Structured Review

Millipore metformin
<t>Metformin</t> improves cell migration in HUVEC exposed to hyperglycemia and CoCl 2 . ( A ) HUVEC were incubated for 24 h with hyperglycemia in the presence or absence of metformin. Scratch lines were created on confluent monolayers. The media containing different glucose concentrations and metformin were replaced. Then cells were incubated with CoCl 2 for 24 h in a 5% CO 2 chamber that was connected to CCD camera. Images were acquired every hour, and three independent biological experiments were performed at which each condition was assessed in duplicate. The scratch area was measured using NIS Elements software. ( B ) Hyperglycemia increased migration after 6, 12, and 18 h; ( C ) whereas hyperglycemia-CoCl 2 significantly reduced migration. Metformin increased cell migration under hyperglycemia-CoCl 2 . Sunitinib was used as a negative control, therefore the line with sunitinab is on x axis as cell migration not affected. Results are expressed as mean ± SEM and were analyzed by one-way ANOVA followed by LSD, ** p
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Images

1) Product Images from "Proangiogenic Effect of Metformin in Endothelial Cells Is via Upregulation of VEGFR1/2 and Their Signaling under Hyperglycemia-Hypoxia"

Article Title: Proangiogenic Effect of Metformin in Endothelial Cells Is via Upregulation of VEGFR1/2 and Their Signaling under Hyperglycemia-Hypoxia

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19010293

Metformin improves cell migration in HUVEC exposed to hyperglycemia and CoCl 2 . ( A ) HUVEC were incubated for 24 h with hyperglycemia in the presence or absence of metformin. Scratch lines were created on confluent monolayers. The media containing different glucose concentrations and metformin were replaced. Then cells were incubated with CoCl 2 for 24 h in a 5% CO 2 chamber that was connected to CCD camera. Images were acquired every hour, and three independent biological experiments were performed at which each condition was assessed in duplicate. The scratch area was measured using NIS Elements software. ( B ) Hyperglycemia increased migration after 6, 12, and 18 h; ( C ) whereas hyperglycemia-CoCl 2 significantly reduced migration. Metformin increased cell migration under hyperglycemia-CoCl 2 . Sunitinib was used as a negative control, therefore the line with sunitinab is on x axis as cell migration not affected. Results are expressed as mean ± SEM and were analyzed by one-way ANOVA followed by LSD, ** p
Figure Legend Snippet: Metformin improves cell migration in HUVEC exposed to hyperglycemia and CoCl 2 . ( A ) HUVEC were incubated for 24 h with hyperglycemia in the presence or absence of metformin. Scratch lines were created on confluent monolayers. The media containing different glucose concentrations and metformin were replaced. Then cells were incubated with CoCl 2 for 24 h in a 5% CO 2 chamber that was connected to CCD camera. Images were acquired every hour, and three independent biological experiments were performed at which each condition was assessed in duplicate. The scratch area was measured using NIS Elements software. ( B ) Hyperglycemia increased migration after 6, 12, and 18 h; ( C ) whereas hyperglycemia-CoCl 2 significantly reduced migration. Metformin increased cell migration under hyperglycemia-CoCl 2 . Sunitinib was used as a negative control, therefore the line with sunitinab is on x axis as cell migration not affected. Results are expressed as mean ± SEM and were analyzed by one-way ANOVA followed by LSD, ** p

Techniques Used: Migration, Incubation, Software, Negative Control

Effect of metformin on VEGFA mRNA and VEGF 165A protein under euglycemia-CoCl 2 , hyperglycemia and hyperglycemia-CoCl 2 . HUVEC were treated with hyperglycemic or euglycemic glucose concentrations as a control. After 24 h, metformin was added to euglycemic and hyperglycemic cultures, then CoCl 2 was added for either 1, 3 or 12 h. ( A ) The variation in mRNA expression levels of VEGFA was assessed by qRT-PCR on three independent biological replicates and ( B ) the variation in protein levels of VEGF 165A was assessed by western blot on three independent biological replicates. Results are presented as mean ± SEM and were analyzed using one-way ANOVA followed by LSD, * p
Figure Legend Snippet: Effect of metformin on VEGFA mRNA and VEGF 165A protein under euglycemia-CoCl 2 , hyperglycemia and hyperglycemia-CoCl 2 . HUVEC were treated with hyperglycemic or euglycemic glucose concentrations as a control. After 24 h, metformin was added to euglycemic and hyperglycemic cultures, then CoCl 2 was added for either 1, 3 or 12 h. ( A ) The variation in mRNA expression levels of VEGFA was assessed by qRT-PCR on three independent biological replicates and ( B ) the variation in protein levels of VEGF 165A was assessed by western blot on three independent biological replicates. Results are presented as mean ± SEM and were analyzed using one-way ANOVA followed by LSD, * p

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

Metformin impairs cell migration in HUVEC exposed to euglycemia and CoCl 2 . ( A ) HUVEC were incubated for 24 h with euglycemia in the presence or absence of metformin. Scratch lines were created on confluent monolayers. The media containing different glucose concentrations and metformin were replaced. Then cells were incubated with CoCl 2 for 24 h in a 5% CO 2 chamber that was connected to CCD camera. Images were acquired every hour, and three independent biological experiments were performed at which each condition was assessed in duplicate. The scratch area was measured using NIS Elements software. ( B ) CoCl 2 induction exhibited no significant effect on cell migration under euglycemia, whereas metformin reduced migration after 18 h. Sunitinib (0.1 µmol/L) was used as a negative control, therefore the line with sunitinab is on x axis as cell migration not affected. Results are expressed as mean ± SEM and were analyzed by one-way ANOVA followed by LSD, ## p
Figure Legend Snippet: Metformin impairs cell migration in HUVEC exposed to euglycemia and CoCl 2 . ( A ) HUVEC were incubated for 24 h with euglycemia in the presence or absence of metformin. Scratch lines were created on confluent monolayers. The media containing different glucose concentrations and metformin were replaced. Then cells were incubated with CoCl 2 for 24 h in a 5% CO 2 chamber that was connected to CCD camera. Images were acquired every hour, and three independent biological experiments were performed at which each condition was assessed in duplicate. The scratch area was measured using NIS Elements software. ( B ) CoCl 2 induction exhibited no significant effect on cell migration under euglycemia, whereas metformin reduced migration after 18 h. Sunitinib (0.1 µmol/L) was used as a negative control, therefore the line with sunitinab is on x axis as cell migration not affected. Results are expressed as mean ± SEM and were analyzed by one-way ANOVA followed by LSD, ## p

Techniques Used: Migration, Incubation, Software, Negative Control

Marimastat antagonizes the effect of metformin on cell migration in HUVEC exposed to hyperglycemia-CoCl 2 . ( A ) HUVEC were incubated with euglycemia or hyperglycemia in the presence or absence of metformin for 24 h. Images were acquired every hour, and three independent biological experiments were performed in duplicate for each condition. The scratch area was measured using NIS Elements software. ( B ) Marimastat had no significant effect on cells treated with euglycemia, metformin and CoCl 2 induction. ( C ) The effect of metformin was inhibited by marimastat treatment. Results are expressed as mean ± SEM and were analyzed using one-way ANOVA followed by LSD, †† p
Figure Legend Snippet: Marimastat antagonizes the effect of metformin on cell migration in HUVEC exposed to hyperglycemia-CoCl 2 . ( A ) HUVEC were incubated with euglycemia or hyperglycemia in the presence or absence of metformin for 24 h. Images were acquired every hour, and three independent biological experiments were performed in duplicate for each condition. The scratch area was measured using NIS Elements software. ( B ) Marimastat had no significant effect on cells treated with euglycemia, metformin and CoCl 2 induction. ( C ) The effect of metformin was inhibited by marimastat treatment. Results are expressed as mean ± SEM and were analyzed using one-way ANOVA followed by LSD, †† p

Techniques Used: Migration, Incubation, Software

Effect of metformin on MMP16 mRNA expression in euglycemic and hyperglycemic conditions with CoCl 2 induction. The variation in RNA expression levels of MMP16 was assessed by qRT-PCR on three independent biological replicates. Results are presented as mean ± SEM and were analyzed using one-way ANOVA followed by LSD. * p
Figure Legend Snippet: Effect of metformin on MMP16 mRNA expression in euglycemic and hyperglycemic conditions with CoCl 2 induction. The variation in RNA expression levels of MMP16 was assessed by qRT-PCR on three independent biological replicates. Results are presented as mean ± SEM and were analyzed using one-way ANOVA followed by LSD. * p

Techniques Used: Expressing, RNA Expression, Quantitative RT-PCR

Comprehensive VEGF signaling network of genes and proteins involved in cell migration and survival. Metformin-treated condition is compared to metformin-untreated condition under hyperglycemia-CoCl 2 for 12 h. The network was created by IPA software rendering VEGF signal transduction pathways. The genes from microarray expression study that are represented with red shades are upregulated, and green shades are downregulated, MMP16 was validated by qRT-PCR. The activity of ERK1/2 was assessed by MAPK activation dual detection assay flow cytometry. The red shade on functional assays denoted activation while green shade inhibition. Solid lines denoted direct interaction; interrupted lines denoted indirect interaction.
Figure Legend Snippet: Comprehensive VEGF signaling network of genes and proteins involved in cell migration and survival. Metformin-treated condition is compared to metformin-untreated condition under hyperglycemia-CoCl 2 for 12 h. The network was created by IPA software rendering VEGF signal transduction pathways. The genes from microarray expression study that are represented with red shades are upregulated, and green shades are downregulated, MMP16 was validated by qRT-PCR. The activity of ERK1/2 was assessed by MAPK activation dual detection assay flow cytometry. The red shade on functional assays denoted activation while green shade inhibition. Solid lines denoted direct interaction; interrupted lines denoted indirect interaction.

Techniques Used: Migration, Indirect Immunoperoxidase Assay, Software, Transduction, Microarray, Expressing, Quantitative RT-PCR, Activity Assay, Activation Assay, Detection Assay, Flow Cytometry, Cytometry, Functional Assay, Inhibition

Metformin improves cell survival with hyperglycemia-CoCl 2 . ( A ) HUVEC were treated with 5.5 or ( B ) 16.5 mmol/L glucose for 48 h exposed to chemical hypoxia for 3, 12 or 24 h in the absence of metformin, ( C ) hyperglycemia treated with metformin (0.01 mmol/L) and supra-physiological concentration of metformin (1.0 mmol/L), and ( D ) hyperglycemia exposed to CoCl 2 for 24 h and parallel cultures were treated with metformin. Apoptosis was assessed by Annexin V staining and flow cytometry. Results are representative of 3 independent experiments and expressed as mean ± SEM and were analyzed by paired t -test, * p
Figure Legend Snippet: Metformin improves cell survival with hyperglycemia-CoCl 2 . ( A ) HUVEC were treated with 5.5 or ( B ) 16.5 mmol/L glucose for 48 h exposed to chemical hypoxia for 3, 12 or 24 h in the absence of metformin, ( C ) hyperglycemia treated with metformin (0.01 mmol/L) and supra-physiological concentration of metformin (1.0 mmol/L), and ( D ) hyperglycemia exposed to CoCl 2 for 24 h and parallel cultures were treated with metformin. Apoptosis was assessed by Annexin V staining and flow cytometry. Results are representative of 3 independent experiments and expressed as mean ± SEM and were analyzed by paired t -test, * p

Techniques Used: Concentration Assay, Staining, Flow Cytometry, Cytometry

2) Product Images from "Differential impact of structurally different anti-diabetic drugs on proliferation and chemosensitivity of acute lymphoblastic leukemia cells"

Article Title: Differential impact of structurally different anti-diabetic drugs on proliferation and chemosensitivity of acute lymphoblastic leukemia cells

Journal: Cell Cycle

doi: 10.4161/cc.20770

Insulin stimulated, while metformin and rosiglitazone inhibited, ALL cells.
Figure Legend Snippet: Insulin stimulated, while metformin and rosiglitazone inhibited, ALL cells.

Techniques Used:

Insulin stimulated, while metformin and rosiglitazone inhibited, ALL cells.
Figure Legend Snippet: Insulin stimulated, while metformin and rosiglitazone inhibited, ALL cells.

Techniques Used:

Insulin stimulated, while metformin and rosiglitazone inhibited, ALL cells.
Figure Legend Snippet: Insulin stimulated, while metformin and rosiglitazone inhibited, ALL cells.

Techniques Used:

Figure 2. Inhibitory effects of metformin and rosiglitazone on leukemic cell DNA replication. (A) The percentage of cells in S phase as determined by PI flow cytometry measuring DNA content were plotted on the bar chart with treatments labeled
Figure Legend Snippet: Figure 2. Inhibitory effects of metformin and rosiglitazone on leukemic cell DNA replication. (A) The percentage of cells in S phase as determined by PI flow cytometry measuring DNA content were plotted on the bar chart with treatments labeled

Techniques Used: Flow Cytometry, Cytometry, Labeling

Insulin stimulated, while metformin and rosiglitazone inhibited, ALL cells.
Figure Legend Snippet: Insulin stimulated, while metformin and rosiglitazone inhibited, ALL cells.

Techniques Used:

Figure 3. Induction of apoptosis by metformin and rosiglitazone in leukemic cells. The percentage of apoptotic cells was measured by dual-staining flow cytometry with annexin V-FITC and propidium iodide. The sum of the percentages indicated in
Figure Legend Snippet: Figure 3. Induction of apoptosis by metformin and rosiglitazone in leukemic cells. The percentage of apoptotic cells was measured by dual-staining flow cytometry with annexin V-FITC and propidium iodide. The sum of the percentages indicated in

Techniques Used: Staining, Flow Cytometry, Cytometry

3) Product Images from "Metformin Inhibits the IL-6-Induced Epithelial-Mesenchymal Transition and Lung Adenocarcinoma Growth and Metastasis"

Article Title: Metformin Inhibits the IL-6-Induced Epithelial-Mesenchymal Transition and Lung Adenocarcinoma Growth and Metastasis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0095884

Metformin suppresses IL-6-induced STAT3 phosphorylation in lung adenocarcinoma cells. (A) Lung cancer cells were treated with IL-6 and the indicated concentrations of metformin, STAT3 phosphorylation status was determined by western blotting. Metformin suppressed STAT3 phosphorylation induced by IL-6 in lung adenocarcinoma cells in a dose-dependent manner. (B) CC827-pSB388 cells were treated with the indicated concentrations of metformin and STAT3 phosphorylation was determined by western blotting. (C) STAT3 phosphorylation in untreated HCC827-pSB388 cells, HCC827-pSB388 cells treated with metformin and HCC827-pSB388 cells treated with cucurbitacin Q were examined by immunofluorescence (800×). (D) STAT3 phosphorylation in tumors from HCC827, HCC827-pSB388 and HCC827-pSB388+Met groups was examined by immunohistochemistry (400×). (E) The average number of p-STAT3 positive cells in each field of view was analyzed. Error bars represent the standard deviation (**, p
Figure Legend Snippet: Metformin suppresses IL-6-induced STAT3 phosphorylation in lung adenocarcinoma cells. (A) Lung cancer cells were treated with IL-6 and the indicated concentrations of metformin, STAT3 phosphorylation status was determined by western blotting. Metformin suppressed STAT3 phosphorylation induced by IL-6 in lung adenocarcinoma cells in a dose-dependent manner. (B) CC827-pSB388 cells were treated with the indicated concentrations of metformin and STAT3 phosphorylation was determined by western blotting. (C) STAT3 phosphorylation in untreated HCC827-pSB388 cells, HCC827-pSB388 cells treated with metformin and HCC827-pSB388 cells treated with cucurbitacin Q were examined by immunofluorescence (800×). (D) STAT3 phosphorylation in tumors from HCC827, HCC827-pSB388 and HCC827-pSB388+Met groups was examined by immunohistochemistry (400×). (E) The average number of p-STAT3 positive cells in each field of view was analyzed. Error bars represent the standard deviation (**, p

Techniques Used: Western Blot, Immunofluorescence, Immunohistochemistry, Standard Deviation

Metformin inhibits IL-6 promotion of lung carcinoma cell invasion and EMT in vitro. (A) Invasion of IL-6 treated lung cancer cells in the presence of various concentrations of metformin. The selective STAT3 inhibitor cucurbitacin Q (Cuc, 2 µmol/L) served as a positive control for inhibition of lung cancer cell invasion. (B) Protein expression levels of E-cadherin, vimentin and snail in cells treated with IL-6 alone or in combination with metformin were analyzed by western blotting. β-actin was used as a loading control. (C) mRNA expression levels of E-cadherin, vimentin and snail in cells treated with IL-6 alone or in combination with metformin were examined by real time PCR and normalized by GAPDH. (D) The expression of E-cadherin and vimentin was detected by immunofluorescence in A549 and HCC827 cells treated with IL-6 alone or in combination with metformin (800×). Error bars represent the standard deviation (*, p
Figure Legend Snippet: Metformin inhibits IL-6 promotion of lung carcinoma cell invasion and EMT in vitro. (A) Invasion of IL-6 treated lung cancer cells in the presence of various concentrations of metformin. The selective STAT3 inhibitor cucurbitacin Q (Cuc, 2 µmol/L) served as a positive control for inhibition of lung cancer cell invasion. (B) Protein expression levels of E-cadherin, vimentin and snail in cells treated with IL-6 alone or in combination with metformin were analyzed by western blotting. β-actin was used as a loading control. (C) mRNA expression levels of E-cadherin, vimentin and snail in cells treated with IL-6 alone or in combination with metformin were examined by real time PCR and normalized by GAPDH. (D) The expression of E-cadherin and vimentin was detected by immunofluorescence in A549 and HCC827 cells treated with IL-6 alone or in combination with metformin (800×). Error bars represent the standard deviation (*, p

Techniques Used: In Vitro, Positive Control, Inhibition, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Standard Deviation

Metformin inhibits tumor growth, EMT, and metastasis induced by IL-6 in vivo. (A) Xenograft at sacrifice. Xenografts from HCC827-pSB388 group were much larger than HCC827 group. Xenografts from HCC827-pSB388+Met group, which treated with metformin, were much smaller than that from HCC827-pSB388 group. (B) Tumor volumes were determined at the time of sacrifice. (C) Metastatic tumor nodules in the lung were examined by H E staining of serial sections. Tumor nodules are marked with red arrows (100× and 400×). (D) The numbers of cancerous metastatic nodules in these lung sections were counted and the average number per field of view is presented. (E) E-cadherin and vimentin expression in tumor tissues from HCC827, HCC827pSB388 and HCC827pSB388+Met groups was analyzed by immunohistochemistry (400×). Error bars represent the standard deviation (*, p
Figure Legend Snippet: Metformin inhibits tumor growth, EMT, and metastasis induced by IL-6 in vivo. (A) Xenograft at sacrifice. Xenografts from HCC827-pSB388 group were much larger than HCC827 group. Xenografts from HCC827-pSB388+Met group, which treated with metformin, were much smaller than that from HCC827-pSB388 group. (B) Tumor volumes were determined at the time of sacrifice. (C) Metastatic tumor nodules in the lung were examined by H E staining of serial sections. Tumor nodules are marked with red arrows (100× and 400×). (D) The numbers of cancerous metastatic nodules in these lung sections were counted and the average number per field of view is presented. (E) E-cadherin and vimentin expression in tumor tissues from HCC827, HCC827pSB388 and HCC827pSB388+Met groups was analyzed by immunohistochemistry (400×). Error bars represent the standard deviation (*, p

Techniques Used: In Vivo, Staining, Expressing, Immunohistochemistry, Standard Deviation

4) Product Images from "Mitochondrial Respiratory Complex I Regulates Neutrophil Activation and Severity of Lung Injury"

Article Title: Mitochondrial Respiratory Complex I Regulates Neutrophil Activation and Severity of Lung Injury

Journal:

doi: 10.1164/rccm.200710-1602OC

Metformin treatment diminishes the severity of LPS-induced acute lung injury. C57BL/6 mice were given LPS (1 mg/kg) intratracheally followed by vehicle intraperitoneally 0.5 and 8 hours post-LPS, or two doses of metformin (125 mg/kg intraperitoneally)
Figure Legend Snippet: Metformin treatment diminishes the severity of LPS-induced acute lung injury. C57BL/6 mice were given LPS (1 mg/kg) intratracheally followed by vehicle intraperitoneally 0.5 and 8 hours post-LPS, or two doses of metformin (125 mg/kg intraperitoneally)

Techniques Used: Mouse Assay

Effects of metformin on LPS-induced neutrophil accumulation in the lungs. Representative histologic sections show decrease in the accumulation of interstitial and intraalveolar neutrophils in LPS/metformin-treated mice compared with mice given LPS and
Figure Legend Snippet: Effects of metformin on LPS-induced neutrophil accumulation in the lungs. Representative histologic sections show decrease in the accumulation of interstitial and intraalveolar neutrophils in LPS/metformin-treated mice compared with mice given LPS and

Techniques Used: Mouse Assay

Metformin inhibits LPS-mediated p65 (Ser-536) phosphorylation, nuclear factor (NF)-κB nuclear translocation, and cytokine production by neutrophils. Neutrophils were incubated with metformin (0 or 250 μM) for 2.5 hours followed by treatment
Figure Legend Snippet: Metformin inhibits LPS-mediated p65 (Ser-536) phosphorylation, nuclear factor (NF)-κB nuclear translocation, and cytokine production by neutrophils. Neutrophils were incubated with metformin (0 or 250 μM) for 2.5 hours followed by treatment

Techniques Used: Translocation Assay, Incubation

Effects of metformin on complex I activity and dihydroethidium (DHE) or 2′,7′-dichlorodihydrofluorescein (DCFH) oxidation in neutrophils. ( A ) Mitochondrial complex I activity was determined in control cell extracts (100 μg/ml)
Figure Legend Snippet: Effects of metformin on complex I activity and dihydroethidium (DHE) or 2′,7′-dichlorodihydrofluorescein (DCFH) oxidation in neutrophils. ( A ) Mitochondrial complex I activity was determined in control cell extracts (100 μg/ml)

Techniques Used: Activity Assay

Effects of metformin on LPS-induced IκB-α degradation. ( A , B ) A representative Western blot and optical densitometry (mean ± SD) of IκB-α and actin from neutrophils incubated with metformin (0–500 μM)
Figure Legend Snippet: Effects of metformin on LPS-induced IκB-α degradation. ( A , B ) A representative Western blot and optical densitometry (mean ± SD) of IκB-α and actin from neutrophils incubated with metformin (0–500 μM)

Techniques Used: Western Blot, Incubation

5) Product Images from "Rescue of mutant rhodopsin traffic by metformin-induced AMPK activation accelerates photoreceptor degeneration"

Article Title: Rescue of mutant rhodopsin traffic by metformin-induced AMPK activation accelerates photoreceptor degeneration

