Structured Review

Mimetics metformin
<t>Metformin</t> improves expansion of CD235+ erythroblasts and BFU-E erythroid colony formation in human RPS19- and RPL11-insufficiency from CB CD34+ progenitors. (A). CD34+ HSPCs were transduced with shRNA against control (shLuc), RPS19 (shRPS19) or RPL11 (shRPL11) and after sorting, were differentiated for 14 days in the presence or absence of 50mM metformin. Cells were counted and the percentage expressing CD235+ erythroid (left) and CD11b+ myeloid (right) was determined by flow cytometry. Obtained values were multiplied to give an overall number that was normalized to the untreated control (grey columns). Values are presented as a percentage of the untreated control. (B) Direct comparison between metformin treated and untreated cultures is facilitated by normalizing the metformin-treated values to the untreated values in control, RPS19- and RPL11-insufficient groups. Values are expressed as a fold induction relative to untreated. (C) Transduced and sorted CD34+ progenitors were cultured in methylcellulose in the presence or absence of metformin for 16 days and BFU-E erythroid (left) and CFU-GM myeloid (right) colonies scored. Values are represented as the percentage of colonies induced in untreated controls (grey columns). (D). To directly compare the effect of metformin in each group, metformin-treated cultures were normalized to untreated cultures and are expressed as a fold induction of the untreated control. (E) Representative images of BFU-E colonies at day 14.Scale bar = 200 μM. Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant * P
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1) Product Images from "Metformin-induced suppression of NLK improves erythropoiesis in pre-clinical models of Diamond Blackfan Anemia through induction of miR-26a"

Article Title: Metformin-induced suppression of NLK improves erythropoiesis in pre-clinical models of Diamond Blackfan Anemia through induction of miR-26a

Journal: Experimental hematology

doi: 10.1016/j.exphem.2020.09.187

Metformin improves expansion of CD235+ erythroblasts and BFU-E erythroid colony formation in human RPS19- and RPL11-insufficiency from CB CD34+ progenitors. (A). CD34+ HSPCs were transduced with shRNA against control (shLuc), RPS19 (shRPS19) or RPL11 (shRPL11) and after sorting, were differentiated for 14 days in the presence or absence of 50mM metformin. Cells were counted and the percentage expressing CD235+ erythroid (left) and CD11b+ myeloid (right) was determined by flow cytometry. Obtained values were multiplied to give an overall number that was normalized to the untreated control (grey columns). Values are presented as a percentage of the untreated control. (B) Direct comparison between metformin treated and untreated cultures is facilitated by normalizing the metformin-treated values to the untreated values in control, RPS19- and RPL11-insufficient groups. Values are expressed as a fold induction relative to untreated. (C) Transduced and sorted CD34+ progenitors were cultured in methylcellulose in the presence or absence of metformin for 16 days and BFU-E erythroid (left) and CFU-GM myeloid (right) colonies scored. Values are represented as the percentage of colonies induced in untreated controls (grey columns). (D). To directly compare the effect of metformin in each group, metformin-treated cultures were normalized to untreated cultures and are expressed as a fold induction of the untreated control. (E) Representative images of BFU-E colonies at day 14.Scale bar = 200 μM. Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant * P
Figure Legend Snippet: Metformin improves expansion of CD235+ erythroblasts and BFU-E erythroid colony formation in human RPS19- and RPL11-insufficiency from CB CD34+ progenitors. (A). CD34+ HSPCs were transduced with shRNA against control (shLuc), RPS19 (shRPS19) or RPL11 (shRPL11) and after sorting, were differentiated for 14 days in the presence or absence of 50mM metformin. Cells were counted and the percentage expressing CD235+ erythroid (left) and CD11b+ myeloid (right) was determined by flow cytometry. Obtained values were multiplied to give an overall number that was normalized to the untreated control (grey columns). Values are presented as a percentage of the untreated control. (B) Direct comparison between metformin treated and untreated cultures is facilitated by normalizing the metformin-treated values to the untreated values in control, RPS19- and RPL11-insufficient groups. Values are expressed as a fold induction relative to untreated. (C) Transduced and sorted CD34+ progenitors were cultured in methylcellulose in the presence or absence of metformin for 16 days and BFU-E erythroid (left) and CFU-GM myeloid (right) colonies scored. Values are represented as the percentage of colonies induced in untreated controls (grey columns). (D). To directly compare the effect of metformin in each group, metformin-treated cultures were normalized to untreated cultures and are expressed as a fold induction of the untreated control. (E) Representative images of BFU-E colonies at day 14.Scale bar = 200 μM. Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant * P

