenesis. After pretreated with or without 10 μM compound C for 1 h, the 3T3-L1 preadipocytes were treated with DMI and metformin for the first two days, and the differentiation was continued for another 3 (Day 5) or 7 (Day 9) days, adipogenic differentiation was then determined by microscopic observation (200×) or Oil Red O staining (100×); ( C ) OD of quantified Oil Red O staining in 3T3-L1 cells ( n = 3); ( D ) Effects of compound C on 5 mM of metformin-induced inhibition of C/EBPα, FASN, and PPARγ expression at day 9 ( n = 3). Cells were treated in the same way as above and the protein expressions were analyzed by Western blot. Blots were quantified using densitometry analysis and results were expressed relatively after normalization to β-Actin. Data represent the means ± SEM from three experiments. * p

Techniques Used: Inhibition, Staining, Expressing, Western Blot

Time course of 5 mM of metformin on phosphorylation of MAPKs, Akt and AMPK in 3T3-L1 cells. Cells were cultured in DMI containing 5 mM of metformin for indicated time points; cell lysates were then collected and the expressions were determined by Western blot. Results were quantified using densitometry and normalized to total MAPKs, Akt, or AMPK accordingly ( n = 3). “0” represents untreated cells (without DMI). Data represent the means ± SEM from three experiments. * p
Figure Legend Snippet: Time course of 5 mM of metformin on phosphorylation of MAPKs, Akt and AMPK in 3T3-L1 cells. Cells were cultured in DMI containing 5 mM of metformin for indicated time points; cell lysates were then collected and the expressions were determined by Western blot. Results were quantified using densitometry and normalized to total MAPKs, Akt, or AMPK accordingly ( n = 3). “0” represents untreated cells (without DMI). Data represent the means ± SEM from three experiments. * p

Techniques Used: Cell Culture, Western Blot

5) Product Images from "Xanthene Derivatives Increase Glucose Utilization through Activation of LKB1-Dependent AMP-Activated Protein Kinase"

Article Title: Xanthene Derivatives Increase Glucose Utilization through Activation of LKB1-Dependent AMP-Activated Protein Kinase

Journal: PLoS ONE

doi: 10.1371/journal.pone.0108771

Xn and Xc activate AMPK in L6 myotubes. The xanthene derivatives Xn (a), Xc (b) and metformin (c) increased phosphorylation of AMPK a 2 thr-172 and ACC ser-79 in a dose-dependent manner. The indicated concentration was administered for 5 min for Xn and Xc, and 2 h for metformin. (d) Concentration–effect curves of three AMPK activators. Each curve is based on quantification of individual experiments allowing comparison of the effective concentration between metformin and xanthene derivatives. The time-dependent phosphorylation trend of AMPK a 2 thr-172 following treatment with the xanthene derivatives Xn (e) and Xc (f). A 5 µM concentration of xanthene derivatives was administered to L6 myotubes at the indicated time point. The numbers indicate min following administration. (g) The graph shows the time-dependent trend in AMPK-phosphorylation induced by Xn and Xc. Western blot data represent one of three independent experiments. Values shown in graphs are means ± SE from three independent experiments.
Figure Legend Snippet: Xn and Xc activate AMPK in L6 myotubes. The xanthene derivatives Xn (a), Xc (b) and metformin (c) increased phosphorylation of AMPK a 2 thr-172 and ACC ser-79 in a dose-dependent manner. The indicated concentration was administered for 5 min for Xn and Xc, and 2 h for metformin. (d) Concentration–effect curves of three AMPK activators. Each curve is based on quantification of individual experiments allowing comparison of the effective concentration between metformin and xanthene derivatives. The time-dependent phosphorylation trend of AMPK a 2 thr-172 following treatment with the xanthene derivatives Xn (e) and Xc (f). A 5 µM concentration of xanthene derivatives was administered to L6 myotubes at the indicated time point. The numbers indicate min following administration. (g) The graph shows the time-dependent trend in AMPK-phosphorylation induced by Xn and Xc. Western blot data represent one of three independent experiments. Values shown in graphs are means ± SE from three independent experiments.

Techniques Used: Concentration Assay, Western Blot

Xn and Xc increase AMPK activity and glucose utilization in high-fat diet-induced diabetic mice. (a) Phosphorylation of AMPK and ACC in the skeletal muscle of high-fat diet-induced diabetic mice model after a single intravenous injection of the indicated concentration of agents. Densitometric analysis of phosphorylation of (b) ACC and (c) AMPK in the skeletal muscle of 4 different individual high-fat diet-induced diabetic mice. Blood glucose levels were measured after intraperitoneal glucose injection (1 g/kg) following a single intravenous administration of (d) Xn and (e) Xc with metformin, at the indicated concentrations to high-fat diet-induced diabetic mice. The graph on the right shows the area under the curve (AUC). (f) Plasma insulin level was measured by orbital eye bleeding after 1 week administration of indicated agents. Results are the mean ± SE of six mice per group (n = 6). One-way analyses of variance and Tukey's multiple comparisons tests were performed to determine the significance of the results of the glucose tolerance tests. *, P
Figure Legend Snippet: Xn and Xc increase AMPK activity and glucose utilization in high-fat diet-induced diabetic mice. (a) Phosphorylation of AMPK and ACC in the skeletal muscle of high-fat diet-induced diabetic mice model after a single intravenous injection of the indicated concentration of agents. Densitometric analysis of phosphorylation of (b) ACC and (c) AMPK in the skeletal muscle of 4 different individual high-fat diet-induced diabetic mice. Blood glucose levels were measured after intraperitoneal glucose injection (1 g/kg) following a single intravenous administration of (d) Xn and (e) Xc with metformin, at the indicated concentrations to high-fat diet-induced diabetic mice. The graph on the right shows the area under the curve (AUC). (f) Plasma insulin level was measured by orbital eye bleeding after 1 week administration of indicated agents. Results are the mean ± SE of six mice per group (n = 6). One-way analyses of variance and Tukey's multiple comparisons tests were performed to determine the significance of the results of the glucose tolerance tests. *, P

Techniques Used: Activity Assay, Mouse Assay, Injection, Concentration Assay

Xn and Xc increase glucose uptake by stimulating GLUT4 translocation. (a) L6 myotubes were incubated in glucose-free Krebs-Henseleit buffer before measurement of glucose levels for 1 h and then incubated with the indicated agents for 1 h. 2-Deoxy [ 14 C] glucose uptake was measured as described in the Materials and Methods . (b, c) To determine GLUT4 translocation to plasma membrane, Xn, Xc, and metformin was administered to L6 myotubes for 1 h at the indicated concentrations, respectively. The o -phenylenediamine assay (b) and immunocytochemistry (c) was performed as described in the Materials and Methods . Values show the mean ± SE. of three independent experiments performed in triplicate. *, P
Figure Legend Snippet: Xn and Xc increase glucose uptake by stimulating GLUT4 translocation. (a) L6 myotubes were incubated in glucose-free Krebs-Henseleit buffer before measurement of glucose levels for 1 h and then incubated with the indicated agents for 1 h. 2-Deoxy [ 14 C] glucose uptake was measured as described in the Materials and Methods . (b, c) To determine GLUT4 translocation to plasma membrane, Xn, Xc, and metformin was administered to L6 myotubes for 1 h at the indicated concentrations, respectively. The o -phenylenediamine assay (b) and immunocytochemistry (c) was performed as described in the Materials and Methods . Values show the mean ± SE. of three independent experiments performed in triplicate. *, P

Techniques Used: Translocation Assay, Incubation, Immunocytochemistry

6) Product Images from "Mechanistic in vitro studies confirm that inhibition of the renal apical efflux transporter multidrug and toxin extrusion ( MATE) 1, and not altered absorption, underlies the increased metformin exposure observed in clinical interactions with cimetidine, trimethoprim or pyrimethamine. Mechanistic in vitro studies confirm that inhibition of the renal apical efflux transporter multidrug and toxin extrusion (MATE) 1, and not altered absorption, underlies the increased metformin exposure observed in clinical interactions with cimetidine, trimethoprim or pyrimethamine"

Article Title: Mechanistic in vitro studies confirm that inhibition of the renal apical efflux transporter multidrug and toxin extrusion ( MATE) 1, and not altered absorption, underlies the increased metformin exposure observed in clinical interactions with cimetidine, trimethoprim or pyrimethamine. Mechanistic in vitro studies confirm that inhibition of the renal apical efflux transporter multidrug and toxin extrusion (MATE) 1, and not altered absorption, underlies the increased metformin exposure observed in clinical interactions with cimetidine, trimethoprim or pyrimethamine

Journal: Pharmacology Research & Perspectives

doi: 10.1002/prp2.357

Figure reproduced from Eyal et al. ( 2010 ) to illustrate how a documented decrease in metformin (500 mg) renal clearance in pregnancy changes (lowers) the initial rising phase of the AUC profile of a drug (metformin) that exhibits “flip‐flop” pharmacokinetics.
Figure Legend Snippet: Figure reproduced from Eyal et al. ( 2010 ) to illustrate how a documented decrease in metformin (500 mg) renal clearance in pregnancy changes (lowers) the initial rising phase of the AUC profile of a drug (metformin) that exhibits “flip‐flop” pharmacokinetics.