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddw387

Improvement of rhodopsin trafficking correlates with disorganised ROS in the retinae of P23H-1 metformin-treated rats. ( A ) Representative retinal images of P23H-1 (P36) rats treated with vehicle or metformin, as indicated. Subcellular localisation of rhodopsin stained with Rho 1D4 antibody. Scale bar: 10 µm. ( B ) Quantification of rhodopsin immunofluorescence intensity in the ROS. A line scan was performed and the mean maximum intensity was assessed within 12 images of ≥ 15 ROS from three animals per treatment. Values are means ± SEM, * P
Figure Legend Snippet: Improvement of rhodopsin trafficking correlates with disorganised ROS in the retinae of P23H-1 metformin-treated rats. ( A ) Representative retinal images of P23H-1 (P36) rats treated with vehicle or metformin, as indicated. Subcellular localisation of rhodopsin stained with Rho 1D4 antibody. Scale bar: 10 µm. ( B ) Quantification of rhodopsin immunofluorescence intensity in the ROS. A line scan was performed and the mean maximum intensity was assessed within 12 images of ≥ 15 ROS from three animals per treatment. Values are means ± SEM, * P

Techniques Used: Staining, Immunofluorescence

Metformin impairs photoreceptor function in P23H-1 rats. P23H-1 rats were treated from P21-P35 with either 300 mg/kg metformin or vehicle-PBS administered daily via IP injection. (A ) Retinae of metformin- (+) or vehicle- treated (-) P23H-1 rats were western blotted with p-AMPKα or an anti-AMPKα antibody to confirm the activation of AMPKα protein in the rat retina after metformin treatment, as indicated. β-Tubulin was used as a loading control. ( B ) Quantified expression levels of AMPKα and p-AMPKα in P23H-1 retina relative to β-tubulin. Densitometric analysis was used to calculate the levels of AMPKα and p-AMPKα relative to the vehicle control; values are means ± SEM, n ≥ 3 biological replicates. (C-E ) Scotopic ERG responses; ( C ) average at 0 log cds/m 2 , ( D ) a-wave, ( e ) b-wave of P23H-1 rats (P36) treated with either 300 mg/kg metformin ( n = 16 biological replicates) or vehicle-PBS ( n = 14 biological replicates), values are means ± SEM. * P
Figure Legend Snippet: Metformin impairs photoreceptor function in P23H-1 rats. P23H-1 rats were treated from P21-P35 with either 300 mg/kg metformin or vehicle-PBS administered daily via IP injection. (A ) Retinae of metformin- (+) or vehicle- treated (-) P23H-1 rats were western blotted with p-AMPKα or an anti-AMPKα antibody to confirm the activation of AMPKα protein in the rat retina after metformin treatment, as indicated. β-Tubulin was used as a loading control. ( B ) Quantified expression levels of AMPKα and p-AMPKα in P23H-1 retina relative to β-tubulin. Densitometric analysis was used to calculate the levels of AMPKα and p-AMPKα relative to the vehicle control; values are means ± SEM, n ≥ 3 biological replicates. (C-E ) Scotopic ERG responses; ( C ) average at 0 log cds/m 2 , ( D ) a-wave, ( e ) b-wave of P23H-1 rats (P36) treated with either 300 mg/kg metformin ( n = 16 biological replicates) or vehicle-PBS ( n = 14 biological replicates), values are means ± SEM. * P

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

Metformin reduces the unfolded protein response (UPR) and does not affect the levels of rhodopsin in the retina of P23H-1 rats. (A ) Retinae of P23H-1 rats treated from P21-P35 with either 300 mg/kg metformin or vehicle-PBS were analysed with markers of the three UPR branches and blotted with antibodies against BiP, p-IRE1, IRE1, p-eIF2A, eIF2a, and ATF6 cleaved or nuclear (N). Actin was used as a loading control. ( B ) Densitometric analysis was used to calculate the levels of BiP, p-IRE1, IRE1, p-eIF2A, eIF2a, and ATF6 (N) relative to vehicle after normalisation to actin; values are means ± SEM, n ≥ 3 (biological replicates). ( C and D ) Retinae of P23H-1 rats treated from P21-P35 with either 300 mg/kg metformin or vehicle-PBS were analysed by a sedimentation assay. Fractions were immunoblotted with the 1D4 antibody against rhodopsin. Densitometric analysis was used to calculate the levels of (C) soluble rhodopsin relative to the vehicle treated and (D) insoluble rhodopsin relative to the vehicle after normalisation to soluble rhodopsin. Values are means ± SEM, n ≥ 4 (biological replicates).
Figure Legend Snippet: Metformin reduces the unfolded protein response (UPR) and does not affect the levels of rhodopsin in the retina of P23H-1 rats. (A ) Retinae of P23H-1 rats treated from P21-P35 with either 300 mg/kg metformin or vehicle-PBS were analysed with markers of the three UPR branches and blotted with antibodies against BiP, p-IRE1, IRE1, p-eIF2A, eIF2a, and ATF6 cleaved or nuclear (N). Actin was used as a loading control. ( B ) Densitometric analysis was used to calculate the levels of BiP, p-IRE1, IRE1, p-eIF2A, eIF2a, and ATF6 (N) relative to vehicle after normalisation to actin; values are means ± SEM, n ≥ 3 (biological replicates). ( C and D ) Retinae of P23H-1 rats treated from P21-P35 with either 300 mg/kg metformin or vehicle-PBS were analysed by a sedimentation assay. Fractions were immunoblotted with the 1D4 antibody against rhodopsin. Densitometric analysis was used to calculate the levels of (C) soluble rhodopsin relative to the vehicle treated and (D) insoluble rhodopsin relative to the vehicle after normalisation to soluble rhodopsin. Values are means ± SEM, n ≥ 4 (biological replicates).

Techniques Used: Sedimentation

Metformin improves P23H rod opsin traffic and folding and reduces P23H-induced cell death. ( A ) SK-N-SH cells transfected with WT-GFP rod opsin (green) or P23H-GFP rod opsin (green) were treated with metformin (1 mM) for 18 h. Fixed, non-permeabilised cells were stained with Rho-4D2 antibody against the extracellular N-terminus (red). Confocal microscopy imaging under identical conditions. Scale bar: 10 μm. Boxed regions show higher magnification. ( B ) Immunoblot with an anti-phospho-AMPKα (p-AMPKα) or an anti-AMPKα antibody. β-Tubulin was used as a loading control. Untreated- (C) and metformin- (+MET) treated P23H-GFP cells were blotted with the Rho-1D4 antibody. ( C ) In-cell western analysis of HA-P23H rod opsin. SK-N-SH cells were fixed and immunostained with an HA antibody against the extracellular N-terminus of rod opsin. The non-permeabilised (cell surface) immunoreactivity was determined as a percentage of total permeabilised immunoreactivity. The data were normalised to the amount of cell surface HA-WT rod opsin, values ± SEM, n ≥ 4, ** P
Figure Legend Snippet: Metformin improves P23H rod opsin traffic and folding and reduces P23H-induced cell death. ( A ) SK-N-SH cells transfected with WT-GFP rod opsin (green) or P23H-GFP rod opsin (green) were treated with metformin (1 mM) for 18 h. Fixed, non-permeabilised cells were stained with Rho-4D2 antibody against the extracellular N-terminus (red). Confocal microscopy imaging under identical conditions. Scale bar: 10 μm. Boxed regions show higher magnification. ( B ) Immunoblot with an anti-phospho-AMPKα (p-AMPKα) or an anti-AMPKα antibody. β-Tubulin was used as a loading control. Untreated- (C) and metformin- (+MET) treated P23H-GFP cells were blotted with the Rho-1D4 antibody. ( C ) In-cell western analysis of HA-P23H rod opsin. SK-N-SH cells were fixed and immunostained with an HA antibody against the extracellular N-terminus of rod opsin. The non-permeabilised (cell surface) immunoreactivity was determined as a percentage of total permeabilised immunoreactivity. The data were normalised to the amount of cell surface HA-WT rod opsin, values ± SEM, n ≥ 4, ** P

Techniques Used: Transfection, Staining, Confocal Microscopy, Imaging, In-Cell ELISA

Increased levels of P23H in the ROS of Rho P23H/P23H mice after metformin treatment. ( A ) Retinal extracts from metformin- (+) or vehicle- treated (-) Rho P23H/P23H (P14) KI mice were western blotted with anti-rhodopsin Rho-1D4 antibody and p-AMPKα or anti-AMPKα antibody. β-Tubulin was used as a loading control. ( B ) Quantification of p-AMPKα and rhodopsin levels in the Rho P23H/P23H KI mouse retina relative to β-tubulin. Densitometric analysis was used to calculate the levels of rhodopsin in metformin-treated mice relative to vehicle; values are means ± SEM, n ≥3 biological replicates. ( C ) Subcellular localisation of rhodopsin (green) and the ER marker calnexin (red) in the retina from Rho P23H/P23H KI mice treated with either vehicle-PBS or metformin. Scale bar 10 µm. (D ) Rhodopsin-calnexin IS staining quantified by calculating the Pearson's and Mander's co-localisation co-efficients using the JaCOP plug-in and ImageJ software, n = 18 images each from 2 vehicle-treated mice and 2 metformin-treated mice. Values are means ± SEM, * P
Figure Legend Snippet: Increased levels of P23H in the ROS of Rho P23H/P23H mice after metformin treatment. ( A ) Retinal extracts from metformin- (+) or vehicle- treated (-) Rho P23H/P23H (P14) KI mice were western blotted with anti-rhodopsin Rho-1D4 antibody and p-AMPKα or anti-AMPKα antibody. β-Tubulin was used as a loading control. ( B ) Quantification of p-AMPKα and rhodopsin levels in the Rho P23H/P23H KI mouse retina relative to β-tubulin. Densitometric analysis was used to calculate the levels of rhodopsin in metformin-treated mice relative to vehicle; values are means ± SEM, n ≥3 biological replicates. ( C ) Subcellular localisation of rhodopsin (green) and the ER marker calnexin (red) in the retina from Rho P23H/P23H KI mice treated with either vehicle-PBS or metformin. Scale bar 10 µm. (D ) Rhodopsin-calnexin IS staining quantified by calculating the Pearson's and Mander's co-localisation co-efficients using the JaCOP plug-in and ImageJ software, n = 18 images each from 2 vehicle-treated mice and 2 metformin-treated mice. Values are means ± SEM, * P

Techniques Used: Mouse Assay, Western Blot, Marker, Staining, Software

Metformin treatment accelerates photoreceptor loss in P23H rodent models. ( A-C ) ONL thickness of P23H-1 rats treated from P21-P35 ( A ) or from P21-P49 ( B ) with either 300 mg/kg metformin or vehicle-PBS daily via IP injection. P23H-1 ONL thickness at P36 ( A ) for metformin-treated ( n = 8 biological replicates) or vehicle-PBS treated rats ( n = 6 biological replicates) or P49 ( B ) (7 weeks of age) for metformin-treated ( n = 5 biological replicates) or vehicle-PBS treated rats ( n = 5 biological replicates) as assessed by OCT measurements across the inferior-superior meridian. Results are either expressed as a spider plot from the optic nerve head (ONH) ( A-B ) or as mean ONL thickness across the whole retina ( C ). Values are means ± SEM. ** P
Figure Legend Snippet: Metformin treatment accelerates photoreceptor loss in P23H rodent models. ( A-C ) ONL thickness of P23H-1 rats treated from P21-P35 ( A ) or from P21-P49 ( B ) with either 300 mg/kg metformin or vehicle-PBS daily via IP injection. P23H-1 ONL thickness at P36 ( A ) for metformin-treated ( n = 8 biological replicates) or vehicle-PBS treated rats ( n = 6 biological replicates) or P49 ( B ) (7 weeks of age) for metformin-treated ( n = 5 biological replicates) or vehicle-PBS treated rats ( n = 5 biological replicates) as assessed by OCT measurements across the inferior-superior meridian. Results are either expressed as a spider plot from the optic nerve head (ONH) ( A-B ) or as mean ONL thickness across the whole retina ( C ). Values are means ± SEM. ** P

Techniques Used: Injection

6) Product Images from "AMPK Downregulates ALK2 via Increasing the Interaction between Smurf1 and Smad6, leading to inhibition of in vitro Osteogenic Differentiation"

Article Title: AMPK Downregulates ALK2 via Increasing the Interaction between Smurf1 and Smad6, leading to inhibition of in vitro Osteogenic Differentiation

Journal: Biochimica et biophysica acta

doi: 10.1016/j.bbamcr.2017.08.009

AMPK promotes proteosomal degradation of ALK2 A–B. Scrambled siRNA (control) and siRNAs for Smad6 (A) or Smurf1 (B) were transfected into FOP fibroblast cells and then treated with or without metformin (10mM) for 24 hours. C–D. FOP fibroblast cells were treated with or without MG132 and/or metformin for 24 hours (C), or after infection with adenovirus expressing the active mutant of AMPK (AMPK-CA), the cells were incubated with MG132 for 24 hours (D). Cell extracts were then blotted with antibodies, as indicated.
Figure Legend Snippet: AMPK promotes proteosomal degradation of ALK2 A–B. Scrambled siRNA (control) and siRNAs for Smad6 (A) or Smurf1 (B) were transfected into FOP fibroblast cells and then treated with or without metformin (10mM) for 24 hours. C–D. FOP fibroblast cells were treated with or without MG132 and/or metformin for 24 hours (C), or after infection with adenovirus expressing the active mutant of AMPK (AMPK-CA), the cells were incubated with MG132 for 24 hours (D). Cell extracts were then blotted with antibodies, as indicated.

Techniques Used: Transfection, Infection, Expressing, Mutagenesis, Incubation

AMPK mediates the inhibitory effect of metformin on ALK2 signaling A. MEF cells, wildtype, LKB1 KO, or AMPKα1 α2 KO, were treated with metformin (10mM) for 24 hours, followed by BMP6 (25ng/ml) for 30 min. B–C. FOP fibroblasts were infected with adenovirus in varying volumes for 48 hours. The virus expresses an active mutant of AMPKα1 (AMPK-CA) (B) or a dominant negative mutant of AMPKα1 (AMPK-DN) (C), both tagged with the flat epitope, or GFP as a control. The cells were treated with or without metformin (10mM) for 24 hours and followed with BMP6 (50ng/ml) for 30 min. Equal amounts of cell extracts (20μg) were blotted with antibodies, as indicated.
Figure Legend Snippet: AMPK mediates the inhibitory effect of metformin on ALK2 signaling A. MEF cells, wildtype, LKB1 KO, or AMPKα1 α2 KO, were treated with metformin (10mM) for 24 hours, followed by BMP6 (25ng/ml) for 30 min. B–C. FOP fibroblasts were infected with adenovirus in varying volumes for 48 hours. The virus expresses an active mutant of AMPKα1 (AMPK-CA) (B) or a dominant negative mutant of AMPKα1 (AMPK-DN) (C), both tagged with the flat epitope, or GFP as a control. The cells were treated with or without metformin (10mM) for 24 hours and followed with BMP6 (50ng/ml) for 30 min. Equal amounts of cell extracts (20μg) were blotted with antibodies, as indicated.

Techniques Used: Infection, Mutagenesis, Dominant Negative Mutation

AMPK upregulates Smad6 and Smurf1 FOP fibroblast cells were treated with metformin (A, B) or aspirin (C) at varying concentrations for 24 hours (A, C) or different times (B, D). Equal cell extracts (20μg) were resolved by Western blot with antibodies, as indicated. Representative blots were presented and graphs represent scan densitometric ratio of bands from three independent blots (average ratio±SD).
Figure Legend Snippet: AMPK upregulates Smad6 and Smurf1 FOP fibroblast cells were treated with metformin (A, B) or aspirin (C) at varying concentrations for 24 hours (A, C) or different times (B, D). Equal cell extracts (20μg) were resolved by Western blot with antibodies, as indicated. Representative blots were presented and graphs represent scan densitometric ratio of bands from three independent blots (average ratio±SD).

Techniques Used: Western Blot

Inhibition of ALK2 signaling in FOP fibroblast cells by AMPK activators A. FOP fibroblast cells were treated with metformin (Met, 10mM), phenformin (Phen, 1mM), A769962 (A76, 10μM), AICAR (1mM) for 8 hours, or left without treatment (Con). B. The fibroblasts were treated with metformin, A769962, or AICAR for 24 hours, followed by BMP6 (50ng/ml) for 30 min. C. The cells were treated with different doses of metformin. D. The cells were treated with metformin (10mM) for different periods of time. Equal amounts of cell extracts (20μg) were blotted with antibodies as indicated. Representative blots were presented and graphs represent scan densitometric ratio of bands from three independent blots (average ratio±SD).
Figure Legend Snippet: Inhibition of ALK2 signaling in FOP fibroblast cells by AMPK activators A. FOP fibroblast cells were treated with metformin (Met, 10mM), phenformin (Phen, 1mM), A769962 (A76, 10μM), AICAR (1mM) for 8 hours, or left without treatment (Con). B. The fibroblasts were treated with metformin, A769962, or AICAR for 24 hours, followed by BMP6 (50ng/ml) for 30 min. C. The cells were treated with different doses of metformin. D. The cells were treated with metformin (10mM) for different periods of time. Equal amounts of cell extracts (20μg) were blotted with antibodies as indicated. Representative blots were presented and graphs represent scan densitometric ratio of bands from three independent blots (average ratio±SD).

Techniques Used: Inhibition

7) Product Images from "Metformin Dysregulates the Unfolded Protein Response and the WNT/β-Catenin Pathway in Endometrial Cancer Cells through an AMPK-Independent Mechanism"

Article Title: Metformin Dysregulates the Unfolded Protein Response and the WNT/β-Catenin Pathway in Endometrial Cancer Cells through an AMPK-Independent Mechanism

Journal: Cells

doi: 10.3390/cells10051067

Inhibition of AMPK by CC does not alter metformin effects on endometrial cancer cells. ( A ) Ishikawa, HEC1B, or AN3CA cells were treated or not for 24 h with 5 mM metformin or 10 µM CC or pretreated for 1 h with 10 µM CC followed by treatment with 5 mM metformin. Total cellular proteins were extracted and Western blot experiments were performed, as described in the Section 2.3 , using antibodies against p-AMPK (left panels) or p-S6 kinase (right panels). Data represent the mean ± SD of three independent experiments. * p
Figure Legend Snippet: Inhibition of AMPK by CC does not alter metformin effects on endometrial cancer cells. ( A ) Ishikawa, HEC1B, or AN3CA cells were treated or not for 24 h with 5 mM metformin or 10 µM CC or pretreated for 1 h with 10 µM CC followed by treatment with 5 mM metformin. Total cellular proteins were extracted and Western blot experiments were performed, as described in the Section 2.3 , using antibodies against p-AMPK (left panels) or p-S6 kinase (right panels). Data represent the mean ± SD of three independent experiments. * p

Techniques Used: Inhibition, Western Blot

Metformin inhibits cell growth and viability in endometrial cancer cells. ( A ) Ishikawa, HEC1B, or AN3CA cells were seeded at a density of 5 × 10 3 cells in a 96-well plate. After 16 h, cells were treated or not with 1, 5, or 10 mM metformin. Cell viability was measured after 48 h using the MTT assay. Values represent the mean absorbance at 570 nm ± SD of triplicates of three independent experiments. * indicates a p -value
Figure Legend Snippet: Metformin inhibits cell growth and viability in endometrial cancer cells. ( A ) Ishikawa, HEC1B, or AN3CA cells were seeded at a density of 5 × 10 3 cells in a 96-well plate. After 16 h, cells were treated or not with 1, 5, or 10 mM metformin. Cell viability was measured after 48 h using the MTT assay. Values represent the mean absorbance at 570 nm ± SD of triplicates of three independent experiments. * indicates a p -value

Techniques Used: MTT Assay

Metformin modulates the mRNA expression of UPR genes in an AMPK-independent manner in endometrial cancer cells. Ishikawa, HEC1B, or AN3CA cells were treated or not for 24 h with 5 mM metformin in the presence or absence of 1 h pretreatment with 10 µM CC. Total RNA was extracted and real-time RT-PCR experiments were performed using oligonucleotides specific for GRP78, ATF6, ATF4, CHOP, and GAPDH as described in the Section 2.4 . Values shown represent the mean (± s.d.) of triplicate samples of three independent experiments. * p
Figure Legend Snippet: Metformin modulates the mRNA expression of UPR genes in an AMPK-independent manner in endometrial cancer cells. Ishikawa, HEC1B, or AN3CA cells were treated or not for 24 h with 5 mM metformin in the presence or absence of 1 h pretreatment with 10 µM CC. Total RNA was extracted and real-time RT-PCR experiments were performed using oligonucleotides specific for GRP78, ATF6, ATF4, CHOP, and GAPDH as described in the Section 2.4 . Values shown represent the mean (± s.d.) of triplicate samples of three independent experiments. * p

Techniques Used: Expressing, Quantitative RT-PCR

Metformin modulates the expression/phosphorylation of UPR proteins and AKT in an AMPK-independent manner in endometrial cancer cells. Ishikawa ( A ), HEC1B ( B ), or AN3CA ( C ) cells were treated or not for 24 h with 5 mM metformin in the presence or absence of 1 h pretreatment with 10 µM CC. Total cellular proteins were extracted and Western blot experiments were performed with antibodies against GRP78, ATF6, ATF4, p-eIF2α, p -AKT, or β-actin (loading control), as described in the Section 2.3 . Values shown represent the mean (± s.d.) of three independent experiments. * p
Figure Legend Snippet: Metformin modulates the expression/phosphorylation of UPR proteins and AKT in an AMPK-independent manner in endometrial cancer cells. Ishikawa ( A ), HEC1B ( B ), or AN3CA ( C ) cells were treated or not for 24 h with 5 mM metformin in the presence or absence of 1 h pretreatment with 10 µM CC. Total cellular proteins were extracted and Western blot experiments were performed with antibodies against GRP78, ATF6, ATF4, p-eIF2α, p -AKT, or β-actin (loading control), as described in the Section 2.3 . Values shown represent the mean (± s.d.) of three independent experiments. * p

Techniques Used: Expressing, Western Blot

Metformin inhibits β-catenin expression independently from AMPK activation in endometrial cancer cells. ( A ) Ishikawa, HEC1B, or AN3CA cells were treated or not for 24 h with 5 mM metformin. Total RNA was extracted and real-time RT-PCR experiments were performed using oligonucleotides specific to β-catenin and GAPDH as described in the Section 2.4 . Values shown represent the mean (± s.d.) of triplicate samples of three independent experiments. ** p
Figure Legend Snippet: Metformin inhibits β-catenin expression independently from AMPK activation in endometrial cancer cells. ( A ) Ishikawa, HEC1B, or AN3CA cells were treated or not for 24 h with 5 mM metformin. Total RNA was extracted and real-time RT-PCR experiments were performed using oligonucleotides specific to β-catenin and GAPDH as described in the Section 2.4 . Values shown represent the mean (± s.d.) of triplicate samples of three independent experiments. ** p

Techniques Used: Expressing, Activation Assay, Quantitative RT-PCR

Metformin inhibits GSK3β phosphorylation in endometrial cancer cells. Ishikawa or AN3CA cells were treated or not for 24 h with 5 mM metformin or 10 µM CC or pretreated for 1 h with 10 µM CC followed by treatment with 5 mM metformin. Total cellular proteins were extracted and Western blot experiments were performed with antibodies against p-GSK3β (Ser9) or vinculin (loading control), as described in the Section 2.3 . Values shown represent the mean (± s.d.) of three independent experiments. ** p
Figure Legend Snippet: Metformin inhibits GSK3β phosphorylation in endometrial cancer cells. Ishikawa or AN3CA cells were treated or not for 24 h with 5 mM metformin or 10 µM CC or pretreated for 1 h with 10 µM CC followed by treatment with 5 mM metformin. Total cellular proteins were extracted and Western blot experiments were performed with antibodies against p-GSK3β (Ser9) or vinculin (loading control), as described in the Section 2.3 . Values shown represent the mean (± s.d.) of three independent experiments. ** p

Techniques Used: Western Blot

Scheme summarizing the observed mechanisms affected by metformin in endometrial cancer cells. Besides AMPK activation, metformin modulates the UPR by activating the PERK/ATF4/CHOP axis and inhibiting the ATF6/GRP78 axis. Furthermore, metformin inhibits the Wnt/β-catenin signaling pathway by reducing β-catenin expression.
Figure Legend Snippet: Scheme summarizing the observed mechanisms affected by metformin in endometrial cancer cells. Besides AMPK activation, metformin modulates the UPR by activating the PERK/ATF4/CHOP axis and inhibiting the ATF6/GRP78 axis. Furthermore, metformin inhibits the Wnt/β-catenin signaling pathway by reducing β-catenin expression.