Techniques Used: Transduction, shRNA, Expressing, Flow Cytometry, Cell Culture, Two Tailed Test

NLK 3’UTR facilitates metformin-mediated NLK suppression. (A) K562 cells stabling expressing luciferase alone (top panel), luciferase expressed behind a minimal promoter and the NLK proximal promoter (2 nd panel), luciferase immediately upstream of the human NLK 3’UTR (3 rd panel), , or luciferase with the SATB1 3’UTR (bottom panel) were grown in the presence of 0, 0.5, 5.0 or 50.0mM of metformin for 72 hrs. Cell were pelleted and either processed for luciferase assay (left) or western blot analysis of endogenous NLK (upper right) or GAPDH (lower right). (B) Luciferase immediately upstream of the human (upper), murine (middle) and zebrafish (lower) NLK 3’UTR were transiently transfected into human K562 (left) and cultured with indicated concentrations of metformin for 72 hrs. Lysates were assessed for luciferase activity and endogenous NLK and GAPDH protein expression by Western blotting. Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant * P
Figure Legend Snippet: NLK 3’UTR facilitates metformin-mediated NLK suppression. (A) K562 cells stabling expressing luciferase alone (top panel), luciferase expressed behind a minimal promoter and the NLK proximal promoter (2 nd panel), luciferase immediately upstream of the human NLK 3’UTR (3 rd panel), , or luciferase with the SATB1 3’UTR (bottom panel) were grown in the presence of 0, 0.5, 5.0 or 50.0mM of metformin for 72 hrs. Cell were pelleted and either processed for luciferase assay (left) or western blot analysis of endogenous NLK (upper right) or GAPDH (lower right). (B) Luciferase immediately upstream of the human (upper), murine (middle) and zebrafish (lower) NLK 3’UTR were transiently transfected into human K562 (left) and cultured with indicated concentrations of metformin for 72 hrs. Lysates were assessed for luciferase activity and endogenous NLK and GAPDH protein expression by Western blotting. Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant * P

Techniques Used: Expressing, Luciferase, Western Blot, Transfection, Cell Culture, Activity Assay, Two Tailed Test

NLK 3’UTR metformin-response element contains 4 potential miRNA binding sites and is highly conserved between human and mouse but divergent in zebrafish. (A) Schematic representing the many potential miRNA binding sites within the human NLK 3’UTR (upper). Diagrammatic representation of the full length and 5 truncated constructs fused to the luciferase gene and engineered to determine the metformin-responsive element within the NLK 3’UTR (lower). (B) After transient transfection of plasmids carrying the various 3’UTR fragments, K562 cells were treated with indicated concentrations of metformin for 72 hrs and assessed for luciferase activity and endogenous NLK and GAPDH protein expression by Western blot. (C) A schematic indicating the potential miRNA binding sites contained within the metformin-responsive element. (D). The corresponding nucleotide sequences of the human, mouse and zebrafish NLK 3’UTRs across the metformin-responsive element. Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant * P
Figure Legend Snippet: NLK 3’UTR metformin-response element contains 4 potential miRNA binding sites and is highly conserved between human and mouse but divergent in zebrafish. (A) Schematic representing the many potential miRNA binding sites within the human NLK 3’UTR (upper). Diagrammatic representation of the full length and 5 truncated constructs fused to the luciferase gene and engineered to determine the metformin-responsive element within the NLK 3’UTR (lower). (B) After transient transfection of plasmids carrying the various 3’UTR fragments, K562 cells were treated with indicated concentrations of metformin for 72 hrs and assessed for luciferase activity and endogenous NLK and GAPDH protein expression by Western blot. (C) A schematic indicating the potential miRNA binding sites contained within the metformin-responsive element. (D). The corresponding nucleotide sequences of the human, mouse and zebrafish NLK 3’UTRs across the metformin-responsive element. Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant * P

Techniques Used: Binding Assay, Construct, Luciferase, Transfection, Activity Assay, Expressing, Western Blot, Two Tailed Test