Techniques Used:

Time linearity (A) and Michaelis–Menten kinetic analysis (B) of metformin transport mediated by OCT 1, OCT 2, MATE 1, and MATE 2‐K. Data are expressed as mean (±SD) of triplicate wells per condition.
Figure Legend Snippet: Time linearity (A) and Michaelis–Menten kinetic analysis (B) of metformin transport mediated by OCT 1, OCT 2, MATE 1, and MATE 2‐K. Data are expressed as mean (±SD) of triplicate wells per condition.

Techniques Used:

Mean concentration‐dependent inhibition of OCT 1‐, OCT 2‐, and MATE 1‐mediated transport of [ 14 C]‐metformin (100 μ mol/L) by cimetidine, trimethoprim, and pyrimethamine. Data are expressed as mean (±SD) from a minimum of triplicate wells over three experimental occasions per inhibitor.
Figure Legend Snippet: Mean concentration‐dependent inhibition of OCT 1‐, OCT 2‐, and MATE 1‐mediated transport of [ 14 C]‐metformin (100 μ mol/L) by cimetidine, trimethoprim, and pyrimethamine. Data are expressed as mean (±SD) from a minimum of triplicate wells over three experimental occasions per inhibitor.

Techniques Used: Concentration Assay, Inhibition

Mean bidirectional apparent permeability of a range of concentrations of metformin (10‐10,000 μ mol/L), and of a single concentration of metformin (10,000 μ mol/L) in the absence and presence of cimetidine (1000 μ mol/L), trimethoprim (500 μ mol/L) or pyrimethamine (200 μ mol/L), across polarized Caco‐2 cell monolayers at pH 6.5/7.4. Incubations with inhibitors were conducted both in buffer alone or buffer containing Fa SSIF and 1 % (w/v) human serum albumin. Data are expressed as mean (±SD) of n =3–8 wells per condition.
Figure Legend Snippet: Mean bidirectional apparent permeability of a range of concentrations of metformin (10‐10,000 μ mol/L), and of a single concentration of metformin (10,000 μ mol/L) in the absence and presence of cimetidine (1000 μ mol/L), trimethoprim (500 μ mol/L) or pyrimethamine (200 μ mol/L), across polarized Caco‐2 cell monolayers at pH 6.5/7.4. Incubations with inhibitors were conducted both in buffer alone or buffer containing Fa SSIF and 1 % (w/v) human serum albumin. Data are expressed as mean (±SD) of n =3–8 wells per condition.

Techniques Used: Permeability, Concentration Assay

7) Product Images from "Constitutive Androstane Receptor-Mediated Inhibition of Metformin on Phase II Metabolic Enzyme SULT2A1"

Article Title: Constitutive Androstane Receptor-Mediated Inhibition of Metformin on Phase II Metabolic Enzyme SULT2A1

Journal: International Journal of Endocrinology

doi: 10.1155/2021/8867218

The effects of metformin with or without CITCO on CAR protein expression. HepaRG cells were treated with metformin (0.1, 0.5, 1 mM) (a) and CITCO (1 μ M) (b) either individually or in combination for 48 h. Cells treated only with DMSO were considered as the control group. Total cell proteins and the nuclear proteins were harvested, respectively. Protein levels were determined by western blotting and were quantified with Image J. Quantitation of the T-CAR protein bands was normalized to β -actin; quantitation of the N-CAR protein bands was normalized to lamin-B1. The data shown are the mean ± SEM ( n = 3) ( ∗ P
Figure Legend Snippet: The effects of metformin with or without CITCO on CAR protein expression. HepaRG cells were treated with metformin (0.1, 0.5, 1 mM) (a) and CITCO (1 μ M) (b) either individually or in combination for 48 h. Cells treated only with DMSO were considered as the control group. Total cell proteins and the nuclear proteins were harvested, respectively. Protein levels were determined by western blotting and were quantified with Image J. Quantitation of the T-CAR protein bands was normalized to β -actin; quantitation of the N-CAR protein bands was normalized to lamin-B1. The data shown are the mean ± SEM ( n = 3) ( ∗ P

Techniques Used: Expressing, Western Blot, Quantitation Assay

The effects of metformin on the levels of RXR α , HNF4 α , and PGC-1 α in HepaRG cells. HepaRG cells were treated with metformin (1 mM) and CITCO (1 μ M), either individually or in combination for 48 h. Cells treated only with DMSO were considered as the control group. Whole-cell extracts were harvested, and the expressions of RXR α , HNF4 α , PGC-1 α, and the internal control β -actin were analyzed using western blotting. Quantitation of the three protein bands was normalized to β -actin, respectively. A representative blot was shown and quantified by Image J software. The data shown are the mean ± SEM ( n = 3).
Figure Legend Snippet: The effects of metformin on the levels of RXR α , HNF4 α , and PGC-1 α in HepaRG cells. HepaRG cells were treated with metformin (1 mM) and CITCO (1 μ M), either individually or in combination for 48 h. Cells treated only with DMSO were considered as the control group. Whole-cell extracts were harvested, and the expressions of RXR α , HNF4 α , PGC-1 α, and the internal control β -actin were analyzed using western blotting. Quantitation of the three protein bands was normalized to β -actin, respectively. A representative blot was shown and quantified by Image J software. The data shown are the mean ± SEM ( n = 3).

Techniques Used: Pyrolysis Gas Chromatography, Western Blot, Quantitation Assay, Software

Regulation of CAR and SULT2A1 by metformin is dependent on the activation of AMPK pathways. HepaRG cells were preincubated with compound C (CC; 20 μ M) for 90 min before the treatment with metformin (1 mM), CITCO (1 μ M), or their combinations. Cells treated only with DMSO were considered as the control group. Cells were then lysed and analyzed for western blotting against SULT2A1, T-CAR, RXR α , PGC-1 α , HNF4 α , β -actin (a), N-CAR, lamin-B1 (b), AMPK, and p-AMPK (c, d). A representative blot was shown and quantified by Image J software. The data shown are the mean ± SEM ( n = 3) ( ∗ P
Figure Legend Snippet: Regulation of CAR and SULT2A1 by metformin is dependent on the activation of AMPK pathways. HepaRG cells were preincubated with compound C (CC; 20 μ M) for 90 min before the treatment with metformin (1 mM), CITCO (1 μ M), or their combinations. Cells treated only with DMSO were considered as the control group. Cells were then lysed and analyzed for western blotting against SULT2A1, T-CAR, RXR α , PGC-1 α , HNF4 α , β -actin (a), N-CAR, lamin-B1 (b), AMPK, and p-AMPK (c, d). A representative blot was shown and quantified by Image J software. The data shown are the mean ± SEM ( n = 3) ( ∗ P

Techniques Used: Activation Assay, Western Blot, Pyrolysis Gas Chromatography, Software