Techniques Used: Activation Assay, Expressing

8) Product Images from "Anti-adipogenic effect of the flavonoids through the activation of AMPK in palmitate (PA)-treated HepG2 cells"

Article Title: Anti-adipogenic effect of the flavonoids through the activation of AMPK in palmitate (PA)-treated HepG2 cells

Journal: Journal of Veterinary Science

doi: 10.4142/jvs.21256

Effects of the flavonoids on the GSK3β phosphorylation level in PA-treated HepG2 cells. HepG2 cells were serum-starved overnight and incubated in a serum-deprived medium containing PA with or without 50 μM flavonoids for 24 h. p-GSK3β was determined by Western blotting. The data are represented as the mean ± SE. GSK3β, glycogen synthase kinase 3 β; PA, palmitate; MET, metformin; HES, hesperidin; NAR, narirutin; NOB, nobiletin; SIN, sinensetin; TAN, tangeretin. * p
Figure Legend Snippet: Effects of the flavonoids on the GSK3β phosphorylation level in PA-treated HepG2 cells. HepG2 cells were serum-starved overnight and incubated in a serum-deprived medium containing PA with or without 50 μM flavonoids for 24 h. p-GSK3β was determined by Western blotting. The data are represented as the mean ± SE. GSK3β, glycogen synthase kinase 3 β; PA, palmitate; MET, metformin; HES, hesperidin; NAR, narirutin; NOB, nobiletin; SIN, sinensetin; TAN, tangeretin. * p

Techniques Used: Incubation, Western Blot

Effect of the flavonoids on glucose uptake in PA-treated HepG2 cells. The glucose uptake assay was carried out using the fluorescent D-glucose analog 2-NBDG. HepG2 cells were serum-starved overnight and incubated in a serum-deprived medium containing PA with or without 50 μM flavonoids for 24 h. followed by incubation with 40 μM 2-NBDG glucose in the presence or absence of 100 nM insulin for 30 min. The cells were rinsed with PBS, and the fluorescence images were captured by IncuCyte ZOOM at 20× magnification (A). The total fluorescence intensities were calculated using IncuCyte ZOOM fluorescence processing software (B). The data are presented as the mean ± SE. 2-NBDG, 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose; CON, control; PA, palmitate; MET, metformin; HES, hesperidin; NAR, narirutin; NOB, nobiletin; SIN, sinensetin; TAN, tangeretin. * p
Figure Legend Snippet: Effect of the flavonoids on glucose uptake in PA-treated HepG2 cells. The glucose uptake assay was carried out using the fluorescent D-glucose analog 2-NBDG. HepG2 cells were serum-starved overnight and incubated in a serum-deprived medium containing PA with or without 50 μM flavonoids for 24 h. followed by incubation with 40 μM 2-NBDG glucose in the presence or absence of 100 nM insulin for 30 min. The cells were rinsed with PBS, and the fluorescence images were captured by IncuCyte ZOOM at 20× magnification (A). The total fluorescence intensities were calculated using IncuCyte ZOOM fluorescence processing software (B). The data are presented as the mean ± SE. 2-NBDG, 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose; CON, control; PA, palmitate; MET, metformin; HES, hesperidin; NAR, narirutin; NOB, nobiletin; SIN, sinensetin; TAN, tangeretin. * p

Techniques Used: Incubation, Fluorescence, Software

9) Product Images from "Metformin Protects Against Diabetes-Induced Cognitive Dysfunction by Inhibiting Mitochondrial Fission Protein DRP1"

Article Title: Metformin Protects Against Diabetes-Induced Cognitive Dysfunction by Inhibiting Mitochondrial Fission Protein DRP1

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2022.832707

The protective effect of metformin is attributed to DRP1 phosphorylation but not AMPK activation in primary neurons. (A) Representative blots and densitometric quantification of AMPK protein in primary neurons. The phosphorylation of AMPK (top blots) was normalized to total AMPK protein (middle), and total AMPK was normalized to β-ACTIN (bottom). (B) Representative blots and densitometric quantification of AMPK protein in primary neurons stimulated high glucose in the presence of GSK 621 or Compound C. The phosphorylation of AMPK (top blots) was normalized to total AMPK protein (middle), and total AMPK was normalized to β-ACTIN (bottom). (C) Representative blots, and densitometric quantification of DRP1 protein in primary neurons stimulated high glucose in the presence of GSK 621, or Compound C. The phosphorylation of DRP1 (top blots) was normalized to total DRP1 protein (middle), and total DRP1 was normalized to β-ACTIN (bottom). Fluorescent signals of Mitotracker-Red CM-H2X ROS (D) (200 ×, Bar = 800 µm) and quantification (E) in primary neurons stimulated with high glucose in the presence of GSK 621, or Compound C. Data presented as means ± SEM, * p
Figure Legend Snippet: The protective effect of metformin is attributed to DRP1 phosphorylation but not AMPK activation in primary neurons. (A) Representative blots and densitometric quantification of AMPK protein in primary neurons. The phosphorylation of AMPK (top blots) was normalized to total AMPK protein (middle), and total AMPK was normalized to β-ACTIN (bottom). (B) Representative blots and densitometric quantification of AMPK protein in primary neurons stimulated high glucose in the presence of GSK 621 or Compound C. The phosphorylation of AMPK (top blots) was normalized to total AMPK protein (middle), and total AMPK was normalized to β-ACTIN (bottom). (C) Representative blots, and densitometric quantification of DRP1 protein in primary neurons stimulated high glucose in the presence of GSK 621, or Compound C. The phosphorylation of DRP1 (top blots) was normalized to total DRP1 protein (middle), and total DRP1 was normalized to β-ACTIN (bottom). Fluorescent signals of Mitotracker-Red CM-H2X ROS (D) (200 ×, Bar = 800 µm) and quantification (E) in primary neurons stimulated with high glucose in the presence of GSK 621, or Compound C. Data presented as means ± SEM, * p

Techniques Used: Activation Assay

The protective effect of metformin is attributed to DRP1 phosphorylation but not AMPK activation in HT22. (A) Representative blots and densitometric quantification of AMPK protein in HT22 stimulated high glucose in the treatment of metformin or Mdivi-1. The phosphorylation of AMPK (top blots) was normalized to total AMPK protein (middle), and total AMPK was normalized to β-ACTIN (bottom). (B) Representative blots and densitometric quantification of AMPK protein in HT22 treated with AMPK agonist GSK 621 in the absence of metformin, or AMPK antagonist Compound C in the presence of metformin. The phosphorylation of AMPK (top blots) was normalized to total AMPK protein (middle), and total AMPK was normalized to β-ACTIN (bottom). (C) Representative blots and densitometric quantification of DRP1 protein in HT22 AMPK agonist GSK 621 in the absence of metformin, or AMPK antagonist Compound C in the presence of metformin. The phosphorylation of DRP1 (top blots) was normalized to total DRP1 protein (middle), and total DRP1 was normalized to β-ACTIN (bottom). Fluorescent signals of Mitotracker-Red CM-H2X ROS (D) (200 ×, Bar = 800 µm) and quantification (E) in HT22 protein in HT22 AMPK agonist GSK 621 in the absence of metformin, or AMPK antagonist Compound C in the presence of metformin. Data presented as means ± SEM, * p
Figure Legend Snippet: The protective effect of metformin is attributed to DRP1 phosphorylation but not AMPK activation in HT22. (A) Representative blots and densitometric quantification of AMPK protein in HT22 stimulated high glucose in the treatment of metformin or Mdivi-1. The phosphorylation of AMPK (top blots) was normalized to total AMPK protein (middle), and total AMPK was normalized to β-ACTIN (bottom). (B) Representative blots and densitometric quantification of AMPK protein in HT22 treated with AMPK agonist GSK 621 in the absence of metformin, or AMPK antagonist Compound C in the presence of metformin. The phosphorylation of AMPK (top blots) was normalized to total AMPK protein (middle), and total AMPK was normalized to β-ACTIN (bottom). (C) Representative blots and densitometric quantification of DRP1 protein in HT22 AMPK agonist GSK 621 in the absence of metformin, or AMPK antagonist Compound C in the presence of metformin. The phosphorylation of DRP1 (top blots) was normalized to total DRP1 protein (middle), and total DRP1 was normalized to β-ACTIN (bottom). Fluorescent signals of Mitotracker-Red CM-H2X ROS (D) (200 ×, Bar = 800 µm) and quantification (E) in HT22 protein in HT22 AMPK agonist GSK 621 in the absence of metformin, or AMPK antagonist Compound C in the presence of metformin. Data presented as means ± SEM, * p

Techniques Used: Activation Assay

Metformin protects against cognitive dysfunction in diabetes by inhibiting DRP1 phosphorylation in the hippocampus. Presences of phosphorylated DRP1 (A) (400 ×) and total DRP1 protein (B) (400 ×) in hippocampal neurons. (C) Presences of oxidative products in hippocampal neurons (200×). (D) Presences of cleaved caspase-3 in hippocampal neurons (400×). (E) Presences of TUNEL signals in hippocampal neurons (200×) DAPI labeled the nuclei (blue), and TUNEL was stained in red (Fluor 594).
Figure Legend Snippet: Metformin protects against cognitive dysfunction in diabetes by inhibiting DRP1 phosphorylation in the hippocampus. Presences of phosphorylated DRP1 (A) (400 ×) and total DRP1 protein (B) (400 ×) in hippocampal neurons. (C) Presences of oxidative products in hippocampal neurons (200×). (D) Presences of cleaved caspase-3 in hippocampal neurons (400×). (E) Presences of TUNEL signals in hippocampal neurons (200×) DAPI labeled the nuclei (blue), and TUNEL was stained in red (Fluor 594).

Techniques Used: TUNEL Assay, Labeling, Staining

(A) Representative picture of mitochondria in hippocampal neurons (upper) and measurement of mitochondria short/long axis ratio (lower) (39 mitochondria from metformin treatment, 45 from Mdivi-1 treatment, n = 3, 200,00×, Bar = 0.5 µm) (B) Representative picture of a synaptic gap in the hippocampus (upper) and measurements of synaptic gap and PSD (lower) (30 synapses from metformin treatment, 26 from Mdivi-1 treatment, n = 3, 500,00×, Bar = 200 nm). (C) Representative picture of dendritic spines in the hippocampus (upper) and counts of dendritic spines per dendrite (lower) (23 dendrites from metformin treatment, 14 from Mdivi-1 treatment, n = 3, 500,00×, Bar = 200 nm). Metformin and Mdivi-1 protect against diabetes-induced cognitive dysfunction in short-term memory (D) , long-term memory (E) , and spatial disorientation (F) . Data presented as means ± SEM, * p
Figure Legend Snippet: (A) Representative picture of mitochondria in hippocampal neurons (upper) and measurement of mitochondria short/long axis ratio (lower) (39 mitochondria from metformin treatment, 45 from Mdivi-1 treatment, n = 3, 200,00×, Bar = 0.5 µm) (B) Representative picture of a synaptic gap in the hippocampus (upper) and measurements of synaptic gap and PSD (lower) (30 synapses from metformin treatment, 26 from Mdivi-1 treatment, n = 3, 500,00×, Bar = 200 nm). (C) Representative picture of dendritic spines in the hippocampus (upper) and counts of dendritic spines per dendrite (lower) (23 dendrites from metformin treatment, 14 from Mdivi-1 treatment, n = 3, 500,00×, Bar = 200 nm). Metformin and Mdivi-1 protect against diabetes-induced cognitive dysfunction in short-term memory (D) , long-term memory (E) , and spatial disorientation (F) . Data presented as means ± SEM, * p

Techniques Used:

10) Product Images from "Metformin causes cancer cell death through downregulation of p53-dependent differentiated embryo chondrocyte 1"

Article Title: Metformin causes cancer cell death through downregulation of p53-dependent differentiated embryo chondrocyte 1

Journal: Journal of Biomedical Science

doi: 10.1186/s12929-018-0478-5

Function of DEC1 in metformin-induced apoptosis in HeLa cells. a HeLa cells were incubated for 20 h with the indicated concentrations of metformin, after which the cell lysates were subjected to western blotting with an antibody against DEC1. ACTN was the loading control. The protein levels of DEC1 after normalization with the loading control protein ACTN are presented as fold change. b HeLa cells were incubated with 5 mM metformin with and without 10 μM MG132 for the indicated times. They were then lysed; divided into cytoplasmic, membrane, and nuclear fractions; and subjected to western blotting with antibodies against DEC1, HSP90α/β (cytoplasmic fraction), EGFR (membrane fraction) and PARP (intact: nuclear fraction; cleaved: nuclear and cytoplasmic fractions). The protein levels of cleaved DEC1, PARP, and cPARP are presented as fold change. c HeLa cells were transiently transfected with 2 μg pEGFP.DEC1 for 5 h and then were observed with fluorescence-microscopy. d HeLa cells were transiently transfected with 2 μg of pEGFP vector and pEGFP.DEC1 and incubated for 13 h with 5 mM metformin. The cell lysates were subjected to western blotting with antibodies against DEC1 and PARP. ACTN was the loading control. The protein levels of PARP and cleaved PARP (cPARP) after normalization with the loading control protein ACTN are presented as fold change. The results are representative of three independent experiments
Figure Legend Snippet: Function of DEC1 in metformin-induced apoptosis in HeLa cells. a HeLa cells were incubated for 20 h with the indicated concentrations of metformin, after which the cell lysates were subjected to western blotting with an antibody against DEC1. ACTN was the loading control. The protein levels of DEC1 after normalization with the loading control protein ACTN are presented as fold change. b HeLa cells were incubated with 5 mM metformin with and without 10 μM MG132 for the indicated times. They were then lysed; divided into cytoplasmic, membrane, and nuclear fractions; and subjected to western blotting with antibodies against DEC1, HSP90α/β (cytoplasmic fraction), EGFR (membrane fraction) and PARP (intact: nuclear fraction; cleaved: nuclear and cytoplasmic fractions). The protein levels of cleaved DEC1, PARP, and cPARP are presented as fold change. c HeLa cells were transiently transfected with 2 μg pEGFP.DEC1 for 5 h and then were observed with fluorescence-microscopy. d HeLa cells were transiently transfected with 2 μg of pEGFP vector and pEGFP.DEC1 and incubated for 13 h with 5 mM metformin. The cell lysates were subjected to western blotting with antibodies against DEC1 and PARP. ACTN was the loading control. The protein levels of PARP and cleaved PARP (cPARP) after normalization with the loading control protein ACTN are presented as fold change. The results are representative of three independent experiments

Techniques Used: Incubation, Western Blot, Transfection, Fluorescence, Microscopy, Plasmid Preparation

Proposed working mechanisms of metformin in cancer cells. Metformin may directly decrease p53 in sensitive cells, which would in turn downregulate expression of its target gene, DEC1 , leading to apoptosis. Metformin not only induces cellular apoptosis but also induces ROS generation through repression of mitochondrial respiration and membrane potential to kill cancer cells. Thus, apoptosis, mitochondrial dysfunction, and ROS generation all contribute to the induction of HeLa cell death by metformin
Figure Legend Snippet: Proposed working mechanisms of metformin in cancer cells. Metformin may directly decrease p53 in sensitive cells, which would in turn downregulate expression of its target gene, DEC1 , leading to apoptosis. Metformin not only induces cellular apoptosis but also induces ROS generation through repression of mitochondrial respiration and membrane potential to kill cancer cells. Thus, apoptosis, mitochondrial dysfunction, and ROS generation all contribute to the induction of HeLa cell death by metformin

Techniques Used: Expressing

Transcriptional and translational regulation of p53 in HeLa cells. a HeLa cells were transiently transfected with 0.5 μg of pSG5.HA vector or the indicated amount of pSG5.HA.p53 and incubated for 12 h with 5 mM metformin. The cell lysates were subjected to western blotting with antibodies against p53, DEC1, and PARP. ACTN was the loading control. The protein levels of p53, DEC1, and cPARP after normalization with the loading control protein ACTN are presented as fold change. b HeLa cells were incubated for 5 h with the indicated concentrations of metformin with or without 10 μM MG132, after which the cell lysates were subjected to western blotting with an antibody against p53. ACTN was the loading control. The protein levels of p53 after normalization with the loading control protein ACTN are presented as fold change. c and d HeLa cells were incubated for 12 h with the indicated concentrations of metformin with and without 0.1 μM actinomycin D (Act D) or 50 μg/ml cycloheximide (CHX). Levels of p53 mRNA and protein were then assayed in the cell lysates using RT-PCR ( c ) and western blotting ( d ), respectively. GAPDH mRNA was the mRNA loading control; ACTN was the protein loading control. e and f HeLa cells were incubated with 5 mM metformin ( e ) or 50 μg/ml CHX ( f ) for the indicated times, after which cell lysates were subjected to western blotting with an antibody against p53. g HeLa cells were incubated for the indicated times with 10 mM metformin with and without 50 ng/ml CHX. The cell lysates were then subjected to western blotting with an antibody against p53. d - g The protein levels of p53 after normalization with the loading control protein ACTN are presented as fold change. The results are representative of three independent experiments
Figure Legend Snippet: Transcriptional and translational regulation of p53 in HeLa cells. a HeLa cells were transiently transfected with 0.5 μg of pSG5.HA vector or the indicated amount of pSG5.HA.p53 and incubated for 12 h with 5 mM metformin. The cell lysates were subjected to western blotting with antibodies against p53, DEC1, and PARP. ACTN was the loading control. The protein levels of p53, DEC1, and cPARP after normalization with the loading control protein ACTN are presented as fold change. b HeLa cells were incubated for 5 h with the indicated concentrations of metformin with or without 10 μM MG132, after which the cell lysates were subjected to western blotting with an antibody against p53. ACTN was the loading control. The protein levels of p53 after normalization with the loading control protein ACTN are presented as fold change. c and d HeLa cells were incubated for 12 h with the indicated concentrations of metformin with and without 0.1 μM actinomycin D (Act D) or 50 μg/ml cycloheximide (CHX). Levels of p53 mRNA and protein were then assayed in the cell lysates using RT-PCR ( c ) and western blotting ( d ), respectively. GAPDH mRNA was the mRNA loading control; ACTN was the protein loading control. e and f HeLa cells were incubated with 5 mM metformin ( e ) or 50 μg/ml CHX ( f ) for the indicated times, after which cell lysates were subjected to western blotting with an antibody against p53. g HeLa cells were incubated for the indicated times with 10 mM metformin with and without 50 ng/ml CHX. The cell lysates were then subjected to western blotting with an antibody against p53. d - g The protein levels of p53 after normalization with the loading control protein ACTN are presented as fold change. The results are representative of three independent experiments

Techniques Used: Transfection, Plasmid Preparation, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction

Verification of p53 and DEC1 into the metformin-induced apoptosis in HeLa cells. a HeLa cells were incubated for 24 h with the indicated concentrations of metformin, 1 mM NAC, and 10 μM pifithrin-α. The cell lysates were then subjected to western blotting with antibodies against PARP and caspase 3. ACTN was the loading control. b Following DEC1 knockdown in HeLa cells, the cells were incubated for 24 h with the indicated concentration of metformin. The cell lysates were then subjected to western blotting with antibodies against PARP and Caspase 3. ACTN was the loading control and DEC1 was the silencing efficiency of shDEC1. The protein levels of cPARP and cCaspase 3 after normalization with the loading control protein ACTN are presented as fold change. The results are representative of three independent experiments
Figure Legend Snippet: Verification of p53 and DEC1 into the metformin-induced apoptosis in HeLa cells. a HeLa cells were incubated for 24 h with the indicated concentrations of metformin, 1 mM NAC, and 10 μM pifithrin-α. The cell lysates were then subjected to western blotting with antibodies against PARP and caspase 3. ACTN was the loading control. b Following DEC1 knockdown in HeLa cells, the cells were incubated for 24 h with the indicated concentration of metformin. The cell lysates were then subjected to western blotting with antibodies against PARP and Caspase 3. ACTN was the loading control and DEC1 was the silencing efficiency of shDEC1. The protein levels of cPARP and cCaspase 3 after normalization with the loading control protein ACTN are presented as fold change. The results are representative of three independent experiments

Techniques Used: Incubation, Western Blot, Concentration Assay

Cytotoxicity of metformin in HeLa and ZR-75-1 cells. a and b HeLa cells ( a ) and ZR-75-1 ( b ) cells were treated with the indicated concentration of metformin for 35 h and 70 h, respectively. Cell viability was measured using the MTT method. c HeLa and ZR-75-1 cells were incubated with indicated concentration of metformin for 20 h and 25 h, respectively. Cell lysates were subjected to western blot analysis using antibodies against cyclin D1, cyclin B1 and H3P. ACTN was the protein loading control. The protein levels of cyclin D1, cyclin B1 and H3P after normalization with the loading control protein ACTN are presented as fold change. d HeLa and ZR-75-1 cells were incubated with the indicated concentration of metformin for 30 h and 57 h, respectively. The cells were then subjected to flow cytometric cell cycle profile analysis. The results are representative of three independent experiments
Figure Legend Snippet: Cytotoxicity of metformin in HeLa and ZR-75-1 cells. a and b HeLa cells ( a ) and ZR-75-1 ( b ) cells were treated with the indicated concentration of metformin for 35 h and 70 h, respectively. Cell viability was measured using the MTT method. c HeLa and ZR-75-1 cells were incubated with indicated concentration of metformin for 20 h and 25 h, respectively. Cell lysates were subjected to western blot analysis using antibodies against cyclin D1, cyclin B1 and H3P. ACTN was the protein loading control. The protein levels of cyclin D1, cyclin B1 and H3P after normalization with the loading control protein ACTN are presented as fold change. d HeLa and ZR-75-1 cells were incubated with the indicated concentration of metformin for 30 h and 57 h, respectively. The cells were then subjected to flow cytometric cell cycle profile analysis. The results are representative of three independent experiments