Metformin improves erythropoiesis in zebrafish model of DBA but not murine models. (A left) Murine fetal liver Kit+ progenitors expressing tetracylin-regulatable shRNA against RPS19 were cultured in the absence (grey columns) or presence (black columns) of doxycycline either alone, or treated with metformin. After 8 days, the number of ter119+ erythroblasts was calculated. (A right) RPL11+/lox mice were left untreated (grey columns) or treated (black columns) with tamoxifen for 5 weeks prior to isolation of bone marrow Lin-Kit+ progenitors. Progenitors were differentiated in the presence or absence of metformin for 8 days before assessing the number of Ter119+ erythroblasts. (B) In conjunction with flow cytometry, cultures were subjected to qRT-PCR for NLK expression and (C) kinase NLK kinase activity was assessed by in vitro kinase assay. (D) Zebrafish were reared and injected with control or rps19 -specific morpholino and treated with 20 mM metformin 4 to 5 hours post fertilization (hpf). At day 3, embryos were stained with o-dianizidine to detect hemoglobin. Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant * P
Figure Legend Snippet: Metformin improves erythropoiesis in zebrafish model of DBA but not murine models. (A left) Murine fetal liver Kit+ progenitors expressing tetracylin-regulatable shRNA against RPS19 were cultured in the absence (grey columns) or presence (black columns) of doxycycline either alone, or treated with metformin. After 8 days, the number of ter119+ erythroblasts was calculated. (A right) RPL11+/lox mice were left untreated (grey columns) or treated (black columns) with tamoxifen for 5 weeks prior to isolation of bone marrow Lin-Kit+ progenitors. Progenitors were differentiated in the presence or absence of metformin for 8 days before assessing the number of Ter119+ erythroblasts. (B) In conjunction with flow cytometry, cultures were subjected to qRT-PCR for NLK expression and (C) kinase NLK kinase activity was assessed by in vitro kinase assay. (D) Zebrafish were reared and injected with control or rps19 -specific morpholino and treated with 20 mM metformin 4 to 5 hours post fertilization (hpf). At day 3, embryos were stained with o-dianizidine to detect hemoglobin. Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant * P

Techniques Used: Expressing, shRNA, Cell Culture, Mouse Assay, Isolation, Flow Cytometry, Quantitative RT-PCR, Activity Assay, In Vitro, Kinase Assay, Injection, Staining, Two Tailed Test

Metformin improves erythropoiesis through suppression of NLK expression. (A) Control (shLuc – grey columns) or RPS19-insufficient (shRPS19 – black columns) progenitors were differentiated in erythroid media alone, vehicle or vehicle containing 50mM metformin or 5uM SD208 for 5 days. 5000 cells per treatment were lysed and immunopurified NLK was subjected to in vitro kinase assay to determine phosphorylation potential against 3 recognized NLK substrates; NLK itself (upper), c-Myb (middle) or raptor (bottom). (B) Simultaneously, qRT-PCR was performed to examine NLK (upper) and RPS19 (lower) mRNA expression. (C) Active NLK was purified from activated Kp53A1 cells and subjected to in vitro kinase assay in the presence of 0, 50 nM, 50 μM, or 50 mM metformin or SD208. The phosphorylation of NLK (upper), c-Myb (middle) and raptor (lower) was determined after 30 mins. (D) CD34+ progenitors were transduced with a combination of either shRNA against luciferase (shLuc – grey columns) or RPS19 (shRPS19 – black columns) and siRNA against a non-targeting sequence (NT) or NLK (siNLK). After 14 days differentiation in the presence or absence of metformin, cells were counted and the percentage of cells with surface expression of CD235 and CD11b were determined by flow cytometry, to yield the number of CD235+ erythroid (upper) and CD11b+ myeloid (lower) cells. The total number of cells is expressed as a percentage of the number of each cell type in the control (untreated/shLuc/NT). Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant * P
Figure Legend Snippet: Metformin improves erythropoiesis through suppression of NLK expression. (A) Control (shLuc – grey columns) or RPS19-insufficient (shRPS19 – black columns) progenitors were differentiated in erythroid media alone, vehicle or vehicle containing 50mM metformin or 5uM SD208 for 5 days. 5000 cells per treatment were lysed and immunopurified NLK was subjected to in vitro kinase assay to determine phosphorylation potential against 3 recognized NLK substrates; NLK itself (upper), c-Myb (middle) or raptor (bottom). (B) Simultaneously, qRT-PCR was performed to examine NLK (upper) and RPS19 (lower) mRNA expression. (C) Active NLK was purified from activated Kp53A1 cells and subjected to in vitro kinase assay in the presence of 0, 50 nM, 50 μM, or 50 mM metformin or SD208. The phosphorylation of NLK (upper), c-Myb (middle) and raptor (lower) was determined after 30 mins. (D) CD34+ progenitors were transduced with a combination of either shRNA against luciferase (shLuc – grey columns) or RPS19 (shRPS19 – black columns) and siRNA against a non-targeting sequence (NT) or NLK (siNLK). After 14 days differentiation in the presence or absence of metformin, cells were counted and the percentage of cells with surface expression of CD235 and CD11b were determined by flow cytometry, to yield the number of CD235+ erythroid (upper) and CD11b+ myeloid (lower) cells. The total number of cells is expressed as a percentage of the number of each cell type in the control (untreated/shLuc/NT). Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant * P