Cell viability of HepaRG cells following exposure to metformin alone and in combination with RIF or CITCO. (a) HepaRG cells were exposed to vehicle control (a similar volume of DMSO), metformin (MET; 0.5, 1 mM), RIF (20 μ M), or metformin in combination with RIF for 24, 48, and 72 h. (b) HepaRG cells were exposed to vehicle control (a similar volume of DMSO), metformin (0.5, 1 mM), CITCO (1 μ M), or metformin in combination with CITCO for 24, 48, and 72 h. Cell viability was monitored by CCK8 assay. The data shown are the mean ± SEM ( n = 3). ( ∗ P
Figure Legend Snippet: Cell viability of HepaRG cells following exposure to metformin alone and in combination with RIF or CITCO. (a) HepaRG cells were exposed to vehicle control (a similar volume of DMSO), metformin (MET; 0.5, 1 mM), RIF (20 μ M), or metformin in combination with RIF for 24, 48, and 72 h. (b) HepaRG cells were exposed to vehicle control (a similar volume of DMSO), metformin (0.5, 1 mM), CITCO (1 μ M), or metformin in combination with CITCO for 24, 48, and 72 h. Cell viability was monitored by CCK8 assay. The data shown are the mean ± SEM ( n = 3). ( ∗ P

Techniques Used: CCK-8 Assay

Metformin represses CITCO-induced SULT2A1 expression. (a–c) HepaRG cells were treated with RIF (20 μ M), CITCO (1 μ M), metformin (0.1, 0.5, 1 mM), or their combination for 24 h. Cells treated only with DMSO were considered as the control group. Total mRNA was collected and the expressions of SULT2A1 and β -actin were analyzed using real-time PCR. Values were normalized to the expression of β -actin. (d–f) HepaRG cells were treated with RIF (20 μ M), CITCO (1 μ M), metformin (0.1, 0.5, 1 mM), or their combination for 48 h. Whole-cell extracts were harvested, and the expression of SULT2A1 and the internal control β -actin were analyzed using western blotting. Quantitation of the SULT2A1 protein bands was normalized to β -actin. A represe ntative blot was shown and quantified by Image J software. The data shown are the mean ± SEM ( n = 3) ( ∗∗ P
Figure Legend Snippet: Metformin represses CITCO-induced SULT2A1 expression. (a–c) HepaRG cells were treated with RIF (20 μ M), CITCO (1 μ M), metformin (0.1, 0.5, 1 mM), or their combination for 24 h. Cells treated only with DMSO were considered as the control group. Total mRNA was collected and the expressions of SULT2A1 and β -actin were analyzed using real-time PCR. Values were normalized to the expression of β -actin. (d–f) HepaRG cells were treated with RIF (20 μ M), CITCO (1 μ M), metformin (0.1, 0.5, 1 mM), or their combination for 48 h. Whole-cell extracts were harvested, and the expression of SULT2A1 and the internal control β -actin were analyzed using western blotting. Quantitation of the SULT2A1 protein bands was normalized to β -actin. A represe ntative blot was shown and quantified by Image J software. The data shown are the mean ± SEM ( n = 3) ( ∗∗ P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Quantitation Assay, Software

8) Product Images from "Differential effects of AMPK agonists on cell growth and metabolism"

Article Title: Differential effects of AMPK agonists on cell growth and metabolism

Journal: Oncogene

doi: 10.1038/onc.2014.301

AMPK agonists inhibit anchorage-independent growth of HCT116 colon cancer cells in an AMPK-independent manner A) HCT116 cells were cultured under anchorage–independent conditions in the presence of metformin (5mM), phenformin (0.625mM), AICAR (1.25mM), 2DG (12.5mM), salicylate (2.5mM) or A-769662 (25μM) as indicated. After 5 days images were taken. B) Immunoblot for AMPKα (Thr172 and total), ACC (Ser79 and total), raptor (Ser792 and total) and actin on lysates from HCT116 cells expressing either control or AMPKα1/2 shRNA. Cells were incubated with metformin (5mM), phenformin (1.25mM), AICAR (1.25mM), 2DG (12.5mM), salicylate (2.5mM) or A-769662 (25μM) for 1h. C) HCT116 cells expressing either control (-) or AMPKα1/α2 shRNA (+) were cultured as in (A), here viable cells were estimated by the fluorescence of the metabolic reduction product of Alamar blue. Each bar represents the mean fluorescence of three replicate wells ± SEM.
Figure Legend Snippet: AMPK agonists inhibit anchorage-independent growth of HCT116 colon cancer cells in an AMPK-independent manner A) HCT116 cells were cultured under anchorage–independent conditions in the presence of metformin (5mM), phenformin (0.625mM), AICAR (1.25mM), 2DG (12.5mM), salicylate (2.5mM) or A-769662 (25μM) as indicated. After 5 days images were taken. B) Immunoblot for AMPKα (Thr172 and total), ACC (Ser79 and total), raptor (Ser792 and total) and actin on lysates from HCT116 cells expressing either control or AMPKα1/2 shRNA. Cells were incubated with metformin (5mM), phenformin (1.25mM), AICAR (1.25mM), 2DG (12.5mM), salicylate (2.5mM) or A-769662 (25μM) for 1h. C) HCT116 cells expressing either control (-) or AMPKα1/α2 shRNA (+) were cultured as in (A), here viable cells were estimated by the fluorescence of the metabolic reduction product of Alamar blue. Each bar represents the mean fluorescence of three replicate wells ± SEM.

Techniques Used: Cell Culture, Expressing, shRNA, Incubation, Fluorescence

AMPK agonists differentially affect mitochondrial respiration Coupled respiration (A) and oxygen consumption rate (OCR) (B) of WT and DKO MEFs treated with metformin, phenformin and salicylate for 24h at the concenrations indicated. OCR (C) and spare respiratory capacity (SRC) (D) of WT and DKO MEFs treated with A-769662 (25μM) for 6h and 24h respectively. E) SRC in WT MEFs upon 6h A-769662 treatment, concentration as indicated. In all experiments each bar represents the mean ± SEM for triplicate cultures and statistical significance is represented by: * ***, p
Figure Legend Snippet: AMPK agonists differentially affect mitochondrial respiration Coupled respiration (A) and oxygen consumption rate (OCR) (B) of WT and DKO MEFs treated with metformin, phenformin and salicylate for 24h at the concenrations indicated. OCR (C) and spare respiratory capacity (SRC) (D) of WT and DKO MEFs treated with A-769662 (25μM) for 6h and 24h respectively. E) SRC in WT MEFs upon 6h A-769662 treatment, concentration as indicated. In all experiments each bar represents the mean ± SEM for triplicate cultures and statistical significance is represented by: * ***, p

Techniques Used: Concentration Assay

AMPK agonists stimulate AMPK-dependent signal transduction in MEFs Immunoblot for AMPKα (Thr172 and total), ACC (Ser79 and total), raptor (Ser792 and total), p70S6K (Thr389 and total), 4E-BP1 (different phosphorylation states marked by arrows) and actin on lysates from WT or DKO MEFs. Cells were incubated with metformin (5mM), phenformin (1.25mM), AICAR (1.25mM), 2DG (12.5mM), salicylate (2.5mM) or A-769662 (25μM) for 1h.
Figure Legend Snippet: AMPK agonists stimulate AMPK-dependent signal transduction in MEFs Immunoblot for AMPKα (Thr172 and total), ACC (Ser79 and total), raptor (Ser792 and total), p70S6K (Thr389 and total), 4E-BP1 (different phosphorylation states marked by arrows) and actin on lysates from WT or DKO MEFs. Cells were incubated with metformin (5mM), phenformin (1.25mM), AICAR (1.25mM), 2DG (12.5mM), salicylate (2.5mM) or A-769662 (25μM) for 1h.

Techniques Used: Transduction, Incubation

The majority of AMPK agonists inhibit proliferation in an AMPK-independent manner A) HEK293 cells were grown for 48h in the presence of metformin, phenformin, AICAR, 2DG, salicylate or A-769662 at the concentrations indicated. Adherent cell growth was determined by crystal violet staining. Data are expressed as mean ± SEM for triplicate cultures. B) Growth of WT or DKO MEFs was determined as in (A). C) WT and DKO MEFs were incubated for 24h with phenformin (0.63mM), AICAR (0.63mM), 2DG (6.25mM) or salicylate (2.5mM) before the percentage of cells in G1, S and G2/M phase were measured by propidium iodide staining. D) Growth of WT and DKO MEFs in the presence of A-769662 was determined as in (A). E) Cell cycle profiles of WT and DKO MEFs following treatment with A-769662 (25μM) was determined as in (C).
Figure Legend Snippet: The majority of AMPK agonists inhibit proliferation in an AMPK-independent manner A) HEK293 cells were grown for 48h in the presence of metformin, phenformin, AICAR, 2DG, salicylate or A-769662 at the concentrations indicated. Adherent cell growth was determined by crystal violet staining. Data are expressed as mean ± SEM for triplicate cultures. B) Growth of WT or DKO MEFs was determined as in (A). C) WT and DKO MEFs were incubated for 24h with phenformin (0.63mM), AICAR (0.63mM), 2DG (6.25mM) or salicylate (2.5mM) before the percentage of cells in G1, S and G2/M phase were measured by propidium iodide staining. D) Growth of WT and DKO MEFs in the presence of A-769662 was determined as in (A). E) Cell cycle profiles of WT and DKO MEFs following treatment with A-769662 (25μM) was determined as in (C).