Techniques Used: Concentration Assay, MTT Assay, Incubation, Western Blot

Metformin-induced apoptosis in HeLa cells. a HeLa cells (8 × 10 4 cells) were incubated with the indicated concentration of metformin for 40 h, after which cell death, apoptosis or necrosis were quantified using FITC-Annexin V and PI and flow cytometry. b Quantitative analysis of percentage of indicated stages is presented as the mean ± S.D. of at least three independent experiments; # p > 0.05 and * p
Figure Legend Snippet: Metformin-induced apoptosis in HeLa cells. a HeLa cells (8 × 10 4 cells) were incubated with the indicated concentration of metformin for 40 h, after which cell death, apoptosis or necrosis were quantified using FITC-Annexin V and PI and flow cytometry. b Quantitative analysis of percentage of indicated stages is presented as the mean ± S.D. of at least three independent experiments; # p > 0.05 and * p

Techniques Used: Incubation, Concentration Assay, Flow Cytometry

Effect of metformin on mitochondrial function in HeLa cells. a HeLa cells were incubated for 20 h with 5 mM metformin, after which the cellular OCR was measured in XF24 bioenergetic assays. b HeLa cells were incubated for 16 h with the indicated concentrations of metformin, after which JC-1 staining was analyzed using flow cytometry. c Changes in FL1-H and FL2-H were further evaluated using a FACS Calibur flow cytometer. The results are representative of two independent experiments
Figure Legend Snippet: Effect of metformin on mitochondrial function in HeLa cells. a HeLa cells were incubated for 20 h with 5 mM metformin, after which the cellular OCR was measured in XF24 bioenergetic assays. b HeLa cells were incubated for 16 h with the indicated concentrations of metformin, after which JC-1 staining was analyzed using flow cytometry. c Changes in FL1-H and FL2-H were further evaluated using a FACS Calibur flow cytometer. The results are representative of two independent experiments

Techniques Used: Incubation, Staining, Flow Cytometry, FACS

Induction of ROS generation by metformin in HeLa cells. a HeLa cells were incubated for 20 h with various concentrations of metformin, after which the live cells was stained with 10 μM DCFH-DA for 40 min at 37 °C and assayed using a flow cytometer. b HeLa cells were incubated for 20 h with 5 mM metformin and/or 1 mM NAC. The cell lysates were then subjected to western blotting with an antibody against PARP. ACTN was the loading control. The protein levels of PARP and cleaved PARP (cPARP) after normalization with the loading control protein ACTN are presented as fold change. c HeLa cells (5 × 10 4 cells) were incubated for 20 h with vehicle (DMSO), 5 mM metformin, 1 mM NAC, or 5 mM metformin plus 1 mM NAC. Quantitative analysis of cell viability is presented as the mean ± S.D. of at least three independent experiments; # p > 0.05, * p
Figure Legend Snippet: Induction of ROS generation by metformin in HeLa cells. a HeLa cells were incubated for 20 h with various concentrations of metformin, after which the live cells was stained with 10 μM DCFH-DA for 40 min at 37 °C and assayed using a flow cytometer. b HeLa cells were incubated for 20 h with 5 mM metformin and/or 1 mM NAC. The cell lysates were then subjected to western blotting with an antibody against PARP. ACTN was the loading control. The protein levels of PARP and cleaved PARP (cPARP) after normalization with the loading control protein ACTN are presented as fold change. c HeLa cells (5 × 10 4 cells) were incubated for 20 h with vehicle (DMSO), 5 mM metformin, 1 mM NAC, or 5 mM metformin plus 1 mM NAC. Quantitative analysis of cell viability is presented as the mean ± S.D. of at least three independent experiments; # p > 0.05, * p

Techniques Used: Incubation, Staining, Flow Cytometry, Western Blot

11) Product Images from "Large-scale in silico identification of drugs exerting sex-specific effects in the heart"

Article Title: Large-scale in silico identification of drugs exerting sex-specific effects in the heart

Journal: Journal of Translational Medicine

doi: 10.1186/s12967-018-1612-6

Sexual differences of therapeutic evaluation for acebutolol and tacrine on heart. a Acute administration acebutolol, the change of heart rate was assessed. Treatment SHR with acebutolol for 4 weeks, the changes of heart rate ( b ) and blood pressure ( c ) were determined. Administration tacrine for 3 weeks, the heart rate changes ( d ), plasma cTNI level ( e ) and superoxide generation of heart by DHE staining were measured in mice, and metformin as a negative control for sexual differences. All data are present as mean ± SD. *P
Figure Legend Snippet: Sexual differences of therapeutic evaluation for acebutolol and tacrine on heart. a Acute administration acebutolol, the change of heart rate was assessed. Treatment SHR with acebutolol for 4 weeks, the changes of heart rate ( b ) and blood pressure ( c ) were determined. Administration tacrine for 3 weeks, the heart rate changes ( d ), plasma cTNI level ( e ) and superoxide generation of heart by DHE staining were measured in mice, and metformin as a negative control for sexual differences. All data are present as mean ± SD. *P

Techniques Used: Staining, Mouse Assay, Negative Control

12) Product Images from "Metformin-activated AMPK regulates β-catenin to reduce cell proliferation in colon carcinoma RKO cells"

Article Title: Metformin-activated AMPK regulates β-catenin to reduce cell proliferation in colon carcinoma RKO cells

Journal: Oncology Letters

doi: 10.3892/ol.2019.9892

Metformin acts as a negative regulator of β-catenin translocation into the nucleus. (A) Cells were fractionated into the cytosol and nucleus, and then subjected to immunoblot analysis using the indicated antibodies. (B) Cell lysates were precipitated using an anti-AMPK antibody. Levels of β-catenin and antibody-bound proteins were measured by immunoblot analysis using anti-β-catenin and anti-AMPK antibodies. (C) Cells were treated with metformin for 24 h and subjected to immunofluorescence staining for β-catenin and AMPK. The number of cells with nuclear β-catenin was quantified by direct counting. Magnification, ×400. ***P
Figure Legend Snippet: Metformin acts as a negative regulator of β-catenin translocation into the nucleus. (A) Cells were fractionated into the cytosol and nucleus, and then subjected to immunoblot analysis using the indicated antibodies. (B) Cell lysates were precipitated using an anti-AMPK antibody. Levels of β-catenin and antibody-bound proteins were measured by immunoblot analysis using anti-β-catenin and anti-AMPK antibodies. (C) Cells were treated with metformin for 24 h and subjected to immunofluorescence staining for β-catenin and AMPK. The number of cells with nuclear β-catenin was quantified by direct counting. Magnification, ×400. ***P

Techniques Used: Translocation Assay, Immunofluorescence, Staining

Metformin inhibits cell proliferation and ATP production in RKO cells. Cells were treated with metformin at different concentrations (5–20 mM) for 24 h and cell viability was measured by (A) BrdU and (B) MTT assay. (C) Cells were subjected to cellular ATP measurement. (D) Cell lysates were subjected to immunoblot analysis for β-catenin, p-AMPK, AMPK and β-actin. Cells were plated into XF24 culture plates and incubated for 24 h using a medium containing glucose, glutamine and pyruvate. (E) OCR responses to oligomycin (2 µM), FCCP (0.1 µM), antimycin A (1 µM) and rotenone (1 µM) were measured and (F) the baseline of ECAR was measured. **P
Figure Legend Snippet: Metformin inhibits cell proliferation and ATP production in RKO cells. Cells were treated with metformin at different concentrations (5–20 mM) for 24 h and cell viability was measured by (A) BrdU and (B) MTT assay. (C) Cells were subjected to cellular ATP measurement. (D) Cell lysates were subjected to immunoblot analysis for β-catenin, p-AMPK, AMPK and β-actin. Cells were plated into XF24 culture plates and incubated for 24 h using a medium containing glucose, glutamine and pyruvate. (E) OCR responses to oligomycin (2 µM), FCCP (0.1 µM), antimycin A (1 µM) and rotenone (1 µM) were measured and (F) the baseline of ECAR was measured. **P

Techniques Used: MTT Assay, Incubation

AMPK acts as a negative regulator of β-catenin in metformin-treated RKO cells. (A) Cells were treated with metformin and/or compound C for 24 h and cell viability was then measured by MTT assay. *P
Figure Legend Snippet: AMPK acts as a negative regulator of β-catenin in metformin-treated RKO cells. (A) Cells were treated with metformin and/or compound C for 24 h and cell viability was then measured by MTT assay. *P

Techniques Used: MTT Assay

Metformin inhibits RKO cell proliferation at high concentrations. (A) Cells were treated with metformin at different concentrations (50 µM to 20 mM) for 24 h and cell viability was measured by MTT assay. (B) Cell lysates were subjected to immunoblot analysis for p-AMPK, AMPK and β-actin. **P
Figure Legend Snippet: Metformin inhibits RKO cell proliferation at high concentrations. (A) Cells were treated with metformin at different concentrations (50 µM to 20 mM) for 24 h and cell viability was measured by MTT assay. (B) Cell lysates were subjected to immunoblot analysis for p-AMPK, AMPK and β-actin. **P

Techniques Used: MTT Assay

AMPK is associated with β-catenin degradation. Cells were treated with 10 mM met and 2.5 µM MG132 for 24 h. Protein levels of p-β-catenin ser33/37 , p-β-catenin ser552 , β-catenin, p-AMPK Thr172 and AMPK were then examined by immunoblot analysis. met, metformin; ser, serine; Thr, threonine; p, phosphorylated; AMPK, 5′-adenosine monophosphate-activated protein kinase.
Figure Legend Snippet: AMPK is associated with β-catenin degradation. Cells were treated with 10 mM met and 2.5 µM MG132 for 24 h. Protein levels of p-β-catenin ser33/37 , p-β-catenin ser552 , β-catenin, p-AMPK Thr172 and AMPK were then examined by immunoblot analysis. met, metformin; ser, serine; Thr, threonine; p, phosphorylated; AMPK, 5′-adenosine monophosphate-activated protein kinase.

Techniques Used:

13) Product Images from "Cell cycle arrest in Metformin treated breast cancer cells involves activation of AMPK, downregulation of cyclin D1, and requires p27Kip1 or p21Cip1"

Article Title: Cell cycle arrest in Metformin treated breast cancer cells involves activation of AMPK, downregulation of cyclin D1, and requires p27Kip1 or p21Cip1

Journal: Journal of Molecular Signaling

doi: 10.1186/1750-2187-3-18

Effect of metformin on cell cycle progression and cell cycle regulatory proteins in MCF7 cells . A . Untreated cells (Con) or cells treated with 8 mM metformin for 1.5 days (Met) were stained with propidium iodide and then analyzed by flow cytometry to estimate the number of cells in each phase of the cell cycle. The experiment was repeated 3 times and the mean and standard error for each cell phase is indicated in the table. B . Equal amounts of protein from untreated cells (Con) or cells treated with metformin for 1.5 days (Met) were analyzed by western blotting using antibodies that recognize the indicated cell cycle regulatory proteins. β-Actin was detected as a loading control C . Total RNA was isolated from untreated cells (C) or cells treated with metformin (8 mM) for 1.5 days (M) and RNase protection assays were performed to detect the mRNAs encoding the indicated cyclins (left panel). Cyclin D1 mRNA levels were normalized using L32 mRNA levels (right panel). The mean of 3 independent experiments is shown and error bars indicate standard deviation. In a t test the value for the metformin treated cells was significantly different from the control with a P value of 0.006. D . MCF7 cells were treated with (Met) or without (Con) metformin (8 mM) and then extracts were prepared for immunoprecipitation (IP) using either a control antibody (C) or an anti-CDK2 antibody (CDK2). The immunoprecipitated proteins were analyzed by western blotting using antibodies that recognize CDK2, p27 Kip1 or p21 Cip1 as indicated on the right.
Figure Legend Snippet: Effect of metformin on cell cycle progression and cell cycle regulatory proteins in MCF7 cells . A . Untreated cells (Con) or cells treated with 8 mM metformin for 1.5 days (Met) were stained with propidium iodide and then analyzed by flow cytometry to estimate the number of cells in each phase of the cell cycle. The experiment was repeated 3 times and the mean and standard error for each cell phase is indicated in the table. B . Equal amounts of protein from untreated cells (Con) or cells treated with metformin for 1.5 days (Met) were analyzed by western blotting using antibodies that recognize the indicated cell cycle regulatory proteins. β-Actin was detected as a loading control C . Total RNA was isolated from untreated cells (C) or cells treated with metformin (8 mM) for 1.5 days (M) and RNase protection assays were performed to detect the mRNAs encoding the indicated cyclins (left panel). Cyclin D1 mRNA levels were normalized using L32 mRNA levels (right panel). The mean of 3 independent experiments is shown and error bars indicate standard deviation. In a t test the value for the metformin treated cells was significantly different from the control with a P value of 0.006. D . MCF7 cells were treated with (Met) or without (Con) metformin (8 mM) and then extracts were prepared for immunoprecipitation (IP) using either a control antibody (C) or an anti-CDK2 antibody (CDK2). The immunoprecipitated proteins were analyzed by western blotting using antibodies that recognize CDK2, p27 Kip1 or p21 Cip1 as indicated on the right.

Techniques Used: Staining, Flow Cytometry, Cytometry, Western Blot, Isolation, Standard Deviation, Immunoprecipitation

Proposed model for the mechanism by which metformin mediates cell cycle arrest . Metformin treatment leads to activation of AMPK which leads to loss of cyclin D1 mRNA and downregulation of cyclin D1 protein. The reduction in cyclin D1 results in the release of sequestered cell cycle inhibitors p27 Kip1 and p21 Cip1 . The released CDK inhibitors bind to and inhibit cyclin E/CDK2, thus preventing cell cycle progression from G1 to S phase. In metformin-resistant MDA-MB-231 cells, levels of the CDK inhibitors are insufficient to block CDK2 even though AMPK is active and cyclin D1 is downregulated.
Figure Legend Snippet: Proposed model for the mechanism by which metformin mediates cell cycle arrest . Metformin treatment leads to activation of AMPK which leads to loss of cyclin D1 mRNA and downregulation of cyclin D1 protein. The reduction in cyclin D1 results in the release of sequestered cell cycle inhibitors p27 Kip1 and p21 Cip1 . The released CDK inhibitors bind to and inhibit cyclin E/CDK2, thus preventing cell cycle progression from G1 to S phase. In metformin-resistant MDA-MB-231 cells, levels of the CDK inhibitors are insufficient to block CDK2 even though AMPK is active and cyclin D1 is downregulated.

Techniques Used: Activation Assay, Multiple Displacement Amplification, Blocking Assay

Metformin inhibits proliferation of cultured breast cancer cells . A . MCF7 cells were treated with metformin at the indicated concentrations for one day and then cell number was determined using a hemocytometer. The mean cell number from 3 independent cultures is shown. Error bars represent standard deviation. Using a t test, all treatments were significantly different from the control with P values of less than 0.01. B . Six different breast cancer cell lines were treated with 8 mM metformin. At the indicated times viable cell number was determined using a hemocytometer. The reported status of estrogen receptor (ER) expression, HER2 amplification, and p53 expression (Wild-type, WT, or mutant, mt) are indicated for each cell line. HER2+ indicates HER2 amplification while HER2- indicates normal HER2 expression. The data points represent the mean cell number from 3 independent cultures and error bars represent standard deviation.
Figure Legend Snippet: Metformin inhibits proliferation of cultured breast cancer cells . A . MCF7 cells were treated with metformin at the indicated concentrations for one day and then cell number was determined using a hemocytometer. The mean cell number from 3 independent cultures is shown. Error bars represent standard deviation. Using a t test, all treatments were significantly different from the control with P values of less than 0.01. B . Six different breast cancer cell lines were treated with 8 mM metformin. At the indicated times viable cell number was determined using a hemocytometer. The reported status of estrogen receptor (ER) expression, HER2 amplification, and p53 expression (Wild-type, WT, or mutant, mt) are indicated for each cell line. HER2+ indicates HER2 amplification while HER2- indicates normal HER2 expression. The data points represent the mean cell number from 3 independent cultures and error bars represent standard deviation.

Techniques Used: Cell Culture, Standard Deviation, Expressing, Amplification, Mutagenesis

Downregulation of cyclin D1 corresponds to activation of AMP-activated protein kinase . A . MCF7 cells were treated with (Met) or without (Con) metformin for 1.5 days and then western blotting was performed to detect active phospho-AMPK, cyclin D1, and β-actin. B . MCF7 cells were treated with antimycin A (AM, 1 μM), AICAR (4 mM), or metformin (8 mM) for 1.5 days. Control cells (Con) were treated with the appropriate vehicle for each reagent. Western blotting was performed to detect active phospho-AMPK, cyclin D1, and β-actin. C . MCF7 cells were pretreated with DMSO (vehicle) or the AMPK-specific inhibitor compound C (20 μM) for 1 day and then treated with (Met) or without (Con) metformin for 1.5 days. Western blotting was performed to detect cyclin D1 or the phosphorylated form of the AMPK substrate ACC. β-actin was used as a loading control.
Figure Legend Snippet: Downregulation of cyclin D1 corresponds to activation of AMP-activated protein kinase . A . MCF7 cells were treated with (Met) or without (Con) metformin for 1.5 days and then western blotting was performed to detect active phospho-AMPK, cyclin D1, and β-actin. B . MCF7 cells were treated with antimycin A (AM, 1 μM), AICAR (4 mM), or metformin (8 mM) for 1.5 days. Control cells (Con) were treated with the appropriate vehicle for each reagent. Western blotting was performed to detect active phospho-AMPK, cyclin D1, and β-actin. C . MCF7 cells were pretreated with DMSO (vehicle) or the AMPK-specific inhibitor compound C (20 μM) for 1 day and then treated with (Met) or without (Con) metformin for 1.5 days. Western blotting was performed to detect cyclin D1 or the phosphorylated form of the AMPK substrate ACC. β-actin was used as a loading control.

Techniques Used: Activation Assay, Western Blot

Overexpression of p27 Kip1 in the metformin-resistant MDA-MB-231 cell line leads to metformin sensitivity . A . (Left panel) MCF7 and MDA-MB-231 cells were treated with (Met) and without (Con) metformin (8 mM) for 2 days. Western blotting was used to detect phospho-AMPK, total AMPK, p27 Kip1 , and β-actin. (Right panel) Same as A except that cell extracts were used to detect phospho-Rb (Serine 795), cyclin D1 or β-actin. B . Extracts from untreated MCF7 and MDA-MB-231 cells were used for western blotting of p27 Kip1 , p21 Cip1 , and β-actin. C . MDA-MB-231 cells were stably transfected with a construct encoding p27 Kip1 that was tagged with a protein C epitope to derive the cell line MDA-MB-231WTp27. Extracts from the parental cell line (231) and the stably transfected cell line (231WTp27) were used for western blotting to detect the epitope-tagged p27 Kip1 (anti-Protein C epitope), p27 Kip1 (p27), and β-actin. D . MDA-MB-231 cells or MDA-MB-231WTp27 cells were treated with (dashed lines) or without (solid lines) metformin (8 mM) for up to 3 days. At the indicated time points cell number was determined using a hemocytometer. Each data point represents the mean cell number from 3 independent cultures and error bars represent standard deviation. E . MDA-MB-231 cells were transiently transfected with a construct encoding p21 Cip1 or empty vector. They were then treated with metformin for the indicated time and cell number was determined as describe in D.
Figure Legend Snippet: Overexpression of p27 Kip1 in the metformin-resistant MDA-MB-231 cell line leads to metformin sensitivity . A . (Left panel) MCF7 and MDA-MB-231 cells were treated with (Met) and without (Con) metformin (8 mM) for 2 days. Western blotting was used to detect phospho-AMPK, total AMPK, p27 Kip1 , and β-actin. (Right panel) Same as A except that cell extracts were used to detect phospho-Rb (Serine 795), cyclin D1 or β-actin. B . Extracts from untreated MCF7 and MDA-MB-231 cells were used for western blotting of p27 Kip1 , p21 Cip1 , and β-actin. C . MDA-MB-231 cells were stably transfected with a construct encoding p27 Kip1 that was tagged with a protein C epitope to derive the cell line MDA-MB-231WTp27. Extracts from the parental cell line (231) and the stably transfected cell line (231WTp27) were used for western blotting to detect the epitope-tagged p27 Kip1 (anti-Protein C epitope), p27 Kip1 (p27), and β-actin. D . MDA-MB-231 cells or MDA-MB-231WTp27 cells were treated with (dashed lines) or without (solid lines) metformin (8 mM) for up to 3 days. At the indicated time points cell number was determined using a hemocytometer. Each data point represents the mean cell number from 3 independent cultures and error bars represent standard deviation. E . MDA-MB-231 cells were transiently transfected with a construct encoding p21 Cip1 or empty vector. They were then treated with metformin for the indicated time and cell number was determined as describe in D.