Techniques Used: Expressing, In Vitro, Kinase Assay, Quantitative RT-PCR, Purification, Transduction, shRNA, Luciferase, Sequencing, Flow Cytometry, Two Tailed Test

Metformin-mediated upregulation of miR-26a in human progenitors suppresses NLK expression. (A) Human CD34+ (upper) and murine Lin-Kit+ (lower) progenitors were differentiated for 8 days in the presence or absence of metformin and the levels of indicated miRNAs were assessed by qRT-PCR. (B) K562 cells stably expressing the luciferase gene coupled to the NLK 3’UTR were mock transfected, transfected with indicated miRNA mimetics, or treated with metformin, and cultured in the presence or absence of metformin for 72 hrs. After lysis, luciferase activity was determined and endogenous NLK and GAPDH protein expression was analyzed by Western blot. (C) K562 cells were mock transfected, or transfected with either miR-34 or miR-26 sponges, prior to being left untreated or treated with metformin for 72 hrs. Lysed cells were subjected to luciferase assay and western blotting to examine NLK and GAPDH protein expression. Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant * P
Figure Legend Snippet: Metformin-mediated upregulation of miR-26a in human progenitors suppresses NLK expression. (A) Human CD34+ (upper) and murine Lin-Kit+ (lower) progenitors were differentiated for 8 days in the presence or absence of metformin and the levels of indicated miRNAs were assessed by qRT-PCR. (B) K562 cells stably expressing the luciferase gene coupled to the NLK 3’UTR were mock transfected, transfected with indicated miRNA mimetics, or treated with metformin, and cultured in the presence or absence of metformin for 72 hrs. After lysis, luciferase activity was determined and endogenous NLK and GAPDH protein expression was analyzed by Western blot. (C) K562 cells were mock transfected, or transfected with either miR-34 or miR-26 sponges, prior to being left untreated or treated with metformin for 72 hrs. Lysed cells were subjected to luciferase assay and western blotting to examine NLK and GAPDH protein expression. Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant * P

Techniques Used: Expressing, Quantitative RT-PCR, Stable Transfection, Luciferase, Transfection, Cell Culture, Lysis, Activity Assay, Western Blot, Two Tailed Test

2) Product Images from "Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts"

Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

Journal: Scientific Reports

doi: 10.1038/s41598-019-41839-1

Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value
Figure Legend Snippet: Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value

Techniques Used:

Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.
Figure Legend Snippet: Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.

Techniques Used: Staining, Software, Significance Assay

Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.
Figure Legend Snippet: Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.

Techniques Used: Activation Assay

Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.
Figure Legend Snippet: Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.

Techniques Used: RNA Sequencing Assay, Transformation Assay

Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).
Figure Legend Snippet: Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).

Techniques Used:

Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.
Figure Legend Snippet: Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.

Techniques Used:

FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P
Figure Legend Snippet: FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P

Techniques Used: Expressing, Binding Assay, Sequencing, Immunofluorescence, Western Blot, Chromatin Immunoprecipitation

3) Product Images from "Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts"

Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

Journal: Scientific Reports

doi: 10.1038/s41598-019-41839-1

Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value
Figure Legend Snippet: Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value

Techniques Used:

Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.
Figure Legend Snippet: Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.

Techniques Used: Staining, Software, Significance Assay

Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.
Figure Legend Snippet: Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.

Techniques Used: Activation Assay

Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.
Figure Legend Snippet: Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.

Techniques Used: RNA Sequencing Assay, Transformation Assay

Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).
Figure Legend Snippet: Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).

Techniques Used:

Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.
Figure Legend Snippet: Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.

Techniques Used:

FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P
Figure Legend Snippet: FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P

Techniques Used: Expressing, Binding Assay, Sequencing, Immunofluorescence, Western Blot, Chromatin Immunoprecipitation

4) Product Images from "Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts"

Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

Journal: Scientific Reports

doi: 10.1038/s41598-019-41839-1

Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value
Figure Legend Snippet: Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value

Techniques Used:

Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.
Figure Legend Snippet: Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.