Techniques Used: Staining, Incubation

The majority of AMPK agonists induce apoptosis and cell death in cells lacking AMPK expression A) HEK293 cells were grown for 48h in the presence of metformin, phenformin, AICAR, 2DG, salicylate or A-769662 at the concentrations indicated and the extent of caspase 3 cleavage was determined. Each bar represents the mean ± SEM for triplicate cultures. B) Caspase 3 cleavage was determined for WT and DKO MEFs in response to phenformin, AICAR, 2DG and salicylate treatment as in (A). C) Viability of WT and DKO MEFs following incubation with phenformin (1.25mM), AICAR (1.25mM), 2DG (12.5mM) or salicylate (2.5mM). Cell death was measured after 48h incubation via propidium iodide uptake. Each bar represents the mean ± SEM for triplicate cultures. D) Caspase 3 cleavage was determined for WT and DKO MEFs in response to A-769662 treatment as in (A). E) Viability of WT and DKO MEFs following incubation with A-769662 (25μM) and phenformin (1.25mM) was determined as in (C).
Figure Legend Snippet: The majority of AMPK agonists induce apoptosis and cell death in cells lacking AMPK expression A) HEK293 cells were grown for 48h in the presence of metformin, phenformin, AICAR, 2DG, salicylate or A-769662 at the concentrations indicated and the extent of caspase 3 cleavage was determined. Each bar represents the mean ± SEM for triplicate cultures. B) Caspase 3 cleavage was determined for WT and DKO MEFs in response to phenformin, AICAR, 2DG and salicylate treatment as in (A). C) Viability of WT and DKO MEFs following incubation with phenformin (1.25mM), AICAR (1.25mM), 2DG (12.5mM) or salicylate (2.5mM). Cell death was measured after 48h incubation via propidium iodide uptake. Each bar represents the mean ± SEM for triplicate cultures. D) Caspase 3 cleavage was determined for WT and DKO MEFs in response to A-769662 treatment as in (A). E) Viability of WT and DKO MEFs following incubation with A-769662 (25μM) and phenformin (1.25mM) was determined as in (C).

Techniques Used: Expressing, Incubation

AMPK agonists differentially affect glycolysis Glucose consumption (A) and lactate production (B) were determined in HEK293 following treatment with metformin (1.25mM), phenformin (0.31mM), AICAR (0.31mM), 2DG (3.13mM), salicylate (1.25mM) or A-769662 (25μM) for 48h. Glucose consumption (C) and lactate production (D) were determined as in (A) and (B) respectively in WT and DKO MEFs following treatment with metformin (1.25mM) and phenformin (0.31mM) for 48h. E) Basal extracellular acidification rate (ECAR) was determined in WT and DKO MEFs following treatment with metformin (5mM) and phenformin (1.25mM) for 6h. Each bar represents the mean ± SEM for triplicate cultures. Glucose consumption (F), lactate production (G) and ECAR (H) were determined in WT and DKO MEFs following treatment with AICAR (0.31mM in F and G, 1.25mM in H) and 2DG (3.13mM in F and G, 12.5mM in H) as in (C), (D) and (E). In all experiments each bar represents the mean ± SEM for triplicate cultures and statistical significance is represented by: *, p
Figure Legend Snippet: AMPK agonists differentially affect glycolysis Glucose consumption (A) and lactate production (B) were determined in HEK293 following treatment with metformin (1.25mM), phenformin (0.31mM), AICAR (0.31mM), 2DG (3.13mM), salicylate (1.25mM) or A-769662 (25μM) for 48h. Glucose consumption (C) and lactate production (D) were determined as in (A) and (B) respectively in WT and DKO MEFs following treatment with metformin (1.25mM) and phenformin (0.31mM) for 48h. E) Basal extracellular acidification rate (ECAR) was determined in WT and DKO MEFs following treatment with metformin (5mM) and phenformin (1.25mM) for 6h. Each bar represents the mean ± SEM for triplicate cultures. Glucose consumption (F), lactate production (G) and ECAR (H) were determined in WT and DKO MEFs following treatment with AICAR (0.31mM in F and G, 1.25mM in H) and 2DG (3.13mM in F and G, 12.5mM in H) as in (C), (D) and (E). In all experiments each bar represents the mean ± SEM for triplicate cultures and statistical significance is represented by: *, p

Techniques Used:

Six known AMPK agonists activate AMPK in Hek293 and MEF cells A) Schematic summarising the mechanism of action of the 6 AMPK agonists used in this study. AICAR is metabolized in cells to ZMP, which binds the γ subunit in place of AMP, leading to AMPK activation. Metformin and phenformin inhibit complex I of the electron transport chain leading to an elevation in the AMP/ATP ratio and subsequent AMPK activation. 2DG also elevates the AMP/ATP ratio by inhibiting glycolysis. Both salicylate and A-769662 activate AMPK directly, binding to the β subunit. B) Immunoblot for ACC (Ser79 and total), AMPKα (Thr172 and total) and actin on lysates from HEK293 cells. HEK293 cells were pretreated with STO-609 (25μM) for 30min before incubation with metformin (5mM), phenformin (1.25mM), AICAR (1.25mM), 2DG (12.5mM), salicylate (2.5mM) or A-769662 (25μM) for 1h. C) Immunoblot for ACC (Ser79 and total), AMPKα (Thr172 and total) and actin on lysates from wild-type (WT) or AMPKα-deficient (DKO) MEFs. Cells were incubated with metformin, phenformin, AICAR, 2DG, salicylate or A-769662 for 1h at the indicated concentrations.
Figure Legend Snippet: Six known AMPK agonists activate AMPK in Hek293 and MEF cells A) Schematic summarising the mechanism of action of the 6 AMPK agonists used in this study. AICAR is metabolized in cells to ZMP, which binds the γ subunit in place of AMP, leading to AMPK activation. Metformin and phenformin inhibit complex I of the electron transport chain leading to an elevation in the AMP/ATP ratio and subsequent AMPK activation. 2DG also elevates the AMP/ATP ratio by inhibiting glycolysis. Both salicylate and A-769662 activate AMPK directly, binding to the β subunit. B) Immunoblot for ACC (Ser79 and total), AMPKα (Thr172 and total) and actin on lysates from HEK293 cells. HEK293 cells were pretreated with STO-609 (25μM) for 30min before incubation with metformin (5mM), phenformin (1.25mM), AICAR (1.25mM), 2DG (12.5mM), salicylate (2.5mM) or A-769662 (25μM) for 1h. C) Immunoblot for ACC (Ser79 and total), AMPKα (Thr172 and total) and actin on lysates from wild-type (WT) or AMPKα-deficient (DKO) MEFs. Cells were incubated with metformin, phenformin, AICAR, 2DG, salicylate or A-769662 for 1h at the indicated concentrations.