Techniques Used: Over Expression, Multiple Displacement Amplification, Western Blot, Stable Transfection, Transfection, Construct, Standard Deviation, Plasmid Preparation

14) Product Images from "Metformin Inhibits Follicle-Stimulating Hormone (FSH) Action in Human Granulosa Cells: Relevance to Polycystic Ovary Syndrome"

Article Title: Metformin Inhibits Follicle-Stimulating Hormone (FSH) Action in Human Granulosa Cells: Relevance to Polycystic Ovary Syndrome

Journal: The Journal of Clinical Endocrinology and Metabolism

doi: 10.1210/jc.2013-1865

A, Effect of metformin and FSH on aromatase mRNA. Cells were treated for 48 hours with testosterone (as an aromatase substrate) and increasing doses of FSH (black solid bars) with or without metformin (Met) (10 −7 M) (checked bars). Aromatase mRNA was measured by real-time qPCR and expressed as a fold change in expression relative to control values (no treatment) and normalized to L19 mRNA expression. Metformin reduced the FSH-mediated increase in CYP19 mRNA levels at 1 and 5 ng/mL FSH (mean ± SEM, n = 5–7; *, P
Figure Legend Snippet: A, Effect of metformin and FSH on aromatase mRNA. Cells were treated for 48 hours with testosterone (as an aromatase substrate) and increasing doses of FSH (black solid bars) with or without metformin (Met) (10 −7 M) (checked bars). Aromatase mRNA was measured by real-time qPCR and expressed as a fold change in expression relative to control values (no treatment) and normalized to L19 mRNA expression. Metformin reduced the FSH-mediated increase in CYP19 mRNA levels at 1 and 5 ng/mL FSH (mean ± SEM, n = 5–7; *, P

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

Effect of metformin on pCREB. A, Representative Western blot using total protein lysates from cells treated with metformin (10 −7 M) for 24 hours and then exposed to either FSH (5 ng/mL) or Fsk (25μM) with or without metformin for 1 hour. Cells were also treated with AICAR (2 mM) with or without FSH/Fsk for 1 hour before lysis and then blotted with anti–α-tubulin (1:2500), anti-pCREB (1:500), and anti-CREB (1:1000). The anti-pCREB antibody also detects the phosphorylated form of the CREB-related protein, ATF-1 (activating transcription factor). The effect of all treatments on pCREB levels is described in B. Lanes L, ladder; lane 1, control; lane 2, metformin; lane 3, FSH; lane 4, FSH plus metformin; lane 5, Fsk; lane 6, Fsk with or without metformin; lane 7, AICAR; lane 8, AICAR plus FSH; lane 9, AICAR plus Fsk. B, Graph of densitometry analysis of Western blots (mean ± SEM, n = 4). FSH (black bar) significantly increased levels of pCREB (**, P = .016, 1-sample t test), which was significantly reduced by metformin (checked bar) (*, P = .03, unpaired t test, n = 4–7 mean ± SEM). Fsk (gray bar) up-regulated pCREB (*, P = .048, 1-sample t test), and this was not affected by the addition of metformin. AICAR (spotted bar) did not alter basal levels of pCREB, but neither did it reduce the FSH- or Fsk-stimulated increase in pCREB. Abbreviations: Con, control; Met, metformin.
Figure Legend Snippet: Effect of metformin on pCREB. A, Representative Western blot using total protein lysates from cells treated with metformin (10 −7 M) for 24 hours and then exposed to either FSH (5 ng/mL) or Fsk (25μM) with or without metformin for 1 hour. Cells were also treated with AICAR (2 mM) with or without FSH/Fsk for 1 hour before lysis and then blotted with anti–α-tubulin (1:2500), anti-pCREB (1:500), and anti-CREB (1:1000). The anti-pCREB antibody also detects the phosphorylated form of the CREB-related protein, ATF-1 (activating transcription factor). The effect of all treatments on pCREB levels is described in B. Lanes L, ladder; lane 1, control; lane 2, metformin; lane 3, FSH; lane 4, FSH plus metformin; lane 5, Fsk; lane 6, Fsk with or without metformin; lane 7, AICAR; lane 8, AICAR plus FSH; lane 9, AICAR plus Fsk. B, Graph of densitometry analysis of Western blots (mean ± SEM, n = 4). FSH (black bar) significantly increased levels of pCREB (**, P = .016, 1-sample t test), which was significantly reduced by metformin (checked bar) (*, P = .03, unpaired t test, n = 4–7 mean ± SEM). Fsk (gray bar) up-regulated pCREB (*, P = .048, 1-sample t test), and this was not affected by the addition of metformin. AICAR (spotted bar) did not alter basal levels of pCREB, but neither did it reduce the FSH- or Fsk-stimulated increase in pCREB. Abbreviations: Con, control; Met, metformin.

Techniques Used: Western Blot, Lysis

Effect of metformin on CRTC2. Panel A, Representative Western blot showing the effect of FSH or Fsk with time on phosphorylated (cytosolic) and nonphosphorylated (nuclear) CRTC2 levels. Cells were treated with 5 ng/mL FSH for 24 hours, 3 hours, 1 hour, and 30 minutes or 25μM Fsk for 3 hours, 1 hour, and 30 minutes before extraction of total protein lysates. Western blotting was performed with anti-CRTC2 antibody (1:2500), which detects both the phosphorylated (pCRTC2) (open arrowhead) and nonphosphorylated (CRTC2) (solid arrowhead) forms. Exposure to Fsk for 30 minutes and 1 hour significantly increased the expression of nonphosphorylated CRTC2 compared with pCRTC2 and after 1 hour FSH also increased CRTC2 levels. Panel B, Cells were treated as described for Figure 4 A and lysates blotted with anti-CRTC2 and α-tubulin. The levels of CRTC2 (nuclear) were related to α-tubulin, and the effect of all treatments is analyzed and described in panel C. Panel C, Graph of densitometry analysis of Western blot experiments described in panel B. Metformin (checked bars) decreased levels of CRTC2 from basal (*, P = .02, 1-sample t test), indicating increased retention of pCRTC2 in the cytoplasm. Fsk (gray bars) significantly increased levels of CRTC2 (*, P = .03, 1-sample t test) as did FSH (black bars), indicating increased movement of dephosphorylated CRTC2 into the nucleus. Addition of metformin (checked bars) to either FSH/Fsk was unable to alter this. Unlike metformin, AICAR (spotted bars) did not alter basal CRTC2 levels, nor was it able to affect the FSH/Fsk-induced increase in nuclear localization of CRTC2. Abbreviations: A, AICAR; Con, control; L, ladder; Met or M, metformin; α-tub, α-tubulin.
Figure Legend Snippet: Effect of metformin on CRTC2. Panel A, Representative Western blot showing the effect of FSH or Fsk with time on phosphorylated (cytosolic) and nonphosphorylated (nuclear) CRTC2 levels. Cells were treated with 5 ng/mL FSH for 24 hours, 3 hours, 1 hour, and 30 minutes or 25μM Fsk for 3 hours, 1 hour, and 30 minutes before extraction of total protein lysates. Western blotting was performed with anti-CRTC2 antibody (1:2500), which detects both the phosphorylated (pCRTC2) (open arrowhead) and nonphosphorylated (CRTC2) (solid arrowhead) forms. Exposure to Fsk for 30 minutes and 1 hour significantly increased the expression of nonphosphorylated CRTC2 compared with pCRTC2 and after 1 hour FSH also increased CRTC2 levels. Panel B, Cells were treated as described for Figure 4 A and lysates blotted with anti-CRTC2 and α-tubulin. The levels of CRTC2 (nuclear) were related to α-tubulin, and the effect of all treatments is analyzed and described in panel C. Panel C, Graph of densitometry analysis of Western blot experiments described in panel B. Metformin (checked bars) decreased levels of CRTC2 from basal (*, P = .02, 1-sample t test), indicating increased retention of pCRTC2 in the cytoplasm. Fsk (gray bars) significantly increased levels of CRTC2 (*, P = .03, 1-sample t test) as did FSH (black bars), indicating increased movement of dephosphorylated CRTC2 into the nucleus. Addition of metformin (checked bars) to either FSH/Fsk was unable to alter this. Unlike metformin, AICAR (spotted bars) did not alter basal CRTC2 levels, nor was it able to affect the FSH/Fsk-induced increase in nuclear localization of CRTC2. Abbreviations: A, AICAR; Con, control; L, ladder; Met or M, metformin; α-tub, α-tubulin.

Techniques Used: Western Blot, Expressing

Effect of metformin on FSH- and Fsk-stimulated CRE activity. Cells were transfected with the CRE-C6-BL reporter construct and treated as described previously. Luciferase reporter assays demonstrated that metformin (checked bars) significantly reduced CRE activity in the presence of 1 and 5 ng/mL FSH (black bars) (*, P
Figure Legend Snippet: Effect of metformin on FSH- and Fsk-stimulated CRE activity. Cells were transfected with the CRE-C6-BL reporter construct and treated as described previously. Luciferase reporter assays demonstrated that metformin (checked bars) significantly reduced CRE activity in the presence of 1 and 5 ng/mL FSH (black bars) (*, P

Techniques Used: Activity Assay, Transfection, Construct, Luciferase

A, Effect of metformin (Met) and FSH on FSHR mRNA. Cells were treated as described in Figure 1 A, and FSHR mRNA was measured by real-time qPCR and expressed as described previously. Metformin (checked bars) reduced basal mRNA levels of FSHR (mean ± SEM, n = 5–7; **, P
Figure Legend Snippet: A, Effect of metformin (Met) and FSH on FSHR mRNA. Cells were treated as described in Figure 1 A, and FSHR mRNA was measured by real-time qPCR and expressed as described previously. Metformin (checked bars) reduced basal mRNA levels of FSHR (mean ± SEM, n = 5–7; **, P

Techniques Used: Real-time Polymerase Chain Reaction

Hypothetical mechanism of metformin's action on FSH activity in granulosa cells. The FSHR is a G protein-coupled receptor, which upon binding by FSH activates adenylyl cyclase (Ad Cy) to produce cAMP from ATP, which then acts to activate PKA downstream. Fsk directly stimulates adenylyl cyclase activity to greatly increase intracellular cAMP levels. Activation of PKA results in phosphorylation of CREB (pCREB), which allows it to bind to a CRE on PII of the aromatase gene ( CYP19 ). In addition, activated PKA dephosphorylates CRTC2, allowing it to enter into the nucleus and bind to CRE in a coactivator complex with CBP and pCREB, resulting in CYP19 transcription. We have shown that metformin reduces the levels of FSHR, which then reduces FSH activity as measured by aromatase expression and E 2 production. It does this without altering intracellular cAMP levels. Metformin decreases levels of pCREB, which may inhibit CYP19 transcription by disrupting the pCREB-CRTC2-CBP coactivator complex that stimulates CRE activation on PII. This is supported by the reduction in FSH-stimulated CRE activity brought about by metformin. This occurs independently of AMPK activation. Metformin is also able to prevent the dephosphorylation of basal CRTC2, thus retaining it in the cytosol. It is, however, unable to prevent the cAMP-driven dephosphorylation and nuclear translocation of CRTC2 induced by exposure to either FSH or Fsk.
Figure Legend Snippet: Hypothetical mechanism of metformin's action on FSH activity in granulosa cells. The FSHR is a G protein-coupled receptor, which upon binding by FSH activates adenylyl cyclase (Ad Cy) to produce cAMP from ATP, which then acts to activate PKA downstream. Fsk directly stimulates adenylyl cyclase activity to greatly increase intracellular cAMP levels. Activation of PKA results in phosphorylation of CREB (pCREB), which allows it to bind to a CRE on PII of the aromatase gene ( CYP19 ). In addition, activated PKA dephosphorylates CRTC2, allowing it to enter into the nucleus and bind to CRE in a coactivator complex with CBP and pCREB, resulting in CYP19 transcription. We have shown that metformin reduces the levels of FSHR, which then reduces FSH activity as measured by aromatase expression and E 2 production. It does this without altering intracellular cAMP levels. Metformin decreases levels of pCREB, which may inhibit CYP19 transcription by disrupting the pCREB-CRTC2-CBP coactivator complex that stimulates CRE activation on PII. This is supported by the reduction in FSH-stimulated CRE activity brought about by metformin. This occurs independently of AMPK activation. Metformin is also able to prevent the dephosphorylation of basal CRTC2, thus retaining it in the cytosol. It is, however, unable to prevent the cAMP-driven dephosphorylation and nuclear translocation of CRTC2 induced by exposure to either FSH or Fsk.

Techniques Used: Activity Assay, Binding Assay, Activation Assay, Expressing, De-Phosphorylation Assay, Translocation Assay

15) Product Images from "Metformin attenuates angiotensin II‐induced TGFβ1 expression by targeting hepatocyte nuclear factor‐4‐α) Metformin attenuates angiotensin II‐induced TGFβ1 expression by targeting hepatocyte nuclear factor‐4‐α"

Article Title: Metformin attenuates angiotensin II‐induced TGFβ1 expression by targeting hepatocyte nuclear factor‐4‐α) Metformin attenuates angiotensin II‐induced TGFβ1 expression by targeting hepatocyte nuclear factor‐4‐α

Journal: British Journal of Pharmacology

doi: 10.1111/bph.13753

Metformin inhibits AngII‐induced TGFβ1 production in cardiac fibroblasts (CFs) via AMPK activation. CFs were pretreated with Compound C (1 μM) for 0.5 h, and metformin (1 mM) was applied for an additional
Figure Legend Snippet: Metformin inhibits AngII‐induced TGFβ1 production in cardiac fibroblasts (CFs) via AMPK activation. CFs were pretreated with Compound C (1 μM) for 0.5 h, and metformin (1 mM) was applied for an additional

Techniques Used: Activation Assay

Metformin inhibits AngII‐induced HNF4α expression, TGFβ1 expression and cardiac fibrosis in an AMPK‐dependent manner. (A–B) Western blot analysis of HNF4α expression in CFs. (A) CFs were pretreated with
Figure Legend Snippet: Metformin inhibits AngII‐induced HNF4α expression, TGFβ1 expression and cardiac fibrosis in an AMPK‐dependent manner. (A–B) Western blot analysis of HNF4α expression in CFs. (A) CFs were pretreated with

Techniques Used: Expressing, Western Blot

Metformin inhibits AngII‐induced HNF4α and TGFβ1 expression. (A) Western blot analysis of HNF4α expression in CFs. Met: metformin, n = 6. (B) MEFs were transfected with the reporter plasmid carrying the
Figure Legend Snippet: Metformin inhibits AngII‐induced HNF4α and TGFβ1 expression. (A) Western blot analysis of HNF4α expression in CFs. Met: metformin, n = 6. (B) MEFs were transfected with the reporter plasmid carrying the

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

Working model showing how metformin inhibits TGFβ1 expression and cardiac fibrosis. AngII induces TGFβ1 expression by increasing HNF4α protein expression and binding activity. Metformin targets the HNF4α protein and then
Figure Legend Snippet: Working model showing how metformin inhibits TGFβ1 expression and cardiac fibrosis. AngII induces TGFβ1 expression by increasing HNF4α protein expression and binding activity. Metformin targets the HNF4α protein and then

Techniques Used: Expressing, Binding Assay, Activity Assay

16) Product Images from "Uncoupling FoxO3A mitochondrial and nuclear functions in cancer cells undergoing metabolic stress and chemotherapy"

Article Title: Uncoupling FoxO3A mitochondrial and nuclear functions in cancer cells undergoing metabolic stress and chemotherapy

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0336-0

FoxO3A represents a survival factor in metabolically stressed cancer cells. Normal cells and tissues under metabolic stress require only the AMPK signal on S30 to direct FoxO3A into the mitochondria. It seems that ERK involvement in FoxO3A mitochondrial localization is exclusive to tumor cells, which reveals a critical difference between normal and cancer cells that could be exploited for cancer therapeutics. In metabolically stressed cancer cells, FoxO3A is recruited to the mitochondria through activation of MEK/ERK and AMPK, which phosphorylate serine 12 and 30, respectively, on FoxO3A N-terminal domain. Subsequently, FoxO3A is imported and cleaved to reach mitochondrial DNA, where it activates expression of the mitochondrial genome to support mitochondrial metabolism. In cancer cells treated with chemotherapeutic agents, accumulation of FoxO3A into the mitochondria promoted survival in a MEK/ERK-dependent manner, while mitochondrial FoxO3A was required for apoptosis induction by metformin
Figure Legend Snippet: FoxO3A represents a survival factor in metabolically stressed cancer cells. Normal cells and tissues under metabolic stress require only the AMPK signal on S30 to direct FoxO3A into the mitochondria. It seems that ERK involvement in FoxO3A mitochondrial localization is exclusive to tumor cells, which reveals a critical difference between normal and cancer cells that could be exploited for cancer therapeutics. In metabolically stressed cancer cells, FoxO3A is recruited to the mitochondria through activation of MEK/ERK and AMPK, which phosphorylate serine 12 and 30, respectively, on FoxO3A N-terminal domain. Subsequently, FoxO3A is imported and cleaved to reach mitochondrial DNA, where it activates expression of the mitochondrial genome to support mitochondrial metabolism. In cancer cells treated with chemotherapeutic agents, accumulation of FoxO3A into the mitochondria promoted survival in a MEK/ERK-dependent manner, while mitochondrial FoxO3A was required for apoptosis induction by metformin

Techniques Used: Metabolic Labelling, Activation Assay, Expressing

mtFoxO3A is involved in cancer cell response to metabolic stress. a HCT116-FoxO3A +/+ and HCT116-FoxO3A −/− cells were subjected to different treatments: glucose restriction (LG, 0.75 mM glucose, 24 h), metformin (MET, 10 μM, 72 h), cisplatin (CDDP, 30 μM, 48 h), irinotecan (CPT-11, 30 μM, 24 h), 5-fluorouracil (5-FU, 2 μM, 24 h) and etoposide (VP-13, 40 μM, 24 h). Relative cell viability and relative cell death were calculated. b Correlation between LG-resistance (days) and mitochondrial FoxO3A (mtFoxO3A) protein levels in different human cell lines (HCT116 and HT29 colorectal cancer cells, HEK293 embryonic kidney cell, DU145 prostate cancer cells, A549 lung cancer cells, MDA-MB-468 breast cancer cells and OVCAR3 ovarian cancer cells). a.u. arbitrary units. c HCT116-FoxO3A −/− cells were transfected with the indicated plasmids (48 h) and subjected to LG (24 h). Upper panel: relative cell viability and relative cell death. Lower panel: immunoblot analysis of total proteins. β-actin: loading control. d Transcription analysis of selected mitochondrial ( ND6 and COX1 ) and nuclear ( BIM ) genes by RT-PCR in HCT116-FoxO3A −/− cells transfected with the indicated plasmids (48 h) and subjected to LG (24 h). e HCT116-FoxO3A −/− cells, transfected with the indicated plasmids (48 h), were subjected to metabolic stress with 2-DG (1 mM, 6 h). The graph reflects the quantification of tetramethylrhodamine ethyl ester (TMRE) fluorescence of active mitochondria in transfected cells. f Clonogenic assay on HCT116-FoxO3A +/+ cells cultured in LG (24 h) and treated with increasing concentrations of trametinib and/or compound C, as indicated, for 24 h. Cell growth percent inhibition at each drug concentration is presented. g Left panel: immunoblot analysis of total and mitochondrial proteins isolated from tumors ( n ≥ 7 for each group) derived from HCT116-xenografted nude mice subjected to 2-DG treatment (100 mg/kg, 6 days). β-actin and HSP60 were used as total and mitochondrial loading control, respectively. Right panel: densitometric analysis of full-length and cleaved FoxO3A normalized against the mitochondrial loading control and the results of the densitometric analysis of the phosphorylated-AMPK and ERK normalized against total AMPK and ERK, respectively, and the loading control. Data are presented as mean ± SEM and significance was calculated with Student’s t test; * p
Figure Legend Snippet: mtFoxO3A is involved in cancer cell response to metabolic stress. a HCT116-FoxO3A +/+ and HCT116-FoxO3A −/− cells were subjected to different treatments: glucose restriction (LG, 0.75 mM glucose, 24 h), metformin (MET, 10 μM, 72 h), cisplatin (CDDP, 30 μM, 48 h), irinotecan (CPT-11, 30 μM, 24 h), 5-fluorouracil (5-FU, 2 μM, 24 h) and etoposide (VP-13, 40 μM, 24 h). Relative cell viability and relative cell death were calculated. b Correlation between LG-resistance (days) and mitochondrial FoxO3A (mtFoxO3A) protein levels in different human cell lines (HCT116 and HT29 colorectal cancer cells, HEK293 embryonic kidney cell, DU145 prostate cancer cells, A549 lung cancer cells, MDA-MB-468 breast cancer cells and OVCAR3 ovarian cancer cells). a.u. arbitrary units. c HCT116-FoxO3A −/− cells were transfected with the indicated plasmids (48 h) and subjected to LG (24 h). Upper panel: relative cell viability and relative cell death. Lower panel: immunoblot analysis of total proteins. β-actin: loading control. d Transcription analysis of selected mitochondrial ( ND6 and COX1 ) and nuclear ( BIM ) genes by RT-PCR in HCT116-FoxO3A −/− cells transfected with the indicated plasmids (48 h) and subjected to LG (24 h). e HCT116-FoxO3A −/− cells, transfected with the indicated plasmids (48 h), were subjected to metabolic stress with 2-DG (1 mM, 6 h). The graph reflects the quantification of tetramethylrhodamine ethyl ester (TMRE) fluorescence of active mitochondria in transfected cells. f Clonogenic assay on HCT116-FoxO3A +/+ cells cultured in LG (24 h) and treated with increasing concentrations of trametinib and/or compound C, as indicated, for 24 h. Cell growth percent inhibition at each drug concentration is presented. g Left panel: immunoblot analysis of total and mitochondrial proteins isolated from tumors ( n ≥ 7 for each group) derived from HCT116-xenografted nude mice subjected to 2-DG treatment (100 mg/kg, 6 days). β-actin and HSP60 were used as total and mitochondrial loading control, respectively. Right panel: densitometric analysis of full-length and cleaved FoxO3A normalized against the mitochondrial loading control and the results of the densitometric analysis of the phosphorylated-AMPK and ERK normalized against total AMPK and ERK, respectively, and the loading control. Data are presented as mean ± SEM and significance was calculated with Student’s t test; * p

Techniques Used: Cycling Probe Technology, Multiple Displacement Amplification, Transfection, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Clonogenic Assay, Cell Culture, Inhibition, Concentration Assay, Isolation, Derivative Assay, Mouse Assay

mtFoxO3A is involved in cancer cell response to chemotherapeutic agents. a – c HCT116-FoxO3A −/− cells were transfected with the indicated plasmids for 48 h and then treated with irinotecan (CPT-11, 30 μM, 24 h). a Upper panel: relative cell viability and relative cell death. Lower panel: immunoblot analysis of total proteins. β-actin: loading control. b Transcription analysis of selected mitochondrial ( ND6 and COX1 ) and nuclear ( BIM ) genes by RT-PCR. c HCT116-FoxO3A −/− cells were transfected with the indicated plasmids for 48 h and then treated with irinotecan (CPT-11, 30 μM, 24 h). Relative cell viability and relative cell death were calculated. d Left panel: immunoblot analysis of total and mitochondrial proteins isolated from tumors ( n ≥ 7 for each group) derived from HCT116-xenografted nude mice subjected to cisplatin treatment (CDDP, 2 mg/kg, 6 days). β-actin and HSP60 were used as total lysate and mitochondrial fraction controls, respectively. Right panel: densitometric analysis of full-length and cleaved FoxO3A normalized against the mitochondrial fractionation loading control and the results of the densitometric analysis of the phosphorylated forms of AMPK and ERK normalized against total AMPK and ERK, respectively, and the loading control. e Clonogenic assay on HCT116-FoxO3A +/+ cells treated with increasing concentrations of trametinib (24 h) and/or irinotecan (24 h), as indicated. f Immunoblot analysis of total proteins isolated from HCT116-FoxO3A +/+ cells upon metformin treatment (MET, 10 μM, 72 h). β-actin: loading control. g HCT116-FoxO3A −/− cells were transfected with the indicated plasmids for 48 h and then treated with metformin (MET, 10 μM, 72 h). Relative cell viability and relative cell death were calculated. h Clonogenic assay on HCT116-FoxO3A +/+ cells treated with increasing concentrations of metformin (24 h) and/or irinotecan (24 h), as indicated. e , h Cell growth percent inhibition at each drug concentration is presented. The data presented are the mean of at least three independent experiments. Where applicable, data are presented as mean ± SEM and significance was calculated with Student’s t test; * p
Figure Legend Snippet: mtFoxO3A is involved in cancer cell response to chemotherapeutic agents. a – c HCT116-FoxO3A −/− cells were transfected with the indicated plasmids for 48 h and then treated with irinotecan (CPT-11, 30 μM, 24 h). a Upper panel: relative cell viability and relative cell death. Lower panel: immunoblot analysis of total proteins. β-actin: loading control. b Transcription analysis of selected mitochondrial ( ND6 and COX1 ) and nuclear ( BIM ) genes by RT-PCR. c HCT116-FoxO3A −/− cells were transfected with the indicated plasmids for 48 h and then treated with irinotecan (CPT-11, 30 μM, 24 h). Relative cell viability and relative cell death were calculated. d Left panel: immunoblot analysis of total and mitochondrial proteins isolated from tumors ( n ≥ 7 for each group) derived from HCT116-xenografted nude mice subjected to cisplatin treatment (CDDP, 2 mg/kg, 6 days). β-actin and HSP60 were used as total lysate and mitochondrial fraction controls, respectively. Right panel: densitometric analysis of full-length and cleaved FoxO3A normalized against the mitochondrial fractionation loading control and the results of the densitometric analysis of the phosphorylated forms of AMPK and ERK normalized against total AMPK and ERK, respectively, and the loading control. e Clonogenic assay on HCT116-FoxO3A +/+ cells treated with increasing concentrations of trametinib (24 h) and/or irinotecan (24 h), as indicated. f Immunoblot analysis of total proteins isolated from HCT116-FoxO3A +/+ cells upon metformin treatment (MET, 10 μM, 72 h). β-actin: loading control. g HCT116-FoxO3A −/− cells were transfected with the indicated plasmids for 48 h and then treated with metformin (MET, 10 μM, 72 h). Relative cell viability and relative cell death were calculated. h Clonogenic assay on HCT116-FoxO3A +/+ cells treated with increasing concentrations of metformin (24 h) and/or irinotecan (24 h), as indicated. e , h Cell growth percent inhibition at each drug concentration is presented. The data presented are the mean of at least three independent experiments. Where applicable, data are presented as mean ± SEM and significance was calculated with Student’s t test; * p