Techniques Used: Staining, Software, Significance Assay

Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.
Figure Legend Snippet: Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.

Techniques Used: Activation Assay

Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.
Figure Legend Snippet: Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.

Techniques Used: RNA Sequencing Assay, Transformation Assay

Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).
Figure Legend Snippet: Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).

Techniques Used:

Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.
Figure Legend Snippet: Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.

Techniques Used:

FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P
Figure Legend Snippet: FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P

Techniques Used: Expressing, Binding Assay, Sequencing, Immunofluorescence, Western Blot, Chromatin Immunoprecipitation

5) Product Images from "Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts"

Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

Journal: Scientific Reports

doi: 10.1038/s41598-019-41839-1

Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value
Figure Legend Snippet: Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value

Techniques Used:

Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.
Figure Legend Snippet: Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.

Techniques Used: Staining, Software, Significance Assay

Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.
Figure Legend Snippet: Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.

Techniques Used: Activation Assay

Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.
Figure Legend Snippet: Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.

Techniques Used: RNA Sequencing Assay, Transformation Assay

Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).
Figure Legend Snippet: Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).

Techniques Used:

Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.
Figure Legend Snippet: Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.

Techniques Used:

FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P
Figure Legend Snippet: FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P

Techniques Used: Expressing, Binding Assay, Sequencing, Immunofluorescence, Western Blot, Chromatin Immunoprecipitation

6) Product Images from "Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts"

Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

Journal: Scientific Reports

doi: 10.1038/s41598-019-41839-1

Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value
Figure Legend Snippet: Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value

Techniques Used:

Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.
Figure Legend Snippet: Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.

Techniques Used: Staining, Software, Significance Assay

Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.
Figure Legend Snippet: Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.

Techniques Used: Activation Assay

Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.
Figure Legend Snippet: Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.

Techniques Used: RNA Sequencing Assay, Transformation Assay

Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).
Figure Legend Snippet: Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).

Techniques Used:

Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.
Figure Legend Snippet: Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.

Techniques Used:

FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P
Figure Legend Snippet: FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P

Techniques Used: Expressing, Binding Assay, Sequencing, Immunofluorescence, Western Blot, Chromatin Immunoprecipitation

7) Product Images from "Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts"

Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

Journal: Scientific Reports

doi: 10.1038/s41598-019-41839-1

Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value
Figure Legend Snippet: Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value

Techniques Used:

Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.
Figure Legend Snippet: Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.

Techniques Used: Staining, Software, Significance Assay

Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.
Figure Legend Snippet: Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.

Techniques Used: Activation Assay

Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.
Figure Legend Snippet: Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.

Techniques Used: RNA Sequencing Assay, Transformation Assay

Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).
Figure Legend Snippet: Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).

Techniques Used:

Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.
Figure Legend Snippet: Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.

Techniques Used:

FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P
Figure Legend Snippet: FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P

Techniques Used: Expressing, Binding Assay, Sequencing, Immunofluorescence, Western Blot, Chromatin Immunoprecipitation

8) Product Images from "Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts"

Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

Journal: Scientific Reports

doi: 10.1038/s41598-019-41839-1

Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value
Figure Legend Snippet: Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value

Techniques Used:

Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.
Figure Legend Snippet: Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.

Techniques Used: Staining, Software, Significance Assay

Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.
Figure Legend Snippet: Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.

Techniques Used: Activation Assay

Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.
Figure Legend Snippet: Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.

Techniques Used: RNA Sequencing Assay, Transformation Assay

Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).
Figure Legend Snippet: Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).

Techniques Used:

Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.
Figure Legend Snippet: Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.

Techniques Used:

FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P
Figure Legend Snippet: FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P

Techniques Used: Expressing, Binding Assay, Sequencing, Immunofluorescence, Western Blot, Chromatin Immunoprecipitation

9) Product Images from "Pharmacological reactivation of MYC-dependent apoptosis induces susceptibility to anti-PD-1 immunotherapy"

Article Title: Pharmacological reactivation of MYC-dependent apoptosis induces susceptibility to anti-PD-1 immunotherapy