Techniques Used: Activation Assay, Binding Assay, Incubation

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    Millipore materials metformin
    Degradation of pSTAT3 ser727 in grade 1 endometrial cancer cells treated with meformin. After Ishikawa cells were treated with control, 10 mM, or 20 mM <t>metformin</t> in high glucose media for 48h, samples were subjected to ubiquitin assay. The ubiquitinated proteins were subjected to immunoblot for pSTAT3 Ser727 and blotted with ubiquitin antibody. A ubiquitination smear of pSTAT3 Ser727 is seen in the metformin treated ishikawa cells under proteasomal inhibition using MG-132.
    Materials Metformin, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore metformin
    Schematic: recruitment of PARP-1-dependent cell death in PC cells by <t>metformin</t> and ARAT (Abi, Enz) combinations. ( A ) Cellular stress induced by metformin/ARATs leads to PARP-1 activation, increase PAR production and cAIF nuclear accumulation. ( B ) Metformin or metformin/ARATs induce cathepsin leak from lysosomes resulting in cleavage of PARP-1, cPARP-1 then binds DNA to prevent uncleaved PARP-1 from accessing DNA for repair.
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    Degradation of pSTAT3 ser727 in grade 1 endometrial cancer cells treated with meformin. After Ishikawa cells were treated with control, 10 mM, or 20 mM metformin in high glucose media for 48h, samples were subjected to ubiquitin assay. The ubiquitinated proteins were subjected to immunoblot for pSTAT3 Ser727 and blotted with ubiquitin antibody. A ubiquitination smear of pSTAT3 Ser727 is seen in the metformin treated ishikawa cells under proteasomal inhibition using MG-132.

    Journal: PLoS ONE

    Article Title: High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer

    doi: 10.1371/journal.pone.0170318

    Figure Lengend Snippet: Degradation of pSTAT3 ser727 in grade 1 endometrial cancer cells treated with meformin. After Ishikawa cells were treated with control, 10 mM, or 20 mM metformin in high glucose media for 48h, samples were subjected to ubiquitin assay. The ubiquitinated proteins were subjected to immunoblot for pSTAT3 Ser727 and blotted with ubiquitin antibody. A ubiquitination smear of pSTAT3 Ser727 is seen in the metformin treated ishikawa cells under proteasomal inhibition using MG-132.

    Article Snippet: Materials Metformin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Ubiquitin Assay, Inhibition

    Metformin inhibits grade 1 endometrial cancer cell proliferation, survival, migration, and induces apoptosis. A . Sulforhodamine B (SRB) assay measured Ishikawa cell proliferation with increasing concentrations of metformin after 24h or 48h, in high-glucose media. B . Proportion of Ishikawa cells surviving (compared to control) after treatment with 10 mM or 20 mM of metformin. C . Cell migration assay, with Ishikawa cells subjected to control, 10 mM, or 20 mM metformin for 24h. Control (0h) is also shown. D . Quantification of % wound closure; the 10 mM and 20 mM treatment groups were each significantly different than control (p

    Journal: PLoS ONE

    Article Title: High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer

    doi: 10.1371/journal.pone.0170318

    Figure Lengend Snippet: Metformin inhibits grade 1 endometrial cancer cell proliferation, survival, migration, and induces apoptosis. A . Sulforhodamine B (SRB) assay measured Ishikawa cell proliferation with increasing concentrations of metformin after 24h or 48h, in high-glucose media. B . Proportion of Ishikawa cells surviving (compared to control) after treatment with 10 mM or 20 mM of metformin. C . Cell migration assay, with Ishikawa cells subjected to control, 10 mM, or 20 mM metformin for 24h. Control (0h) is also shown. D . Quantification of % wound closure; the 10 mM and 20 mM treatment groups were each significantly different than control (p

    Article Snippet: Materials Metformin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Migration, Sulforhodamine B Assay, Cell Migration Assay

    Xenograft endometrial tumor weight and expression of STAT3 in mice treated with metformin. A xenograft study was done in which nude mice were injected with 1 x 10 6 Ishikawa endometrial cancer cells subcutaneously in the right flank. After tumors were at least 3–5 mm in diameter, treatment with control, metformin 100 mg/kg, or metformin 200 mg/kg was started. Mice were sacrificed after 4 weeks of treatment. A . Tumor weight (in grams) from the mice with the 3 largest tumors in each group. B . Average body weight (g) of the mice in each group at conclusion of the study. C . Western blot results of STAT3 and associated proteins after treatment with control, 100 mg/kg, or 200 mg/kg of metformin.

    Journal: PLoS ONE

    Article Title: High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer

    doi: 10.1371/journal.pone.0170318

    Figure Lengend Snippet: Xenograft endometrial tumor weight and expression of STAT3 in mice treated with metformin. A xenograft study was done in which nude mice were injected with 1 x 10 6 Ishikawa endometrial cancer cells subcutaneously in the right flank. After tumors were at least 3–5 mm in diameter, treatment with control, metformin 100 mg/kg, or metformin 200 mg/kg was started. Mice were sacrificed after 4 weeks of treatment. A . Tumor weight (in grams) from the mice with the 3 largest tumors in each group. B . Average body weight (g) of the mice in each group at conclusion of the study. C . Western blot results of STAT3 and associated proteins after treatment with control, 100 mg/kg, or 200 mg/kg of metformin.

    Article Snippet: Materials Metformin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Expressing, Mouse Assay, Injection, Western Blot

    Expression of apoptosis and cell proliferation-related proteins in metformin-treated grade 1 endometrial cancer cells overexpressing STAT3. Ishikawa endometrial cancer cells were transfected with a STAT3-overexpressing plasmid. A . Western blot confirming overexpression of pSTAT3 ser727 and total STAT3. B . Western blot of proteins involved in apoptosis or cell proliferation in control or STAT3-overexpressing Ishikawa cells treated with control or 20 mM metformin in high-glucose medium for 48h. (C: control, TR: transfection reagent only, OE: transfected with STAT3-overexpressing plasmid, Ctrl: control, Met: metformin).

    Journal: PLoS ONE

    Article Title: High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer

    doi: 10.1371/journal.pone.0170318

    Figure Lengend Snippet: Expression of apoptosis and cell proliferation-related proteins in metformin-treated grade 1 endometrial cancer cells overexpressing STAT3. Ishikawa endometrial cancer cells were transfected with a STAT3-overexpressing plasmid. A . Western blot confirming overexpression of pSTAT3 ser727 and total STAT3. B . Western blot of proteins involved in apoptosis or cell proliferation in control or STAT3-overexpressing Ishikawa cells treated with control or 20 mM metformin in high-glucose medium for 48h. (C: control, TR: transfection reagent only, OE: transfected with STAT3-overexpressing plasmid, Ctrl: control, Met: metformin).

    Article Snippet: Materials Metformin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Over Expression

    Metformin inhibits STAT3, its regulatory proteins and upregulated apoptosis-related proteins, in grade 1 endometrial cancer cells. A . Western blot of STAT3 and its regulatory proteins in Ishikawa cells after treatment with control, 10 mM, or 20 mM metformin for 48h in high-glucose conditions. B . qPCR of STAT3 and some of its regulatory genes in Ishikawa cells after treatment with control, 10 mM, or 20 mM metformin for 48h. Groups significantly different than control (p

    Journal: PLoS ONE

    Article Title: High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer

    doi: 10.1371/journal.pone.0170318

    Figure Lengend Snippet: Metformin inhibits STAT3, its regulatory proteins and upregulated apoptosis-related proteins, in grade 1 endometrial cancer cells. A . Western blot of STAT3 and its regulatory proteins in Ishikawa cells after treatment with control, 10 mM, or 20 mM metformin for 48h in high-glucose conditions. B . qPCR of STAT3 and some of its regulatory genes in Ishikawa cells after treatment with control, 10 mM, or 20 mM metformin for 48h. Groups significantly different than control (p

    Article Snippet: Materials Metformin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Western Blot, Real-time Polymerase Chain Reaction

    A combined treatment of phenformin and TMZ exerts an enhanced cytotoxic effect on GSCs in vitro and in vivo ( A ) MTT assay determined cytotoxicity of combined treatments of phenformin or metformin with TMZ (100 μM) for 24 hr. Statistical analysis compares TMZ treated (pink bars) vs control (blue bars). * p

    Journal: Oncotarget

    Article Title: Repurposing phenformin for the targeting of glioma stem cells and the treatment of glioblastoma

    doi: 10.18632/oncotarget.10919

    Figure Lengend Snippet: A combined treatment of phenformin and TMZ exerts an enhanced cytotoxic effect on GSCs in vitro and in vivo ( A ) MTT assay determined cytotoxicity of combined treatments of phenformin or metformin with TMZ (100 μM) for 24 hr. Statistical analysis compares TMZ treated (pink bars) vs control (blue bars). * p

    Article Snippet: Materials Metformin and phenformin were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: In Vitro, In Vivo, MTT Assay