Techniques Used: Transfection, Cycling Probe Technology, Reverse Transcription Polymerase Chain Reaction, Isolation, Derivative Assay, Mouse Assay, Fractionation, Clonogenic Assay, Inhibition, Concentration Assay

17) Product Images from "Effect of the combination of metformin and fenofibrate on glucose homeostasis in diabetic Goto-Kakizaki rats"

Article Title: Effect of the combination of metformin and fenofibrate on glucose homeostasis in diabetic Goto-Kakizaki rats

Journal: Experimental & Molecular Medicine

doi: 10.1038/emm.2013.58

The sensitivity to an exogenous GLP-1 analog. The blood glucose levels ( a ) and AUCs ( b ), and the blood insulin levels ( c ) and iAUCs ( d ). Filled circles and black bars: Ctrl, control group; open circles and white bars: MA, metformin alone group; filled squares and upward dashed bars: FA, fenofibrate alone group; open squares and downward dashed bars: MF, metformin plus fenofibrate group. * P
Figure Legend Snippet: The sensitivity to an exogenous GLP-1 analog. The blood glucose levels ( a ) and AUCs ( b ), and the blood insulin levels ( c ) and iAUCs ( d ). Filled circles and black bars: Ctrl, control group; open circles and white bars: MA, metformin alone group; filled squares and upward dashed bars: FA, fenofibrate alone group; open squares and downward dashed bars: MF, metformin plus fenofibrate group. * P

Techniques Used:

Immunohistochemical staining for insulin ( a ), percent beta cell area in the pancreas ( b ), immunofluorescence staining for the GLP-1 receptor (green) and insulin (red) ( c ), and ratio of the GLP-1 receptor-positive area to the insulin-positive area ( d ). Scale bars, 50 μm. Black bars: Ctrl, control group; white bars: MA, metformin alone group; upward dashed bars: FA, fenofibrate alone group; downward dashed bars: MF, metformin plus fenofibrate group. *** P
Figure Legend Snippet: Immunohistochemical staining for insulin ( a ), percent beta cell area in the pancreas ( b ), immunofluorescence staining for the GLP-1 receptor (green) and insulin (red) ( c ), and ratio of the GLP-1 receptor-positive area to the insulin-positive area ( d ). Scale bars, 50 μm. Black bars: Ctrl, control group; white bars: MA, metformin alone group; upward dashed bars: FA, fenofibrate alone group; downward dashed bars: MF, metformin plus fenofibrate group. *** P

Techniques Used: Immunohistochemistry, Staining, Immunofluorescence

Tracking data for body weights ( a ), food intake ( b ), random blood glucose concentrations ( c ) and plasma levels of total cholesterol and triglycerides ( d ) after 4 weeks of treatment. Filled circles and black bars: Ctrl, control group; open circles and white bars: MA, metformin alone group; filled squares and upward dashed bars: FA, fenofibrate alone group; open squares and downward dashed bars: MF, metformin plus fenofibrate group ( a – c ). Black bars: total cholesterol; white bars: triglycerides ( d ). * P
Figure Legend Snippet: Tracking data for body weights ( a ), food intake ( b ), random blood glucose concentrations ( c ) and plasma levels of total cholesterol and triglycerides ( d ) after 4 weeks of treatment. Filled circles and black bars: Ctrl, control group; open circles and white bars: MA, metformin alone group; filled squares and upward dashed bars: FA, fenofibrate alone group; open squares and downward dashed bars: MF, metformin plus fenofibrate group ( a – c ). Black bars: total cholesterol; white bars: triglycerides ( d ). * P

Techniques Used:

The oral glucose tolerance test results. The blood glucose levels ( a ) and AUCs ( b ). Filled circles and black bars: Ctrl, control group; open circles and white bars: MA, metformin alone group; filled squares and upward dashed bars: FA, fenofibrate alone group; open squares and downward dashed bars: MF, metformin plus fenofibrate group. * P
Figure Legend Snippet: The oral glucose tolerance test results. The blood glucose levels ( a ) and AUCs ( b ). Filled circles and black bars: Ctrl, control group; open circles and white bars: MA, metformin alone group; filled squares and upward dashed bars: FA, fenofibrate alone group; open squares and downward dashed bars: MF, metformin plus fenofibrate group. * P

Techniques Used:

18) Product Images from "Anaphase Promoting Complex activity mediates clinical responsiveness to recurrent canine lymphoma"

Article Title: Anaphase Promoting Complex activity mediates clinical responsiveness to recurrent canine lymphoma

Journal: bioRxiv

doi: 10.1101/2020.05.26.115337

ABC transporters do not play a critical role in the development of canine MDR lymphoma. A. Differential gene expression of the family of ABC transporters on the canine microarray was determined for each MDR canine sample. Two samples from canine 1 were evaluated by microarray. B. ABC transporter gene expression changes were determined for the 2 canines (2 and 4) that were treated with metformin and analyzed by microarray. The 2 values for each gene were averaged and the SEM was plotted.
Figure Legend Snippet: ABC transporters do not play a critical role in the development of canine MDR lymphoma. A. Differential gene expression of the family of ABC transporters on the canine microarray was determined for each MDR canine sample. Two samples from canine 1 were evaluated by microarray. B. ABC transporter gene expression changes were determined for the 2 canines (2 and 4) that were treated with metformin and analyzed by microarray. The 2 values for each gene were averaged and the SEM was plotted.

Techniques Used: Expressing, Microarray

Changes in APC target gene expression correlate with altered clinical responses. A. Cervical lymph node FNA samples were obtained from canine 4 before metformin treatment when the tumor was chemoresistant, during remission following metformin treatment, and then after relapse, with differential gene expression compared to normal skin samples using canine microarray. A Venn diagram was used to determine similarities in genes overexpressed 3 FC in the tumor, 2 FC down-regulated following remission, then 3 FC upregulated following relapse. This is predicted to identify genes specifically involved in the MDR phenotype. From this analysis, 27 genes were found to be overexpressed in chemoresistant cells, reduced upon remission, then overexpressed once again when remission failed. B. A STRING analysis indicates that the 27 genes were highly interconnected, with the majority of the genes encoding APC substrates (red nodes, circled in black), and genes encoding proteins required for chromosome maintenance (green and blue nodes, APC substrates circled in black). C. Differential gene expression was determined for the APC substrates identified in the array (shown in Figure 3 ) in chemoresistant tumors and following remission and relapse, compared to control skin samples. D. Microarray results were validated by qRT-PCR of original FNA aspirate samples for DLGAP5 (encoding HURP) and PTTG1 (encoding Securin) in canine subject 4 at study entry and remission.
Figure Legend Snippet: Changes in APC target gene expression correlate with altered clinical responses. A. Cervical lymph node FNA samples were obtained from canine 4 before metformin treatment when the tumor was chemoresistant, during remission following metformin treatment, and then after relapse, with differential gene expression compared to normal skin samples using canine microarray. A Venn diagram was used to determine similarities in genes overexpressed 3 FC in the tumor, 2 FC down-regulated following remission, then 3 FC upregulated following relapse. This is predicted to identify genes specifically involved in the MDR phenotype. From this analysis, 27 genes were found to be overexpressed in chemoresistant cells, reduced upon remission, then overexpressed once again when remission failed. B. A STRING analysis indicates that the 27 genes were highly interconnected, with the majority of the genes encoding APC substrates (red nodes, circled in black), and genes encoding proteins required for chromosome maintenance (green and blue nodes, APC substrates circled in black). C. Differential gene expression was determined for the APC substrates identified in the array (shown in Figure 3 ) in chemoresistant tumors and following remission and relapse, compared to control skin samples. D. Microarray results were validated by qRT-PCR of original FNA aspirate samples for DLGAP5 (encoding HURP) and PTTG1 (encoding Securin) in canine subject 4 at study entry and remission.

Techniques Used: Expressing, Microarray, Quantitative RT-PCR

Changes in MDR protein biomarker levels within tumor samples from canine subjects before and after oral metformin addition. A. Protein lysates were prepared from canines 2, 6, 7 and 8 for analysis by Western blotting using antibodies against MDR-1 and tubulin. The control was derived from skin samples from canine 2. Canines 2 and 6 were recruited with drug resistant lymphoma, while canines 7 and 8 were drug sensitive. Tumor samples were obtained by fine needle aspirates (FNAs) following 1 and 2 weeks of metformin treatment from canine 2. B. Lysates prepared from skin and tumor samples obtained from drug resistant canine 3, before and after metformin therapy, were analyzed with antibodies against the MDR markers shown, with tubulin serving as the load control. C. and D. Protein lysates prepared from skin and tumor samples from drug resistant canines 4 and 5, before and after metformin treatment, were assessed using antibodies against MDR-1, with Ponceau S representing relative load controls.
Figure Legend Snippet: Changes in MDR protein biomarker levels within tumor samples from canine subjects before and after oral metformin addition. A. Protein lysates were prepared from canines 2, 6, 7 and 8 for analysis by Western blotting using antibodies against MDR-1 and tubulin. The control was derived from skin samples from canine 2. Canines 2 and 6 were recruited with drug resistant lymphoma, while canines 7 and 8 were drug sensitive. Tumor samples were obtained by fine needle aspirates (FNAs) following 1 and 2 weeks of metformin treatment from canine 2. B. Lysates prepared from skin and tumor samples obtained from drug resistant canine 3, before and after metformin therapy, were analyzed with antibodies against the MDR markers shown, with tubulin serving as the load control. C. and D. Protein lysates prepared from skin and tumor samples from drug resistant canines 4 and 5, before and after metformin treatment, were assessed using antibodies against MDR-1, with Ponceau S representing relative load controls.

Techniques Used: Biomarker Assay, Western Blot, Derivative Assay

19) Product Images from "Nuclear respiratory factor 1 and endurance exercise promote human telomere transcription"

Article Title: Nuclear respiratory factor 1 and endurance exercise promote human telomere transcription

Journal: Science Advances

doi: 10.1126/sciadv.1600031

AMPK activation in human myotubes induces NRF1-dependent increase in TERRA levels. ( A ) TERRA-FISH (green) combined with telomeric DNA FISH (red) in myotubes. Blue, DAPI. Scale bar, 5 μm. ( B ) Quantification of (A) on 25 nuclei. ( C ) ACC phosphorylation in myotubes treated with either AICAR, metformin, or phenformin. ( D ) qRT-PCR analysis of TERRA levels in treated myotubes normalized to β 2M cDNA and to untreated cells. Error bars indicate SD ( n = 4). ( E ) Western blot analysis of NRF1 knockdown in myotubes. ( F ) qRT-PCR analysis of TERRA in siNRF1-treated myotubes and upon phenformin treatment. Values were normalized to β 2M cDNA and to siLuci-treated cells without phenformin. Error bars indicate SD ( n = 3). ( G ) Unified theory of aging ( 34 ) revisited with data from this study (green). See text for details.
Figure Legend Snippet: AMPK activation in human myotubes induces NRF1-dependent increase in TERRA levels. ( A ) TERRA-FISH (green) combined with telomeric DNA FISH (red) in myotubes. Blue, DAPI. Scale bar, 5 μm. ( B ) Quantification of (A) on 25 nuclei. ( C ) ACC phosphorylation in myotubes treated with either AICAR, metformin, or phenformin. ( D ) qRT-PCR analysis of TERRA levels in treated myotubes normalized to β 2M cDNA and to untreated cells. Error bars indicate SD ( n = 4). ( E ) Western blot analysis of NRF1 knockdown in myotubes. ( F ) qRT-PCR analysis of TERRA in siNRF1-treated myotubes and upon phenformin treatment. Values were normalized to β 2M cDNA and to siLuci-treated cells without phenformin. Error bars indicate SD ( n = 3). ( G ) Unified theory of aging ( 34 ) revisited with data from this study (green). See text for details.

Techniques Used: Activation Assay, Fluorescence In Situ Hybridization, Quantitative RT-PCR, Western Blot

20) Product Images from "Simultaneous Induction of Glycolysis and Oxidative Phosphorylation during Activation of Hepatic Stellate Cells Reveals Novel Mitochondrial Targets to Treat Liver Fibrosis"

Article Title: Simultaneous Induction of Glycolysis and Oxidative Phosphorylation during Activation of Hepatic Stellate Cells Reveals Novel Mitochondrial Targets to Treat Liver Fibrosis

Journal: Cells

doi: 10.3390/cells9112456

Glycolysis, glutaminolysis and OXPHOS are independently necessary for maintaining HSC activated phenotype. Primary human activated HSCs were treated with glycolysis inhibitor 2DG (1 mmol/L), glutaminase inhibitor CB-839 (5 µmol/L) and complex I of ETC inhibitor, metformin (2 mmol/L) for 72 h. ( A ) The mRNA of aSMA ( ACTA2 ) and collagen I ( COL1A1 ), ( B ) real-time proliferation (arrow indicates the start of the treatment) and bar graph summarizing the confluency percentage after 3 days of treatment calculated using an automated cell imager and corresponding software (Incucyte, Zoom); n = 3, mean ± SEM * p
Figure Legend Snippet: Glycolysis, glutaminolysis and OXPHOS are independently necessary for maintaining HSC activated phenotype. Primary human activated HSCs were treated with glycolysis inhibitor 2DG (1 mmol/L), glutaminase inhibitor CB-839 (5 µmol/L) and complex I of ETC inhibitor, metformin (2 mmol/L) for 72 h. ( A ) The mRNA of aSMA ( ACTA2 ) and collagen I ( COL1A1 ), ( B ) real-time proliferation (arrow indicates the start of the treatment) and bar graph summarizing the confluency percentage after 3 days of treatment calculated using an automated cell imager and corresponding software (Incucyte, Zoom); n = 3, mean ± SEM * p

Techniques Used: Software

21) Product Images from "Metformin Inhibits Expression and Secretion of PEDF in Adipocyte and Hepatocyte via Promoting AMPK Phosphorylation"

Article Title: Metformin Inhibits Expression and Secretion of PEDF in Adipocyte and Hepatocyte via Promoting AMPK Phosphorylation

Journal: Mediators of Inflammation

doi: 10.1155/2013/429207

The effects of metformin on the secretion and expression of PEDF during IR improvement in vitro. (a) IR models of 3T3-L1 cell and HepG2 cell were treated with metformin (0.01, 0.1, and 1 mM) for 24 h, and 2-NBDG uptake was examined. Finally, 0.1 mM metformin was chosen for later experiments. (b) Supernatants of the cells were collected, and PEDF concentrations were determined by ELISA. (c) RT-PCR assay results of PEDF mRNA levels. (d) Western blot analysis of PEDF protein expression and phosphorylation of AMPK. The histogram represents mean ± SD of the densitometric scans for protein bands from three experiments, normalized by comparison with β -actin and expressed as a percentage of control. NC: normal control; IR: insulin resistance; MET: metformin; iA: inhibitor of AMPK. * P
Figure Legend Snippet: The effects of metformin on the secretion and expression of PEDF during IR improvement in vitro. (a) IR models of 3T3-L1 cell and HepG2 cell were treated with metformin (0.01, 0.1, and 1 mM) for 24 h, and 2-NBDG uptake was examined. Finally, 0.1 mM metformin was chosen for later experiments. (b) Supernatants of the cells were collected, and PEDF concentrations were determined by ELISA. (c) RT-PCR assay results of PEDF mRNA levels. (d) Western blot analysis of PEDF protein expression and phosphorylation of AMPK. The histogram represents mean ± SD of the densitometric scans for protein bands from three experiments, normalized by comparison with β -actin and expressed as a percentage of control. NC: normal control; IR: insulin resistance; MET: metformin; iA: inhibitor of AMPK. * P

Techniques Used: Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, IA

The mRNA expression of the key enzymes regulating gluconeogenesis and fatty acid synthesis in the liver. SD rats were fed a normal chow diet (NC group, n = 6) or a high fat diet ( n = 12) for 15 weeks; then the high-fat-diet rats were gavaged saline (HF group, n = 6) or metformin 400 mg/kg/d (MET group, n = 6) for 4 weeks. * P
Figure Legend Snippet: The mRNA expression of the key enzymes regulating gluconeogenesis and fatty acid synthesis in the liver. SD rats were fed a normal chow diet (NC group, n = 6) or a high fat diet ( n = 12) for 15 weeks; then the high-fat-diet rats were gavaged saline (HF group, n = 6) or metformin 400 mg/kg/d (MET group, n = 6) for 4 weeks. * P

Techniques Used: Expressing

Effects of metformin on the serum PEDF levels and PEDF expression in the adipose tissue and liver of the obese rats. SD rats were fed a normal chow diet (NC group, n = 6) or a high fat diet ( n = 12) for 15 weeks; then the high-fat-diet rats were gavaged saline (HF group, n = 6) or metformin 400 mg/kg/d (MET group, n = 6) for 4 weeks. At the end of the 19th week, hyperinsulinemic-euglycemic clamp were performed, and the serum, liver, and adipose tissues were collected. (a) Error bar charts showed the serum PEDF concentrations in different groups; (b) Scatter plots showed a negative correlation between serum PEDF levels and insulin sensitivity; (c) RT-PCR assay results of PEDF mRNA levels in WAT and liver; (d) Western blot analysis of PEDF protein expression and phosphorylation of AMPK. The histogram represents mean ± SD of the densitomeric scans for protein bands from three experiments, normalized by comparison with β -actin and expressed as a percentage of control. * P
Figure Legend Snippet: Effects of metformin on the serum PEDF levels and PEDF expression in the adipose tissue and liver of the obese rats. SD rats were fed a normal chow diet (NC group, n = 6) or a high fat diet ( n = 12) for 15 weeks; then the high-fat-diet rats were gavaged saline (HF group, n = 6) or metformin 400 mg/kg/d (MET group, n = 6) for 4 weeks. At the end of the 19th week, hyperinsulinemic-euglycemic clamp were performed, and the serum, liver, and adipose tissues were collected. (a) Error bar charts showed the serum PEDF concentrations in different groups; (b) Scatter plots showed a negative correlation between serum PEDF levels and insulin sensitivity; (c) RT-PCR assay results of PEDF mRNA levels in WAT and liver; (d) Western blot analysis of PEDF protein expression and phosphorylation of AMPK. The histogram represents mean ± SD of the densitomeric scans for protein bands from three experiments, normalized by comparison with β -actin and expressed as a percentage of control. * P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

22) Product Images from "Metformin inhibits growth of lung adenocarcinoma cells by inducing apoptosis via the mitochondria-mediated pathway"

Article Title: Metformin inhibits growth of lung adenocarcinoma cells by inducing apoptosis via the mitochondria-mediated pathway

Journal: Oncology Letters

doi: 10.3892/ol.2015.3450

Effects of metformin on the proliferation of A549 cells. (A) Metformin inhibited the proliferation of the cells in a dose- and time-dependent manner. The A549 cells were exposed to increasing doses of metformin for 24, 48 and 72 h respectively, and cell
Figure Legend Snippet: Effects of metformin on the proliferation of A549 cells. (A) Metformin inhibited the proliferation of the cells in a dose- and time-dependent manner. The A549 cells were exposed to increasing doses of metformin for 24, 48 and 72 h respectively, and cell

Techniques Used:

Metformin induces apoptosis of A549 cells. (A) A549 cells were treated with 10 mM metformin for 48 h. Images of the cells were captured under a light microscope, or the cells were stained with Hoechst 33342 and visualized under a fluorescence microscope.
Figure Legend Snippet: Metformin induces apoptosis of A549 cells. (A) A549 cells were treated with 10 mM metformin for 48 h. Images of the cells were captured under a light microscope, or the cells were stained with Hoechst 33342 and visualized under a fluorescence microscope.

Techniques Used: Light Microscopy, Staining, Fluorescence, Microscopy

Metformin blocks the cell cycle in the G 0 /G 1 phase. Flow cytometry analysis of proliferating A549 cells 48 h after the treatment with metformin (10 and 20 mM). Fractions of cells in the G 0 /G 1 , S and G 2 /M phases of the cell cycle are indicted. Untreated
Figure Legend Snippet: Metformin blocks the cell cycle in the G 0 /G 1 phase. Flow cytometry analysis of proliferating A549 cells 48 h after the treatment with metformin (10 and 20 mM). Fractions of cells in the G 0 /G 1 , S and G 2 /M phases of the cell cycle are indicted. Untreated

Techniques Used: Flow Cytometry, Cytometry

Metformin decreases A549 xenograft tumor proliferation. (A) The final tumor weight at necropsy at 18 days post-administration. (B) Effect of metformin on the body weight of mice during 18 days of treatment. (C) Examples of tumor regression in metformin-treated
Figure Legend Snippet: Metformin decreases A549 xenograft tumor proliferation. (A) The final tumor weight at necropsy at 18 days post-administration. (B) Effect of metformin on the body weight of mice during 18 days of treatment. (C) Examples of tumor regression in metformin-treated

Techniques Used: Mouse Assay

Metformin induces apoptosis of A549 cells mainly through the mitochondia-mediated pathway. (A) Effect of metformin on the expression of apoptosis-regulating proteins, as assessed by western blotting. Protein fractions of total cells were assessed for
Figure Legend Snippet: Metformin induces apoptosis of A549 cells mainly through the mitochondia-mediated pathway. (A) Effect of metformin on the expression of apoptosis-regulating proteins, as assessed by western blotting. Protein fractions of total cells were assessed for

Techniques Used: Expressing, Western Blot

23) Product Images from "miR-378a-3p Participates in Metformin’s Mechanism of Action on C2C12 Cells under Hyperglycemia"

Article Title: miR-378a-3p Participates in Metformin’s Mechanism of Action on C2C12 Cells under Hyperglycemia

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms22020541

Higher concentrations of metformin upregulate miR-378a-3p and Ppargc1b in C2C12 myoblasts independently of AMPK. ( A ) miR-378a-3p expression in C2C12 cells after being exposed to 25 mM glucose for 3 and 7 days. ( B ) miR-378a-3p and ( C ) Ppargc1b expression in C2C12 myoblasts incubated with 0.05, 0.40, 10 and 25 mM of metformin. ( D ) miR-378a-3p expression and ( E ) Ppargc1b expression after incubation with 0.5 mM AICAR. All data is given as mean ± S.E.M. ( n = 3). * p
Figure Legend Snippet: Higher concentrations of metformin upregulate miR-378a-3p and Ppargc1b in C2C12 myoblasts independently of AMPK. ( A ) miR-378a-3p expression in C2C12 cells after being exposed to 25 mM glucose for 3 and 7 days. ( B ) miR-378a-3p and ( C ) Ppargc1b expression in C2C12 myoblasts incubated with 0.05, 0.40, 10 and 25 mM of metformin. ( D ) miR-378a-3p expression and ( E ) Ppargc1b expression after incubation with 0.5 mM AICAR. All data is given as mean ± S.E.M. ( n = 3). * p

Techniques Used: Expressing, Incubation

Metformin upregulates Tfam in C2C12 myoblasts but not when miR-378a-3p is inhibited. ( A – C ) Expression level of Tfam in C2C12 myoblasts. ( D ) Representative images of Western Blot showing TFAM and COXIV protein content. Mitochondria-related factors, such as ( E – G ) TFAM and ( H – J ) COXIV, were evaluated through Western Blot. All data is given as mean ± S.E.M. ( n = 3). * p = 0.014 versus Glc 25 mM, ** p = 0.002 versus Glc 25 mM.
Figure Legend Snippet: Metformin upregulates Tfam in C2C12 myoblasts but not when miR-378a-3p is inhibited. ( A – C ) Expression level of Tfam in C2C12 myoblasts. ( D ) Representative images of Western Blot showing TFAM and COXIV protein content. Mitochondria-related factors, such as ( E – G ) TFAM and ( H – J ) COXIV, were evaluated through Western Blot. All data is given as mean ± S.E.M. ( n = 3). * p = 0.014 versus Glc 25 mM, ** p = 0.002 versus Glc 25 mM.

Techniques Used: Expressing, Western Blot

Metformin decreases PEPCK activity but not when miR-378a-3p is inhibited. PEPCK activity ( A ) in cells exposed to 0.05, 0.40 and 25 mM of metformin, ( B ) in cells overexpressing miR-378a-3p, and ( C ) in cells incubated with miR-378a-3p inhibitors and with 0.05, 0.40 and 25 mM of metformin. All data is given as mean ± S.E.M. ( n = 3). * p = 0.0164 versus Glc 25 mM, ** p = 0.0052 versus Glc 25 mM, *** p = 0.0004 versus Glc 25 mM, **** p
Figure Legend Snippet: Metformin decreases PEPCK activity but not when miR-378a-3p is inhibited. PEPCK activity ( A ) in cells exposed to 0.05, 0.40 and 25 mM of metformin, ( B ) in cells overexpressing miR-378a-3p, and ( C ) in cells incubated with miR-378a-3p inhibitors and with 0.05, 0.40 and 25 mM of metformin. All data is given as mean ± S.E.M. ( n = 3). * p = 0.0164 versus Glc 25 mM, ** p = 0.0052 versus Glc 25 mM, *** p = 0.0004 versus Glc 25 mM, **** p

Techniques Used: Activity Assay, Incubation

Lower concentrations of metformin increase ATP content in C2C12 myoblasts but this effect is impaired in cells with miR-378a-3p inhibited. ( A ) Expression levels of miR-378a-3p after transfection with mimic and inhibitor negative controls (Mimic NC and Inh NC, respectively), and with miR-378a-3p mimic and inhibitor in cells exposed to glucose 25 mM for 3 days. ( B ) ATP content in C2C12 myoblasts. ( C ) Cell viability assay using the Live/Dead viability/cytotoxicity assay kit. All data is given as mean ± S.E.M. ( n = 3). * p
Figure Legend Snippet: Lower concentrations of metformin increase ATP content in C2C12 myoblasts but this effect is impaired in cells with miR-378a-3p inhibited. ( A ) Expression levels of miR-378a-3p after transfection with mimic and inhibitor negative controls (Mimic NC and Inh NC, respectively), and with miR-378a-3p mimic and inhibitor in cells exposed to glucose 25 mM for 3 days. ( B ) ATP content in C2C12 myoblasts. ( C ) Cell viability assay using the Live/Dead viability/cytotoxicity assay kit. All data is given as mean ± S.E.M. ( n = 3). * p

Techniques Used: Expressing, Transfection, Viability Assay, Cytotoxicity Assay

24) Product Images from "Effect of Metformin on Metabolic Improvement and Gut Microbiota"

Article Title: Effect of Metformin on Metabolic Improvement and Gut Microbiota

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01357-14

Effects of dietary change and metformin treatment on mucin expression in small intestine tissue and the histology of small intestine and liver tissue. (A) The levels of mRNA for MUC2 and MUC5 were increased after metformin treatment in female mice on
Figure Legend Snippet: Effects of dietary change and metformin treatment on mucin expression in small intestine tissue and the histology of small intestine and liver tissue. (A) The levels of mRNA for MUC2 and MUC5 were increased after metformin treatment in female mice on

Techniques Used: Expressing, Mouse Assay

Effects of metformin and phenformin on growth of Akkermansia muciniphila as a proportion of all bacteria in BHI medium. Mixed stool, original stool samples from HFD-Met mice prior to culture. The amount of Akkermansia muciniphila bacteria as a proportion
Figure Legend Snippet: Effects of metformin and phenformin on growth of Akkermansia muciniphila as a proportion of all bacteria in BHI medium. Mixed stool, original stool samples from HFD-Met mice prior to culture. The amount of Akkermansia muciniphila bacteria as a proportion

Techniques Used: Mouse Assay

Comparison of KEGG pathways predicted using PICRUSt according to diet and metformin treatment. Groups categorized according to metformin treatment and diet were clearly clustered on the basis of the KEGG pathways predicted using PICRUSt, as well as the
Figure Legend Snippet: Comparison of KEGG pathways predicted using PICRUSt according to diet and metformin treatment. Groups categorized according to metformin treatment and diet were clearly clustered on the basis of the KEGG pathways predicted using PICRUSt, as well as the

Techniques Used:

Microbial diversity and difference in the bacterial community between groups categorized according to diet and metformin treatment. (A) Rarefaction curve of bacterial diversity according to dietary change and metformin treatment in mice on an HFD and
Figure Legend Snippet: Microbial diversity and difference in the bacterial community between groups categorized according to diet and metformin treatment. (A) Rarefaction curve of bacterial diversity according to dietary change and metformin treatment in mice on an HFD and

Techniques Used: Mouse Assay

Effects of dietary changes and metformin treatment on body weight and glucose, TC, and HDL levels. Mice were induced to develop metabolic disorders while on an HFD for 18 weeks and then subjected to metformin treatment and a dietary change to an ND for
Figure Legend Snippet: Effects of dietary changes and metformin treatment on body weight and glucose, TC, and HDL levels. Mice were induced to develop metabolic disorders while on an HFD for 18 weeks and then subjected to metformin treatment and a dietary change to an ND for

Techniques Used: Mouse Assay

Correlation between metabolic biomarkers and bacterial abundance during metformin treatment in mice with HFD-induced obesity. *, statistical significance based on a P value of
Figure Legend Snippet: Correlation between metabolic biomarkers and bacterial abundance during metformin treatment in mice with HFD-induced obesity. *, statistical significance based on a P value of

Techniques Used: Mouse Assay

25) Product Images from "Metformin inhibits gastric cancer via the inhibition of HIF1α/PKM2 signaling"

Article Title: Metformin inhibits gastric cancer via the inhibition of HIF1α/PKM2 signaling

Journal: American Journal of Cancer Research

doi:

Metformin inhibits the invasion and migration of gastric cancer cells. SGC7901 and BGC823 cells were seeded on transwell for invasion and migration analysis. The numbers of invaded and migrated cells were counted in five separate fields using light microscopy.
Figure Legend Snippet: Metformin inhibits the invasion and migration of gastric cancer cells. SGC7901 and BGC823 cells were seeded on transwell for invasion and migration analysis. The numbers of invaded and migrated cells were counted in five separate fields using light microscopy.

Techniques Used: Migration, Light Microscopy

Metformin reduces HIF1α, PARP and COX protein expression in gastric cancer cells. (A, B) SGC7901 cells were treated with metformin (0, 40 mM) for 24 h and then analyzed for the expression of HIF1α (A) and COX (B) by immunofluorescence.
Figure Legend Snippet: Metformin reduces HIF1α, PARP and COX protein expression in gastric cancer cells. (A, B) SGC7901 cells were treated with metformin (0, 40 mM) for 24 h and then analyzed for the expression of HIF1α (A) and COX (B) by immunofluorescence.

Techniques Used: Expressing, Immunofluorescence

Metformin induces apoptosis and cell cycle arrest in gastric cancer cells. SGC7901 cells (A, C) and BGC823 cells (B, D) were treated with metformin (0, 20, 50 mM) for 24 h. The proportions of early apoptosis cells (A, B) and cells in different phases
Figure Legend Snippet: Metformin induces apoptosis and cell cycle arrest in gastric cancer cells. SGC7901 cells (A, C) and BGC823 cells (B, D) were treated with metformin (0, 20, 50 mM) for 24 h. The proportions of early apoptosis cells (A, B) and cells in different phases

Techniques Used:

Metformin inhibits the viability of gastric cancer cells. SGC7901 cells (A) and BGC823cells (B) were treated with metformin (0-50 mM) for 24, 48, 72 h. Cell viability was evaluated by CCK-8 assy.
Figure Legend Snippet: Metformin inhibits the viability of gastric cancer cells. SGC7901 cells (A) and BGC823cells (B) were treated with metformin (0-50 mM) for 24, 48, 72 h. Cell viability was evaluated by CCK-8 assy.

Techniques Used: CCK-8 Assay

26) Product Images from "Synergistic cell death in FLT3-ITD positive acute myeloid leukemia by combined treatment with metformin and 6-benzylthioinosine"

Article Title: Synergistic cell death in FLT3-ITD positive acute myeloid leukemia by combined treatment with metformin and 6-benzylthioinosine

Journal: Leukemia research

doi: 10.1016/j.leukres.2016.10.004

6-BT and metformin reduces intracellular ATP levels and extracellular glucose uptake A) MV4-11, MOLM-14 and OCI-AML3 cells were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM), 6-BT and metformin for 24 hours and intracellular ATP levels were measured in live cells using the Perkin Elmer ATP Bioluminescence kit. B) MV4-11, MOLM-14 and OCI-AML3 cells were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM), 6-BT and metformin for 24 hours and extracellular glucose levels were measured using the Life Technologies Amplex Red Glucose Assay. (** p
Figure Legend Snippet: 6-BT and metformin reduces intracellular ATP levels and extracellular glucose uptake A) MV4-11, MOLM-14 and OCI-AML3 cells were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM), 6-BT and metformin for 24 hours and intracellular ATP levels were measured in live cells using the Perkin Elmer ATP Bioluminescence kit. B) MV4-11, MOLM-14 and OCI-AML3 cells were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM), 6-BT and metformin for 24 hours and extracellular glucose levels were measured using the Life Technologies Amplex Red Glucose Assay. (** p

Techniques Used: Glucose Assay

6-BT and metformin have partial effects on inhibition of mTOR activity as measured by phosphorylation of downstream substrates MV4-11, MOLM-14 and OCI-AML3 cells were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM), 6-BT and metformin or with AZD-8055 (500 nM) for 24 hours and protein lysates were probed with antibodies to total and phosphorylated (Ser 65 and Thr 37/46) 4EBP1 and total and phosphorylated (Thr 389) p70S6 Kinase. GAPDH was used as the loading control.
Figure Legend Snippet: 6-BT and metformin have partial effects on inhibition of mTOR activity as measured by phosphorylation of downstream substrates MV4-11, MOLM-14 and OCI-AML3 cells were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM), 6-BT and metformin or with AZD-8055 (500 nM) for 24 hours and protein lysates were probed with antibodies to total and phosphorylated (Ser 65 and Thr 37/46) 4EBP1 and total and phosphorylated (Thr 389) p70S6 Kinase. GAPDH was used as the loading control.

Techniques Used: Inhibition, Activity Assay

mTOR inhibition by metformin or nucleotide synthesis inhibition by 6-BT does not account for the synergistic cytotoxicity of the combination A) MV4-11 and MOLM-14 cells were treated with vehicle (DMSO), 6-BT (10 μM), AZD-8055 (250 and 500 nM) or combinations and cytotoxicity was measured at 48 hours using trypan blue. B) MV4-11, MOLM-14 and OCI-AML3 cells were treated with vehicle (DMSO), 6-BT (10 μM) and metformin (10 mM) with or without pre-treatment with AZD-8055 (250 nM) for 2 hours and cell death was estimated using trypan blue. C) MV4-11 and MOLM-14 cells were treated with vehicle (DMSO), 6-mercaptopurine (6-MP) (100 and 1000 nM), 6-BT (10 μM) or combinations and cytotoxicity was measured at 48 hours using trypan blue. (** p
Figure Legend Snippet: mTOR inhibition by metformin or nucleotide synthesis inhibition by 6-BT does not account for the synergistic cytotoxicity of the combination A) MV4-11 and MOLM-14 cells were treated with vehicle (DMSO), 6-BT (10 μM), AZD-8055 (250 and 500 nM) or combinations and cytotoxicity was measured at 48 hours using trypan blue. B) MV4-11, MOLM-14 and OCI-AML3 cells were treated with vehicle (DMSO), 6-BT (10 μM) and metformin (10 mM) with or without pre-treatment with AZD-8055 (250 nM) for 2 hours and cell death was estimated using trypan blue. C) MV4-11 and MOLM-14 cells were treated with vehicle (DMSO), 6-mercaptopurine (6-MP) (100 and 1000 nM), 6-BT (10 μM) or combinations and cytotoxicity was measured at 48 hours using trypan blue. (** p

Techniques Used: Inhibition

6-BT and metformin reduces reactive oxygen species (ROS) levels and expression of pSTAT5 A) MV4-11, MOLM-14 and OCI-AML3 cells were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM), 6-BT and metformin for 24 hours and ROS levels within live cells were measured with H 2 DCFDA staining via flow cytometry B ) MV4-11, MOLM-14 and OCI-AML3 were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM) and 6-BT and metformin for 24 hours and protein lysates were probed with antibodies to total and phosphorylated (Tyr 694) STAT5. GAPDH was used as the loading control.
Figure Legend Snippet: 6-BT and metformin reduces reactive oxygen species (ROS) levels and expression of pSTAT5 A) MV4-11, MOLM-14 and OCI-AML3 cells were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM), 6-BT and metformin for 24 hours and ROS levels within live cells were measured with H 2 DCFDA staining via flow cytometry B ) MV4-11, MOLM-14 and OCI-AML3 were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM) and 6-BT and metformin for 24 hours and protein lysates were probed with antibodies to total and phosphorylated (Tyr 694) STAT5. GAPDH was used as the loading control.

Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry

6-BT and metformin combination reduces glycolytic flux in response to metformin inhibited oxidative phosphorylation A) B) MV4-11 and OCI-AML3 cells were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM), 6-BT and metformin for 24 hours and oxidative phosphorylation rate (oxygen consumption rate – OCR) and glycolytic rate (extracellular acidification rate – ECAR) was measured using the Seahorse XF24 extracellular flux analyzer.
Figure Legend Snippet: 6-BT and metformin combination reduces glycolytic flux in response to metformin inhibited oxidative phosphorylation A) B) MV4-11 and OCI-AML3 cells were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM), 6-BT and metformin for 24 hours and oxidative phosphorylation rate (oxygen consumption rate – OCR) and glycolytic rate (extracellular acidification rate – ECAR) was measured using the Seahorse XF24 extracellular flux analyzer.

Techniques Used:

Synergistic cytotoxicity of 6-BT and metformin in FLT3-ITD + AML cell lines A) MV4-11, MOLM-14, OCI-AML3, Nomo-1 and THP-1 cell lines were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM) or 6-BT and metformin for 48 hours. Cell death was measured by trypan blue staining. B ) Growth inhibition following treatment with 6-BT and metformin was measured in MV4-11, MOLM-14, OCI-AML3, Nomo-1 and THP-1 cell lines following treatment with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM) or 6-BT and metformin for 48 hours. Fold inhibition in growth was measured by counting viable cells using the trypan blue exclusion method. (** p
Figure Legend Snippet: Synergistic cytotoxicity of 6-BT and metformin in FLT3-ITD + AML cell lines A) MV4-11, MOLM-14, OCI-AML3, Nomo-1 and THP-1 cell lines were treated with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM) or 6-BT and metformin for 48 hours. Cell death was measured by trypan blue staining. B ) Growth inhibition following treatment with 6-BT and metformin was measured in MV4-11, MOLM-14, OCI-AML3, Nomo-1 and THP-1 cell lines following treatment with vehicle (DMSO), 6-BT (10 μM), metformin (10 mM) or 6-BT and metformin for 48 hours. Fold inhibition in growth was measured by counting viable cells using the trypan blue exclusion method. (** p

Techniques Used: Staining, Inhibition

27) Product Images from "Effects of atorvastatin and metformin on development of pentylenetetrazole-induced seizure in mice"

Article Title: Effects of atorvastatin and metformin on development of pentylenetetrazole-induced seizure in mice

Journal: Heliyon

doi: 10.1016/j.heliyon.2020.e03761

Comparison of the effect of ‘Metformin’, ‘Atorvastatin’ and ‘Metformin + Atorvastatin’ treatments on stage 5 duration in PTZ kindling mice. Values are expressed as mean ± SD (n = 7 in each group), and are shown for each injection (⁄p
Figure Legend Snippet: Comparison of the effect of ‘Metformin’, ‘Atorvastatin’ and ‘Metformin + Atorvastatin’ treatments on stage 5 duration in PTZ kindling mice. Values are expressed as mean ± SD (n = 7 in each group), and are shown for each injection (⁄p

Techniques Used: Mouse Assay, Injection

Comparison of the effect of ‘Metformin’, ‘Atorvastatin’ and ‘Metformin + Atorvastatin’ treatments on seizure stage in different injections in PTZ kindling mice. Values are expressed as mean ± SD (n = 7 in each group), and are shown for each injection (⁄p
Figure Legend Snippet: Comparison of the effect of ‘Metformin’, ‘Atorvastatin’ and ‘Metformin + Atorvastatin’ treatments on seizure stage in different injections in PTZ kindling mice. Values are expressed as mean ± SD (n = 7 in each group), and are shown for each injection (⁄p

Techniques Used: Mouse Assay, Injection

Comparison of the effect of ‘Metformin’, ‘Atorvastatin’ and ‘Metformin + Atorvastatin’ treatments on stage 2 latency in PTZ kindling mice. Values are expressed as mean ± SD (n = 7 in each group), and are shown for each injection (⁄p
Figure Legend Snippet: Comparison of the effect of ‘Metformin’, ‘Atorvastatin’ and ‘Metformin + Atorvastatin’ treatments on stage 2 latency in PTZ kindling mice. Values are expressed as mean ± SD (n = 7 in each group), and are shown for each injection (⁄p

Techniques Used: Mouse Assay, Injection

Comparison of the effect of ‘Metformin’, ‘Atorvastatin’ and ‘Metformin + Atorvastatin’ treatments on stage 5 latency in PTZ kindling mice. Values are expressed as mean ± SD (n = 7 in each group), and are shown for each injection (⁄p
Figure Legend Snippet: Comparison of the effect of ‘Metformin’, ‘Atorvastatin’ and ‘Metformin + Atorvastatin’ treatments on stage 5 latency in PTZ kindling mice. Values are expressed as mean ± SD (n = 7 in each group), and are shown for each injection (⁄p

Techniques Used: Mouse Assay, Injection

28) Product Images from "Metformin-treated cancer cells modulate macrophage polarization through AMPK-NF-κB signaling"

Article Title: Metformin-treated cancer cells modulate macrophage polarization through AMPK-NF-κB signaling

Journal: Oncotarget

doi: 10.18632/oncotarget.14982

AMPK-NF-κB signaling participated in macrophage polarization Breast cancer cells (MDA-MB231) were treated with metformin 60 μM combined with an AMPK inhibitor (Compound C, CC) or NF-κB inhibitor (BAY-117082, BAY) for 6 h. The supernatant was collected to treat macrophages for 48 h, followed by flow cytometry analysis of CD206, M2 phenotype ( A ) and CD16, M1 phenotype ( B ). Data are expressed as mean ± SD, * p
Figure Legend Snippet: AMPK-NF-κB signaling participated in macrophage polarization Breast cancer cells (MDA-MB231) were treated with metformin 60 μM combined with an AMPK inhibitor (Compound C, CC) or NF-κB inhibitor (BAY-117082, BAY) for 6 h. The supernatant was collected to treat macrophages for 48 h, followed by flow cytometry analysis of CD206, M2 phenotype ( A ) and CD16, M1 phenotype ( B ). Data are expressed as mean ± SD, * p

Techniques Used: Multiple Displacement Amplification, Flow Cytometry, Cytometry

Metformin treated cancer cells increased M1 cytokine and decreased M2 cytokine expression in macrophage THP-1 cells were stimulated with PMA (200 nM) for 24 h, then incubated with breast cancer conditioned medium (CM) with or without metformin (60 μM) for 6 h, followed by analysis of the secretion of IL-8, IL-10, TGF-β, IL-12 and TNF-α using quantitative PCR ( A–E ). Data are expressed as mean ± SD, * p
Figure Legend Snippet: Metformin treated cancer cells increased M1 cytokine and decreased M2 cytokine expression in macrophage THP-1 cells were stimulated with PMA (200 nM) for 24 h, then incubated with breast cancer conditioned medium (CM) with or without metformin (60 μM) for 6 h, followed by analysis of the secretion of IL-8, IL-10, TGF-β, IL-12 and TNF-α using quantitative PCR ( A–E ). Data are expressed as mean ± SD, * p

Techniques Used: Expressing, Incubation, Real-time Polymerase Chain Reaction

Metformin decreased IL-4, IL-10, IL-13 and increased IFN-γ expression in breast cancer cells Breast cancer cells (MDA-MB231/MDA-MB453) were treated with metformin (60 μM) for 6 h, followed by analysis of the secretion of IL-4, IL-10, IL-13 and IFN-γ using quantitative PCR ( A – D ). Data are expressed as mean ± SD, * p
Figure Legend Snippet: Metformin decreased IL-4, IL-10, IL-13 and increased IFN-γ expression in breast cancer cells Breast cancer cells (MDA-MB231/MDA-MB453) were treated with metformin (60 μM) for 6 h, followed by analysis of the secretion of IL-4, IL-10, IL-13 and IFN-γ using quantitative PCR ( A – D ). Data are expressed as mean ± SD, * p