    DCA treatment increases the inhibitory effect of phenformin on the stemness and survival of GSCs in vitro and in vivo ( A ) A combined treatment of phenformin or metformin with DCA on the inhibition of GSC self-renewal in 10 days. * represents the statistical analysis based on the DCA concentration, i.e, comparing 5 mM or 10 mM DCA treated groups with control untreated group ; + represents the statistical analysis that compares biguanide treatment vs. control. * or + p

    Journal: Oncotarget

    Article Title: Repurposing phenformin for the targeting of glioma stem cells and the treatment of glioblastoma

    doi: 10.18632/oncotarget.10919

    Figure Lengend Snippet: DCA treatment increases the inhibitory effect of phenformin on the stemness and survival of GSCs in vitro and in vivo ( A ) A combined treatment of phenformin or metformin with DCA on the inhibition of GSC self-renewal in 10 days. * represents the statistical analysis based on the DCA concentration, i.e, comparing 5 mM or 10 mM DCA treated groups with control untreated group ; + represents the statistical analysis that compares biguanide treatment vs. control. * or + p

    Article Snippet: Materials Metformin and phenformin were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: In Vitro, In Vivo, Inhibition, Concentration Assay

    Schematic: recruitment of PARP-1-dependent cell death in PC cells by metformin and ARAT (Abi, Enz) combinations. ( A ) Cellular stress induced by metformin/ARATs leads to PARP-1 activation, increase PAR production and cAIF nuclear accumulation. ( B ) Metformin or metformin/ARATs induce cathepsin leak from lysosomes resulting in cleavage of PARP-1, cPARP-1 then binds DNA to prevent uncleaved PARP-1 from accessing DNA for repair.

    Journal: Cancers

    Article Title: Metformin and Androgen Receptor-Axis-Targeted (ARAT) Agents Induce Two PARP-1-Dependent Cell Death Pathways in Androgen-Sensitive Human Prostate Cancer Cells

    doi: 10.3390/cancers13040633

    Figure Lengend Snippet: Schematic: recruitment of PARP-1-dependent cell death in PC cells by metformin and ARAT (Abi, Enz) combinations. ( A ) Cellular stress induced by metformin/ARATs leads to PARP-1 activation, increase PAR production and cAIF nuclear accumulation. ( B ) Metformin or metformin/ARATs induce cathepsin leak from lysosomes resulting in cleavage of PARP-1, cPARP-1 then binds DNA to prevent uncleaved PARP-1 from accessing DNA for repair.

    Article Snippet: Materials and DrugsAcridine orange, metformin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), chloroquine, and E-64d were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA).

    Techniques: Activation Assay

    Treatment of PC cells with metformin in combination with ARAT agents (Enz, and Abi). ( A , B ) Trypan blue staining. Cells were treated for 5 days with metformin 1 µM or Enz (10 µM) ± metformin or Abi (5 µM) ± metformin, then stained with trypan blue. Both floating and attached cells were collected. ( A ) Left. LNCaP floating cells shown as a percentage of total treated cells (floating + attached) in each plate. Right. Treated LNCaP attached cells are shown as a fraction of attached control (DMSO-treated) cells (set as 1) ( B ) Left. VCaP floating cells shown as a percentage of total treated cells (floating + attached) in each plate. Right. Treated VCaP attached cells are shown as a fraction of attached control (DMSO-treated) cells (set as 1). Results represent the mean ± S.D. from two separate experiments, each done with triplicate determinations per data point. (* p

    Journal: Cancers

    Article Title: Metformin and Androgen Receptor-Axis-Targeted (ARAT) Agents Induce Two PARP-1-Dependent Cell Death Pathways in Androgen-Sensitive Human Prostate Cancer Cells

    doi: 10.3390/cancers13040633

    Figure Lengend Snippet: Treatment of PC cells with metformin in combination with ARAT agents (Enz, and Abi). ( A , B ) Trypan blue staining. Cells were treated for 5 days with metformin 1 µM or Enz (10 µM) ± metformin or Abi (5 µM) ± metformin, then stained with trypan blue. Both floating and attached cells were collected. ( A ) Left. LNCaP floating cells shown as a percentage of total treated cells (floating + attached) in each plate. Right. Treated LNCaP attached cells are shown as a fraction of attached control (DMSO-treated) cells (set as 1) ( B ) Left. VCaP floating cells shown as a percentage of total treated cells (floating + attached) in each plate. Right. Treated VCaP attached cells are shown as a fraction of attached control (DMSO-treated) cells (set as 1). Results represent the mean ± S.D. from two separate experiments, each done with triplicate determinations per data point. (* p

    Article Snippet: Materials and DrugsAcridine orange, metformin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), chloroquine, and E-64d were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA).

    Techniques: Staining

    Lysosome- and cathepsin G-mediated effects in PC cells. ( A , B ) Acridine orange staining of LNCaP cells ( A ) and VCaP cells ( B ) after treatment with metformin (1 mM), Enz (10 µM) ± metformin, or Abi (5 µM) ± metformin for 5 days demonstrate enhanced lysosome permeability with metformin or combination treatments. ( C , D ) Western blot. Effect of lysosome inhibition by chloroquine (100 µM) ( C ) or E-64d ( D ) on metformin (1 mM for 5 days)-mediated PARP-1 cleavage in LNCaP or VCaP cells. ( E , F ) Effect of silencing cathepsin G ( E ) or cathepsin D ( F ) by siRNA on PARP-1 cleavage in LNCaP cells treated with metformin 1 mM ± Enz 10 µM for 5 days. The original western blot figures are available in a separate Supplementary Materials document. ( G ) Trypan blue staining. LNCaP cells were treated for 5 days with inhibitors (chloroquine, E-64d) or drug (metformin, Enz) or both as shown, stained with trypan blue, and floating and attached cells collected. Shown are floating cells as a percentage of the total (floating + attached) treated cells. Results represent the mean ± S.D. from three separate experiments, each done with triplicate determinations per data point. a, (* p

    Journal: Cancers

    Article Title: Metformin and Androgen Receptor-Axis-Targeted (ARAT) Agents Induce Two PARP-1-Dependent Cell Death Pathways in Androgen-Sensitive Human Prostate Cancer Cells

    doi: 10.3390/cancers13040633

    Figure Lengend Snippet: Lysosome- and cathepsin G-mediated effects in PC cells. ( A , B ) Acridine orange staining of LNCaP cells ( A ) and VCaP cells ( B ) after treatment with metformin (1 mM), Enz (10 µM) ± metformin, or Abi (5 µM) ± metformin for 5 days demonstrate enhanced lysosome permeability with metformin or combination treatments. ( C , D ) Western blot. Effect of lysosome inhibition by chloroquine (100 µM) ( C ) or E-64d ( D ) on metformin (1 mM for 5 days)-mediated PARP-1 cleavage in LNCaP or VCaP cells. ( E , F ) Effect of silencing cathepsin G ( E ) or cathepsin D ( F ) by siRNA on PARP-1 cleavage in LNCaP cells treated with metformin 1 mM ± Enz 10 µM for 5 days. The original western blot figures are available in a separate Supplementary Materials document. ( G ) Trypan blue staining. LNCaP cells were treated for 5 days with inhibitors (chloroquine, E-64d) or drug (metformin, Enz) or both as shown, stained with trypan blue, and floating and attached cells collected. Shown are floating cells as a percentage of the total (floating + attached) treated cells. Results represent the mean ± S.D. from three separate experiments, each done with triplicate determinations per data point. a, (* p

    Article Snippet: Materials and DrugsAcridine orange, metformin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), chloroquine, and E-64d were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA).

    Techniques: Staining, Permeability, Western Blot, Inhibition

    Effect of metformin on LNCaP and VCaP cells. ( A ) BrdU assay. Effect of metformin dose on cell proliferation. Shown is BrdU incorporation in 3 days metformin-treated cells as a fraction of control (DMSO-treated) cells. Results represent the mean ± S.D. from three separate experiments, each done with triplicate determinations per data point. (* p

    Journal: Cancers

    Article Title: Metformin and Androgen Receptor-Axis-Targeted (ARAT) Agents Induce Two PARP-1-Dependent Cell Death Pathways in Androgen-Sensitive Human Prostate Cancer Cells

    doi: 10.3390/cancers13040633

    Figure Lengend Snippet: Effect of metformin on LNCaP and VCaP cells. ( A ) BrdU assay. Effect of metformin dose on cell proliferation. Shown is BrdU incorporation in 3 days metformin-treated cells as a fraction of control (DMSO-treated) cells. Results represent the mean ± S.D. from three separate experiments, each done with triplicate determinations per data point. (* p

    Article Snippet: Materials and DrugsAcridine orange, metformin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), chloroquine, and E-64d were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA).

    Techniques: BrdU Staining, BrdU Incorporation Assay

    Metformin inhibits androgen-mediated IGF-IR protein, mRNA up-regulation, and IGF-IR promoter activity. A, Time-course analysis of IGF-IR protein expression. Cells were serum starved for 24 hours and then preincubated with 10mM metformin (Met) for 4 hours before adding 10nM R1881 for the indicated time points. Cells were therefore solubilized and analyzed by Western blotting. ns, nonstatistically significant ( P > .05); *, P

    Journal: Endocrinology

    Article Title: Metformin Inhibits Androgen-Induced IGF-IR Up-Regulation in Prostate Cancer Cells by Disrupting Membrane-Initiated Androgen Signaling

    doi: 10.1210/en.2013-1925

    Figure Lengend Snippet: Metformin inhibits androgen-mediated IGF-IR protein, mRNA up-regulation, and IGF-IR promoter activity. A, Time-course analysis of IGF-IR protein expression. Cells were serum starved for 24 hours and then preincubated with 10mM metformin (Met) for 4 hours before adding 10nM R1881 for the indicated time points. Cells were therefore solubilized and analyzed by Western blotting. ns, nonstatistically significant ( P > .05); *, P

    Article Snippet: The following materials were purchased from the indicated manufacturers: metformin, methyltrienolone (R1881), Src activity inhibitor (4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), and rapamycin from Sigma Chemical Co; fetal calf serum and Opti-MEM from Gibco Laboratories; temsirolimus from DBA; and Metafectene PRO from Biontex Laboratories GmbH.

    Techniques: Activity Assay, Expressing, Western Blot

    Involvement of AMPK and p53/REDD1 pathways in the inhibitory effect of metformin. A, Androgen-stimulated IGF-IR up-regulation after AMPK silencing. Cells were transiently transfected with siRNAs direct to AMPK or scrambled siRNAs. After 24 hours, cells were serum starved for 24 hours and then treated with metformin (Met) (10mM) 4 hours before the incubation or not with R1881 (10nM) for further 24 hours. Then cells were lysed and analyzed by Western blotting. Filters were probed with antibodies specific for IGF-IR and AMPK. Anti-β-tubulin immunoblotting was used to control protein loading. The histogram represents the mean ± SEM (error bars) of densitometric analyses of 3 separate experiments. *, P

    Journal: Endocrinology

    Article Title: Metformin Inhibits Androgen-Induced IGF-IR Up-Regulation in Prostate Cancer Cells by Disrupting Membrane-Initiated Androgen Signaling

    doi: 10.1210/en.2013-1925

    Figure Lengend Snippet: Involvement of AMPK and p53/REDD1 pathways in the inhibitory effect of metformin. A, Androgen-stimulated IGF-IR up-regulation after AMPK silencing. Cells were transiently transfected with siRNAs direct to AMPK or scrambled siRNAs. After 24 hours, cells were serum starved for 24 hours and then treated with metformin (Met) (10mM) 4 hours before the incubation or not with R1881 (10nM) for further 24 hours. Then cells were lysed and analyzed by Western blotting. Filters were probed with antibodies specific for IGF-IR and AMPK. Anti-β-tubulin immunoblotting was used to control protein loading. The histogram represents the mean ± SEM (error bars) of densitometric analyses of 3 separate experiments. *, P

    Article Snippet: The following materials were purchased from the indicated manufacturers: metformin, methyltrienolone (R1881), Src activity inhibitor (4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), and rapamycin from Sigma Chemical Co; fetal calf serum and Opti-MEM from Gibco Laboratories; temsirolimus from DBA; and Metafectene PRO from Biontex Laboratories GmbH.

    Techniques: Transfection, Incubation, Western Blot

    Metformin inhibition of androgen-dependent IGF-IR up-regulation requires CREB cofactor CRTC2. A, CRTC2 localization by immunofluorescence. LNCaP cells were serum starved for 24 hours and then pretreated with metformin (Met) (10mM) for 4 hours followed by R1881 exposure (10nM for 24 h). Cells were then plated on cover slips, fixed, and stained for CRCT2 (red) and filamentous actin (phalloidin, green). Nuclei were visualized with Hoechst (blue). The images presented are representative of most cells examined for that treatment. B, Effect of metformin treatment in presence or absence of R1881 on CRCT2 phosphorylation at Ser171. LNCaP cells were serum starved for 24 hours and then pretreated with metformin (30mM) for 4 hours followed by 30 minutes of R1881 (10nM) exposure. Cells were, therefore, solubilized and analyzed by Western blotting. The top panel shows a representative experiment. The histogram represents the mean ± SEM (error bars) of densitometric analysis of 3 separate experiments. Activity of IGF-IR promoter (C) and CRE (D) after CRCT2 silencing. LNCaP cells were transiently transfected with siRNAs to CRCT2 or scrambled shRNAs and, after 24 hours, were transfected again with plasmids encoding the full-length IGF-IR promoter-luciferase vector (C) or CRE-Luc construct (D). Twelve hours after transfection, cells were serum starved for 10 hours and then pretreated with metformin (10mM) before adding R1881 (10nM) for further 24 hours. Columns, means of 3 separate experiments, normalized for transfection efficiency with a GFP vector. In C and D, ns, not statistically significant ( P > .05); *, P

    Journal: Endocrinology

    Article Title: Metformin Inhibits Androgen-Induced IGF-IR Up-Regulation in Prostate Cancer Cells by Disrupting Membrane-Initiated Androgen Signaling

    doi: 10.1210/en.2013-1925

    Figure Lengend Snippet: Metformin inhibition of androgen-dependent IGF-IR up-regulation requires CREB cofactor CRTC2. A, CRTC2 localization by immunofluorescence. LNCaP cells were serum starved for 24 hours and then pretreated with metformin (Met) (10mM) for 4 hours followed by R1881 exposure (10nM for 24 h). Cells were then plated on cover slips, fixed, and stained for CRCT2 (red) and filamentous actin (phalloidin, green). Nuclei were visualized with Hoechst (blue). The images presented are representative of most cells examined for that treatment. B, Effect of metformin treatment in presence or absence of R1881 on CRCT2 phosphorylation at Ser171. LNCaP cells were serum starved for 24 hours and then pretreated with metformin (30mM) for 4 hours followed by 30 minutes of R1881 (10nM) exposure. Cells were, therefore, solubilized and analyzed by Western blotting. The top panel shows a representative experiment. The histogram represents the mean ± SEM (error bars) of densitometric analysis of 3 separate experiments. Activity of IGF-IR promoter (C) and CRE (D) after CRCT2 silencing. LNCaP cells were transiently transfected with siRNAs to CRCT2 or scrambled shRNAs and, after 24 hours, were transfected again with plasmids encoding the full-length IGF-IR promoter-luciferase vector (C) or CRE-Luc construct (D). Twelve hours after transfection, cells were serum starved for 10 hours and then pretreated with metformin (10mM) before adding R1881 (10nM) for further 24 hours. Columns, means of 3 separate experiments, normalized for transfection efficiency with a GFP vector. In C and D, ns, not statistically significant ( P > .05); *, P

    Article Snippet: The following materials were purchased from the indicated manufacturers: metformin, methyltrienolone (R1881), Src activity inhibitor (4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), and rapamycin from Sigma Chemical Co; fetal calf serum and Opti-MEM from Gibco Laboratories; temsirolimus from DBA; and Metafectene PRO from Biontex Laboratories GmbH.