Techniques Used: Expressing, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction

Metformin treatment activated AMPK and inhibited NF-κB signaling in cancer cells Breast cancer cells (MDA-MB231/MDA-MB453) were treated with metformin 60 μM for 6 h. The protein lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against phospho-AMPK, AMPK, phospho-p65, p65 and GAPDH ( A ). Breast cancer cells (MDA-MB231/MDA-MB453) were treated with metformin 60 μM combined with an AMPK inhibitor (Compound C, CC) or NF-κB inhibitor (BAY-117082, BAY) for 6 h. The protein lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against phospho-AMPK, AMPK, phospho-p65, p65 and GAPDH ( B, C ). The addition of the Compound C or BAY-117082 to metformin-treated cells and the secretion of IL-4, IL-10 and IL-13 from the breast cancer cells were assayed by quantitative PCR ( D, E, F ). Data are expressed as mean ± SD, * p
Figure Legend Snippet: Metformin treatment activated AMPK and inhibited NF-κB signaling in cancer cells Breast cancer cells (MDA-MB231/MDA-MB453) were treated with metformin 60 μM for 6 h. The protein lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against phospho-AMPK, AMPK, phospho-p65, p65 and GAPDH ( A ). Breast cancer cells (MDA-MB231/MDA-MB453) were treated with metformin 60 μM combined with an AMPK inhibitor (Compound C, CC) or NF-κB inhibitor (BAY-117082, BAY) for 6 h. The protein lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against phospho-AMPK, AMPK, phospho-p65, p65 and GAPDH ( B, C ). The addition of the Compound C or BAY-117082 to metformin-treated cells and the secretion of IL-4, IL-10 and IL-13 from the breast cancer cells were assayed by quantitative PCR ( D, E, F ). Data are expressed as mean ± SD, * p

Techniques Used: Multiple Displacement Amplification, SDS Page, Real-time Polymerase Chain Reaction

Administration of metformin affected tumor growth and TAM polarization in a xenograft model Schematic diagram of the experimental process in the xenograft model ( A ). The tumor volumes were determined ( B ). The weights of the mice were measured in all groups before and after metformin treatment ( C ). The tumor tissues were removed and subjected to immunohistochemistry (IHC) analysis. The infiltrated macrophages were analyzed for overall macrophage marker F4/80, M1 marker CD16, and M2 marker CD206, and the quantified data is shown. Scale bar 50 μm ( D ). Data are expressed as mean ± SD, * p
Figure Legend Snippet: Administration of metformin affected tumor growth and TAM polarization in a xenograft model Schematic diagram of the experimental process in the xenograft model ( A ). The tumor volumes were determined ( B ). The weights of the mice were measured in all groups before and after metformin treatment ( C ). The tumor tissues were removed and subjected to immunohistochemistry (IHC) analysis. The infiltrated macrophages were analyzed for overall macrophage marker F4/80, M1 marker CD16, and M2 marker CD206, and the quantified data is shown. Scale bar 50 μm ( D ). Data are expressed as mean ± SD, * p

Techniques Used: Mouse Assay, Immunohistochemistry, Marker

Metformin treated cancer cells polarized macrophage toward M1 phenotype THP-1 cells were stimulated with PMA (200 nM) for 24 h, then incubated with breast cancer (MDA-MB231/MDA-MB453) conditioned medium (CM) with or without metformin (60 μM) for 6 h, followed by flow cytometry analysis to quantify the amount of CD206, an M2 macrophage marker, and CD16, an M1 marker ( A, B ). Data are expressed as mean ± SD, * p
Figure Legend Snippet: Metformin treated cancer cells polarized macrophage toward M1 phenotype THP-1 cells were stimulated with PMA (200 nM) for 24 h, then incubated with breast cancer (MDA-MB231/MDA-MB453) conditioned medium (CM) with or without metformin (60 μM) for 6 h, followed by flow cytometry analysis to quantify the amount of CD206, an M2 macrophage marker, and CD16, an M1 marker ( A, B ). Data are expressed as mean ± SD, * p

Techniques Used: Incubation, Multiple Displacement Amplification, Flow Cytometry, Cytometry, Marker

29) Product Images from "Increase in apoptosis by combination of metformin with silibinin in human colorectal cancer cells"

Article Title: Increase in apoptosis by combination of metformin with silibinin in human colorectal cancer cells

Journal: World Journal of Gastroenterology : WJG

doi: 10.3748/wjg.v21.i14.4169

Combined metformin and silibinin treatment inhibits protein kinase B phosphorylation by enhancing phosphatase and tensin homolog expression. Proteins isolated from COLO 205 cells were probed using an antibody against phosphatase and tensin homolog (PTEN)
Figure Legend Snippet: Combined metformin and silibinin treatment inhibits protein kinase B phosphorylation by enhancing phosphatase and tensin homolog expression. Proteins isolated from COLO 205 cells were probed using an antibody against phosphatase and tensin homolog (PTEN)

Techniques Used: Expressing, Isolation

Treatment with silibinin and metformin in combination or alone reduces the viability of COLO 205 cells. Reduced cell viability was observed following 24-h incubation with silibinin (0-200 μmol/L) and metformin (0-20 mmol/L) in combination or alone.
Figure Legend Snippet: Treatment with silibinin and metformin in combination or alone reduces the viability of COLO 205 cells. Reduced cell viability was observed following 24-h incubation with silibinin (0-200 μmol/L) and metformin (0-20 mmol/L) in combination or alone.

Techniques Used: Incubation

Combined metformin and silibinin treatment enhances AMP-activated protein kinase phosphorylation. COLO 205 cells were treated with silibinin and metformin in combination or alone. The protein samples were then probed using an antibody against phosphorylated
Figure Legend Snippet: Combined metformin and silibinin treatment enhances AMP-activated protein kinase phosphorylation. COLO 205 cells were treated with silibinin and metformin in combination or alone. The protein samples were then probed using an antibody against phosphorylated

Techniques Used:

Combined metformin and silibinin treatment induces COLO 205 cell apoptosis. COLO 205 cells were treated with silibinin and metformin in combination or alone. Apo-Glo assay showing apoptosis of COLO 205 cells treated with silibinin (100 μmol/L)
Figure Legend Snippet: Combined metformin and silibinin treatment induces COLO 205 cell apoptosis. COLO 205 cells were treated with silibinin and metformin in combination or alone. Apo-Glo assay showing apoptosis of COLO 205 cells treated with silibinin (100 μmol/L)

Techniques Used: Glo Assay

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    Millipore metformin
    <t>Metformin</t> improves cell migration in HUVEC exposed to hyperglycemia and CoCl 2 . ( A ) HUVEC were incubated for 24 h with hyperglycemia in the presence or absence of metformin. Scratch lines were created on confluent monolayers. The media containing different glucose concentrations and metformin were replaced. Then cells were incubated with CoCl 2 for 24 h in a 5% CO 2 chamber that was connected to CCD camera. Images were acquired every hour, and three independent biological experiments were performed at which each condition was assessed in duplicate. The scratch area was measured using NIS Elements software. ( B ) Hyperglycemia increased migration after 6, 12, and 18 h; ( C ) whereas hyperglycemia-CoCl 2 significantly reduced migration. Metformin increased cell migration under hyperglycemia-CoCl 2 . Sunitinib was used as a negative control, therefore the line with sunitinab is on x axis as cell migration not affected. Results are expressed as mean ± SEM and were analyzed by one-way ANOVA followed by LSD, ** p
    Metformin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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
    https://www.bioz.com/result/materials metformin/product/Millipore
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    Price from $9.99 to $1999.99
    materials metformin - by Bioz Stars, 2022-08
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    Metformin improves cell migration in HUVEC exposed to hyperglycemia and CoCl 2 . ( A ) HUVEC were incubated for 24 h with hyperglycemia in the presence or absence of metformin. Scratch lines were created on confluent monolayers. The media containing different glucose concentrations and metformin were replaced. Then cells were incubated with CoCl 2 for 24 h in a 5% CO 2 chamber that was connected to CCD camera. Images were acquired every hour, and three independent biological experiments were performed at which each condition was assessed in duplicate. The scratch area was measured using NIS Elements software. ( B ) Hyperglycemia increased migration after 6, 12, and 18 h; ( C ) whereas hyperglycemia-CoCl 2 significantly reduced migration. Metformin increased cell migration under hyperglycemia-CoCl 2 . Sunitinib was used as a negative control, therefore the line with sunitinab is on x axis as cell migration not affected. Results are expressed as mean ± SEM and were analyzed by one-way ANOVA followed by LSD, ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Proangiogenic Effect of Metformin in Endothelial Cells Is via Upregulation of VEGFR1/2 and Their Signaling under Hyperglycemia-Hypoxia

    doi: 10.3390/ijms19010293

    Figure Lengend Snippet: Metformin improves cell migration in HUVEC exposed to hyperglycemia and CoCl 2 . ( A ) HUVEC were incubated for 24 h with hyperglycemia in the presence or absence of metformin. Scratch lines were created on confluent monolayers. The media containing different glucose concentrations and metformin were replaced. Then cells were incubated with CoCl 2 for 24 h in a 5% CO 2 chamber that was connected to CCD camera. Images were acquired every hour, and three independent biological experiments were performed at which each condition was assessed in duplicate. The scratch area was measured using NIS Elements software. ( B ) Hyperglycemia increased migration after 6, 12, and 18 h; ( C ) whereas hyperglycemia-CoCl 2 significantly reduced migration. Metformin increased cell migration under hyperglycemia-CoCl 2 . Sunitinib was used as a negative control, therefore the line with sunitinab is on x axis as cell migration not affected. Results are expressed as mean ± SEM and were analyzed by one-way ANOVA followed by LSD, ** p

    Article Snippet: The medium was replaced with fresh medium containing respective concentrations of glucose and metformin, and then the cells were exposed to CoCl2 for up to 24 h. As a negative control for cell migration, HUVEC were treated with marimastat, an matrix metalloproteinase (MMP) inhibitor (Sigma-Aldrich, Dorset, UK) to a final concentration of 10 µmol/L in DMSO [ ] or 0.1 µmol/L sunitinib malate a VEGF inhibitor (Sigma-Aldrich, Dorset, UK) [ ].

    Techniques: Migration, Incubation, Software, Negative Control

    Effect of metformin on VEGFA mRNA and VEGF 165A protein under euglycemia-CoCl 2 , hyperglycemia and hyperglycemia-CoCl 2 . HUVEC were treated with hyperglycemic or euglycemic glucose concentrations as a control. After 24 h, metformin was added to euglycemic and hyperglycemic cultures, then CoCl 2 was added for either 1, 3 or 12 h. ( A ) The variation in mRNA expression levels of VEGFA was assessed by qRT-PCR on three independent biological replicates and ( B ) the variation in protein levels of VEGF 165A was assessed by western blot on three independent biological replicates. Results are presented as mean ± SEM and were analyzed using one-way ANOVA followed by LSD, * p

    Journal: International Journal of Molecular Sciences

    Article Title: Proangiogenic Effect of Metformin in Endothelial Cells Is via Upregulation of VEGFR1/2 and Their Signaling under Hyperglycemia-Hypoxia

    doi: 10.3390/ijms19010293

    Figure Lengend Snippet: Effect of metformin on VEGFA mRNA and VEGF 165A protein under euglycemia-CoCl 2 , hyperglycemia and hyperglycemia-CoCl 2 . HUVEC were treated with hyperglycemic or euglycemic glucose concentrations as a control. After 24 h, metformin was added to euglycemic and hyperglycemic cultures, then CoCl 2 was added for either 1, 3 or 12 h. ( A ) The variation in mRNA expression levels of VEGFA was assessed by qRT-PCR on three independent biological replicates and ( B ) the variation in protein levels of VEGF 165A was assessed by western blot on three independent biological replicates. Results are presented as mean ± SEM and were analyzed using one-way ANOVA followed by LSD, * p

    Article Snippet: The medium was replaced with fresh medium containing respective concentrations of glucose and metformin, and then the cells were exposed to CoCl2 for up to 24 h. As a negative control for cell migration, HUVEC were treated with marimastat, an matrix metalloproteinase (MMP) inhibitor (Sigma-Aldrich, Dorset, UK) to a final concentration of 10 µmol/L in DMSO [ ] or 0.1 µmol/L sunitinib malate a VEGF inhibitor (Sigma-Aldrich, Dorset, UK) [ ].

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    Metformin impairs cell migration in HUVEC exposed to euglycemia and CoCl 2 . ( A ) HUVEC were incubated for 24 h with euglycemia in the presence or absence of metformin. Scratch lines were created on confluent monolayers. The media containing different glucose concentrations and metformin were replaced. Then cells were incubated with CoCl 2 for 24 h in a 5% CO 2 chamber that was connected to CCD camera. Images were acquired every hour, and three independent biological experiments were performed at which each condition was assessed in duplicate. The scratch area was measured using NIS Elements software. ( B ) CoCl 2 induction exhibited no significant effect on cell migration under euglycemia, whereas metformin reduced migration after 18 h. Sunitinib (0.1 µmol/L) was used as a negative control, therefore the line with sunitinab is on x axis as cell migration not affected. Results are expressed as mean ± SEM and were analyzed by one-way ANOVA followed by LSD, ## p

    Journal: International Journal of Molecular Sciences

    Article Title: Proangiogenic Effect of Metformin in Endothelial Cells Is via Upregulation of VEGFR1/2 and Their Signaling under Hyperglycemia-Hypoxia

    doi: 10.3390/ijms19010293

    Figure Lengend Snippet: Metformin impairs cell migration in HUVEC exposed to euglycemia and CoCl 2 . ( A ) HUVEC were incubated for 24 h with euglycemia in the presence or absence of metformin. Scratch lines were created on confluent monolayers. The media containing different glucose concentrations and metformin were replaced. Then cells were incubated with CoCl 2 for 24 h in a 5% CO 2 chamber that was connected to CCD camera. Images were acquired every hour, and three independent biological experiments were performed at which each condition was assessed in duplicate. The scratch area was measured using NIS Elements software. ( B ) CoCl 2 induction exhibited no significant effect on cell migration under euglycemia, whereas metformin reduced migration after 18 h. Sunitinib (0.1 µmol/L) was used as a negative control, therefore the line with sunitinab is on x axis as cell migration not affected. Results are expressed as mean ± SEM and were analyzed by one-way ANOVA followed by LSD, ## p

    Article Snippet: The medium was replaced with fresh medium containing respective concentrations of glucose and metformin, and then the cells were exposed to CoCl2 for up to 24 h. As a negative control for cell migration, HUVEC were treated with marimastat, an matrix metalloproteinase (MMP) inhibitor (Sigma-Aldrich, Dorset, UK) to a final concentration of 10 µmol/L in DMSO [ ] or 0.1 µmol/L sunitinib malate a VEGF inhibitor (Sigma-Aldrich, Dorset, UK) [ ].

    Techniques: Migration, Incubation, Software, Negative Control

    Marimastat antagonizes the effect of metformin on cell migration in HUVEC exposed to hyperglycemia-CoCl 2 . ( A ) HUVEC were incubated with euglycemia or hyperglycemia in the presence or absence of metformin for 24 h. Images were acquired every hour, and three independent biological experiments were performed in duplicate for each condition. The scratch area was measured using NIS Elements software. ( B ) Marimastat had no significant effect on cells treated with euglycemia, metformin and CoCl 2 induction. ( C ) The effect of metformin was inhibited by marimastat treatment. Results are expressed as mean ± SEM and were analyzed using one-way ANOVA followed by LSD, †† p

    Journal: International Journal of Molecular Sciences

    Article Title: Proangiogenic Effect of Metformin in Endothelial Cells Is via Upregulation of VEGFR1/2 and Their Signaling under Hyperglycemia-Hypoxia

    doi: 10.3390/ijms19010293

    Figure Lengend Snippet: Marimastat antagonizes the effect of metformin on cell migration in HUVEC exposed to hyperglycemia-CoCl 2 . ( A ) HUVEC were incubated with euglycemia or hyperglycemia in the presence or absence of metformin for 24 h. Images were acquired every hour, and three independent biological experiments were performed in duplicate for each condition. The scratch area was measured using NIS Elements software. ( B ) Marimastat had no significant effect on cells treated with euglycemia, metformin and CoCl 2 induction. ( C ) The effect of metformin was inhibited by marimastat treatment. Results are expressed as mean ± SEM and were analyzed using one-way ANOVA followed by LSD, †† p

    Article Snippet: The medium was replaced with fresh medium containing respective concentrations of glucose and metformin, and then the cells were exposed to CoCl2 for up to 24 h. As a negative control for cell migration, HUVEC were treated with marimastat, an matrix metalloproteinase (MMP) inhibitor (Sigma-Aldrich, Dorset, UK) to a final concentration of 10 µmol/L in DMSO [ ] or 0.1 µmol/L sunitinib malate a VEGF inhibitor (Sigma-Aldrich, Dorset, UK) [ ].

    Techniques: Migration, Incubation, Software

    Effect of metformin on MMP16 mRNA expression in euglycemic and hyperglycemic conditions with CoCl 2 induction. The variation in RNA expression levels of MMP16 was assessed by qRT-PCR on three independent biological replicates. Results are presented as mean ± SEM and were analyzed using one-way ANOVA followed by LSD. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Proangiogenic Effect of Metformin in Endothelial Cells Is via Upregulation of VEGFR1/2 and Their Signaling under Hyperglycemia-Hypoxia

    doi: 10.3390/ijms19010293

    Figure Lengend Snippet: Effect of metformin on MMP16 mRNA expression in euglycemic and hyperglycemic conditions with CoCl 2 induction. The variation in RNA expression levels of MMP16 was assessed by qRT-PCR on three independent biological replicates. Results are presented as mean ± SEM and were analyzed using one-way ANOVA followed by LSD. * p

    Article Snippet: The medium was replaced with fresh medium containing respective concentrations of glucose and metformin, and then the cells were exposed to CoCl2 for up to 24 h. As a negative control for cell migration, HUVEC were treated with marimastat, an matrix metalloproteinase (MMP) inhibitor (Sigma-Aldrich, Dorset, UK) to a final concentration of 10 µmol/L in DMSO [ ] or 0.1 µmol/L sunitinib malate a VEGF inhibitor (Sigma-Aldrich, Dorset, UK) [ ].

    Techniques: Expressing, RNA Expression, Quantitative RT-PCR

    Comprehensive VEGF signaling network of genes and proteins involved in cell migration and survival. Metformin-treated condition is compared to metformin-untreated condition under hyperglycemia-CoCl 2 for 12 h. The network was created by IPA software rendering VEGF signal transduction pathways. The genes from microarray expression study that are represented with red shades are upregulated, and green shades are downregulated, MMP16 was validated by qRT-PCR. The activity of ERK1/2 was assessed by MAPK activation dual detection assay flow cytometry. The red shade on functional assays denoted activation while green shade inhibition. Solid lines denoted direct interaction; interrupted lines denoted indirect interaction.

    Journal: International Journal of Molecular Sciences

    Article Title: Proangiogenic Effect of Metformin in Endothelial Cells Is via Upregulation of VEGFR1/2 and Their Signaling under Hyperglycemia-Hypoxia

    doi: 10.3390/ijms19010293

    Figure Lengend Snippet: Comprehensive VEGF signaling network of genes and proteins involved in cell migration and survival. Metformin-treated condition is compared to metformin-untreated condition under hyperglycemia-CoCl 2 for 12 h. The network was created by IPA software rendering VEGF signal transduction pathways. The genes from microarray expression study that are represented with red shades are upregulated, and green shades are downregulated, MMP16 was validated by qRT-PCR. The activity of ERK1/2 was assessed by MAPK activation dual detection assay flow cytometry. The red shade on functional assays denoted activation while green shade inhibition. Solid lines denoted direct interaction; interrupted lines denoted indirect interaction.

    Article Snippet: The medium was replaced with fresh medium containing respective concentrations of glucose and metformin, and then the cells were exposed to CoCl2 for up to 24 h. As a negative control for cell migration, HUVEC were treated with marimastat, an matrix metalloproteinase (MMP) inhibitor (Sigma-Aldrich, Dorset, UK) to a final concentration of 10 µmol/L in DMSO [ ] or 0.1 µmol/L sunitinib malate a VEGF inhibitor (Sigma-Aldrich, Dorset, UK) [ ].

    Techniques: Migration, Indirect Immunoperoxidase Assay, Software, Transduction, Microarray, Expressing, Quantitative RT-PCR, Activity Assay, Activation Assay, Detection Assay, Flow Cytometry, Cytometry, Functional Assay, Inhibition

    Metformin improves cell survival with hyperglycemia-CoCl 2 . ( A ) HUVEC were treated with 5.5 or ( B ) 16.5 mmol/L glucose for 48 h exposed to chemical hypoxia for 3, 12 or 24 h in the absence of metformin, ( C ) hyperglycemia treated with metformin (0.01 mmol/L) and supra-physiological concentration of metformin (1.0 mmol/L), and ( D ) hyperglycemia exposed to CoCl 2 for 24 h and parallel cultures were treated with metformin. Apoptosis was assessed by Annexin V staining and flow cytometry. Results are representative of 3 independent experiments and expressed as mean ± SEM and were analyzed by paired t -test, * p

    Journal: International Journal of Molecular Sciences

    Article Title: Proangiogenic Effect of Metformin in Endothelial Cells Is via Upregulation of VEGFR1/2 and Their Signaling under Hyperglycemia-Hypoxia

    doi: 10.3390/ijms19010293

    Figure Lengend Snippet: Metformin improves cell survival with hyperglycemia-CoCl 2 . ( A ) HUVEC were treated with 5.5 or ( B ) 16.5 mmol/L glucose for 48 h exposed to chemical hypoxia for 3, 12 or 24 h in the absence of metformin, ( C ) hyperglycemia treated with metformin (0.01 mmol/L) and supra-physiological concentration of metformin (1.0 mmol/L), and ( D ) hyperglycemia exposed to CoCl 2 for 24 h and parallel cultures were treated with metformin. Apoptosis was assessed by Annexin V staining and flow cytometry. Results are representative of 3 independent experiments and expressed as mean ± SEM and were analyzed by paired t -test, * p

    Article Snippet: The medium was replaced with fresh medium containing respective concentrations of glucose and metformin, and then the cells were exposed to CoCl2 for up to 24 h. As a negative control for cell migration, HUVEC were treated with marimastat, an matrix metalloproteinase (MMP) inhibitor (Sigma-Aldrich, Dorset, UK) to a final concentration of 10 µmol/L in DMSO [ ] or 0.1 µmol/L sunitinib malate a VEGF inhibitor (Sigma-Aldrich, Dorset, UK) [ ].

    Techniques: Concentration Assay, Staining, Flow Cytometry, Cytometry

    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