    Techniques: Inhibition, Immunofluorescence, Staining, Western Blot, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Construct

    Metformin inhibits androgen-induced IGF-IR up-regulation by inhibiting CREB activity and phosphorylation. A, Androgen-induced IGF-IR up-regulation after CREB silencing. LNCaP cells were transiently transfected with shRNAs direct to CREB or scrambled shRNAs. After 24 hours, cells were serum starved for 24 hours and then were treated with metformin (10mM) in association or not with R1881 (10nM) for further 24 hours. Cells were lysed and analyzed by Western blotting. Filters were probed with antibodies specific for IGF-IR and CREB. Immunoblotting anti-β-tubulin was used to control protein loading. The histogram represents the mean ± SEM (error bars) of densitometric analyses of 3 separate experiments. ns, not statistically significant ( P > .05); ***, P

    Journal: Endocrinology

    Article Title: Metformin Inhibits Androgen-Induced IGF-IR Up-Regulation in Prostate Cancer Cells by Disrupting Membrane-Initiated Androgen Signaling

    doi: 10.1210/en.2013-1925

    Figure Lengend Snippet: Metformin inhibits androgen-induced IGF-IR up-regulation by inhibiting CREB activity and phosphorylation. A, Androgen-induced IGF-IR up-regulation after CREB silencing. LNCaP cells were transiently transfected with shRNAs direct to CREB or scrambled shRNAs. After 24 hours, cells were serum starved for 24 hours and then were treated with metformin (10mM) in association or not with R1881 (10nM) for further 24 hours. Cells were lysed and analyzed by Western blotting. Filters were probed with antibodies specific for IGF-IR and CREB. Immunoblotting anti-β-tubulin was used to control protein loading. The histogram represents the mean ± SEM (error bars) of densitometric analyses of 3 separate experiments. ns, not statistically significant ( P > .05); ***, P

    Article Snippet: The following materials were purchased from the indicated manufacturers: metformin, methyltrienolone (R1881), Src activity inhibitor (4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), and rapamycin from Sigma Chemical Co; fetal calf serum and Opti-MEM from Gibco Laboratories; temsirolimus from DBA; and Metafectene PRO from Biontex Laboratories GmbH.

    Techniques: Activity Assay, Transfection, Western Blot

    Cross talk between metformin, androgens, and IGF system and molecular mechanisms of metformin inhibitory effects. In prostate cancer cells, androgens up-regulate IGF-IR by inducing CREB transcriptional activation through membrane-initiated activation of the Src/MAPK/phosphoinositide 3-kinase (PI3K) pathway. The molecular mechanisms by which metformin may disrupt this androgen effect include: 1) inhibition of androgen-induced TORC2 dephosphorylation and nuclear translocation with consequent reduction in CREB/TORC2 complex transcription activity; 2) inhibition of mTOR/p70S6K signaling, partially through the AMPK and REDD1 pathways; and 3) Disruption of AR/c-Src interaction and inhibition of AR phosphorylation. Ras, rat sarcoma; Raf, rapidly accelerated fibrosarcoma; Mek, mitogen-activated protein kinase kinase.

    Journal: Endocrinology

    Article Title: Metformin Inhibits Androgen-Induced IGF-IR Up-Regulation in Prostate Cancer Cells by Disrupting Membrane-Initiated Androgen Signaling

    doi: 10.1210/en.2013-1925

    Figure Lengend Snippet: Cross talk between metformin, androgens, and IGF system and molecular mechanisms of metformin inhibitory effects. In prostate cancer cells, androgens up-regulate IGF-IR by inducing CREB transcriptional activation through membrane-initiated activation of the Src/MAPK/phosphoinositide 3-kinase (PI3K) pathway. The molecular mechanisms by which metformin may disrupt this androgen effect include: 1) inhibition of androgen-induced TORC2 dephosphorylation and nuclear translocation with consequent reduction in CREB/TORC2 complex transcription activity; 2) inhibition of mTOR/p70S6K signaling, partially through the AMPK and REDD1 pathways; and 3) Disruption of AR/c-Src interaction and inhibition of AR phosphorylation. Ras, rat sarcoma; Raf, rapidly accelerated fibrosarcoma; Mek, mitogen-activated protein kinase kinase.

    Article Snippet: The following materials were purchased from the indicated manufacturers: metformin, methyltrienolone (R1881), Src activity inhibitor (4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), and rapamycin from Sigma Chemical Co; fetal calf serum and Opti-MEM from Gibco Laboratories; temsirolimus from DBA; and Metafectene PRO from Biontex Laboratories GmbH.

    Techniques: Activation Assay, Inhibition, De-Phosphorylation Assay, Translocation Assay, Activity Assay

    Metformin inhibits the androgen-mediated enhancement of IGF-I biological effects. A, Cell proliferation. LNCaP cells were grown in 96-well plates, serum starved for 24 hours, and preincubated with or without R1881 (10nM) in the presence or absence of metformin (Met) (10mM). After 24 hours, the medium was then replaced with R1881-free medium containing 1% stripped serum, and cells were incubated with or without 10nM IGF-I for additional 24 hours, in presence or absence of metformin (10mM). Cell viability was evaluated by MTT assay. Values are expressed as percentages of untreated cells and represent the mean ± SEM (errors bars) of 2 separate experiments performed in triplicate. **, P

    Journal: Endocrinology

    Article Title: Metformin Inhibits Androgen-Induced IGF-IR Up-Regulation in Prostate Cancer Cells by Disrupting Membrane-Initiated Androgen Signaling

    doi: 10.1210/en.2013-1925

    Figure Lengend Snippet: Metformin inhibits the androgen-mediated enhancement of IGF-I biological effects. A, Cell proliferation. LNCaP cells were grown in 96-well plates, serum starved for 24 hours, and preincubated with or without R1881 (10nM) in the presence or absence of metformin (Met) (10mM). After 24 hours, the medium was then replaced with R1881-free medium containing 1% stripped serum, and cells were incubated with or without 10nM IGF-I for additional 24 hours, in presence or absence of metformin (10mM). Cell viability was evaluated by MTT assay. Values are expressed as percentages of untreated cells and represent the mean ± SEM (errors bars) of 2 separate experiments performed in triplicate. **, P

    Article Snippet: The following materials were purchased from the indicated manufacturers: metformin, methyltrienolone (R1881), Src activity inhibitor (4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), and rapamycin from Sigma Chemical Co; fetal calf serum and Opti-MEM from Gibco Laboratories; temsirolimus from DBA; and Metafectene PRO from Biontex Laboratories GmbH.

    Techniques: Incubation, MTT Assay

    c-Src recruitment to AR. A and B, c-Src binding with AR after metformin (Met) treatment with or without R1881. Cells were serum starved for 24 hours and then treated with metformin (10mM) for 1 hour (A) or for 24 hours (B) followed by the exposure to 10nM R1881 for 2 minutes. Cells were then solubilized, and the lysates were immunoprecipitated with an anti-Src-specific antibody and analyzed by Western blotting. Total lysates (input) were also evaluated as a control. Filters were probed with anti-AR, anti-Src, anti-pp70S6K, and anti-β-tubulin antibodies as indicated. The panel shows a representative experiment of 3. The histogram represents the mean ± SEM (error bars) of densitometric analyses of AR and pp70S6K for the 3 experiments. *, P

    Journal: Endocrinology

    Article Title: Metformin Inhibits Androgen-Induced IGF-IR Up-Regulation in Prostate Cancer Cells by Disrupting Membrane-Initiated Androgen Signaling

    doi: 10.1210/en.2013-1925

    Figure Lengend Snippet: c-Src recruitment to AR. A and B, c-Src binding with AR after metformin (Met) treatment with or without R1881. Cells were serum starved for 24 hours and then treated with metformin (10mM) for 1 hour (A) or for 24 hours (B) followed by the exposure to 10nM R1881 for 2 minutes. Cells were then solubilized, and the lysates were immunoprecipitated with an anti-Src-specific antibody and analyzed by Western blotting. Total lysates (input) were also evaluated as a control. Filters were probed with anti-AR, anti-Src, anti-pp70S6K, and anti-β-tubulin antibodies as indicated. The panel shows a representative experiment of 3. The histogram represents the mean ± SEM (error bars) of densitometric analyses of AR and pp70S6K for the 3 experiments. *, P

    Article Snippet: The following materials were purchased from the indicated manufacturers: metformin, methyltrienolone (R1881), Src activity inhibitor (4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), and rapamycin from Sigma Chemical Co; fetal calf serum and Opti-MEM from Gibco Laboratories; temsirolimus from DBA; and Metafectene PRO from Biontex Laboratories GmbH.

    Techniques: Binding Assay, Immunoprecipitation, Western Blot