Journal: Nature Communications

doi: 10.1038/s41467-019-08541-2

ABn treatment reduces viability of MYC-high triple-negative breast cancer cell lines, inhibits tumor growth, and extends survival in patient-derived xenografts. a . The drugs were navitoclax, venetoclax, a BCL-X L -selective inhibitor A-1155463 and an MCL-1-selective inhibitor A-1210477. b AB treatment-induces apoptosis in MYC-high PDEc. The cultures were treated with DMSO, 1 μM navitoclax, 10 mM metformin or combination for 24 h. Blinded scoring of apoptosis was carried out for all samples. c MYC expression in 17 triple-negative breast cancer cell lines. MYC index: MYC intensity normalized to a blot-to-blot reference sample, highest intensity band (HCC1599) and loading control. N.B. HCC1599 excluded from the final analysis due to poor growth. Tubulin: Loading control. d The effect of AB treatment on the viability of MYC-low and MYC-high TNBC cell lines. The upper panel shows kill curves and the dashed line marks the EC 20 . The lower panel shows metformin effect at EC 20 of navitoclax. Student’s t -test (unpaired), SD. e Summary table of drug treatments in TNBC cell lines. The cell lines were categorized according to MYC index (0 = undetectable MYC expression; + to +++ = relative MYC expression level). Blue boxes indicate statistically non-significant and pink boxes statistically significant differences between each single-agent treatment and the corresponding combination. Student’s t -test (unpaired). f Representative images of tumors developing in TNBC-PDX mice. Black arrows: Primary tumors developing at the site of the tumor grafts; blue arrows: Metastases. g TNBC-PDX tumors retain MYC expression during in vivo passaging. Tumor generations G1–G3. h Effect of AB treatment on tumor growth and survival in cohorts of TNBC-PDX mice. The mice were treated with vehicle, 100 mg/kg/d navitoclax, 600 mg/kg/d metformin or the combination for 21 days, and followed up until day 60. Student’s t -test (unpaired), SEM. In the survival graph: P -value: Significant difference between vehicle and AB-treated cohorts
Figure Legend Snippet: ABn treatment reduces viability of MYC-high triple-negative breast cancer cell lines, inhibits tumor growth, and extends survival in patient-derived xenografts. a . The drugs were navitoclax, venetoclax, a BCL-X L -selective inhibitor A-1155463 and an MCL-1-selective inhibitor A-1210477. b AB treatment-induces apoptosis in MYC-high PDEc. The cultures were treated with DMSO, 1 μM navitoclax, 10 mM metformin or combination for 24 h. Blinded scoring of apoptosis was carried out for all samples. c MYC expression in 17 triple-negative breast cancer cell lines. MYC index: MYC intensity normalized to a blot-to-blot reference sample, highest intensity band (HCC1599) and loading control. N.B. HCC1599 excluded from the final analysis due to poor growth. Tubulin: Loading control. d The effect of AB treatment on the viability of MYC-low and MYC-high TNBC cell lines. The upper panel shows kill curves and the dashed line marks the EC 20 . The lower panel shows metformin effect at EC 20 of navitoclax. Student’s t -test (unpaired), SD. e Summary table of drug treatments in TNBC cell lines. The cell lines were categorized according to MYC index (0 = undetectable MYC expression; + to +++ = relative MYC expression level). Blue boxes indicate statistically non-significant and pink boxes statistically significant differences between each single-agent treatment and the corresponding combination. Student’s t -test (unpaired). f Representative images of tumors developing in TNBC-PDX mice. Black arrows: Primary tumors developing at the site of the tumor grafts; blue arrows: Metastases. g TNBC-PDX tumors retain MYC expression during in vivo passaging. Tumor generations G1–G3. h Effect of AB treatment on tumor growth and survival in cohorts of TNBC-PDX mice. The mice were treated with vehicle, 100 mg/kg/d navitoclax, 600 mg/kg/d metformin or the combination for 21 days, and followed up until day 60. Student’s t -test (unpaired), SEM. In the survival graph: P -value: Significant difference between vehicle and AB-treated cohorts

Techniques Used: Derivative Assay, Expressing, Mouse Assay, In Vivo, Passaging

ABn treatment inhibits tumor growth and extends survival of mice syngrafted with WapMyc tumors. a Combination treatment protocol for orthotopically syngrafted WapMyc tumors. The mice were treated with a vehicle, navitoclax, and two concentrations of metformin alone or in combination with navitoclax for 21 days. Immunoprofiling samples were collected right after the drug treatments or after a follow-up period. In the follow-up period the samples were collected when the tumors reached Ø 2 cm and the mice had to be killed. b ABn treatment-induces BIM activation in vivo. The level of BIM immunostaining was scored in IHC samples from treated tumors using a scale of 1–4 (representative examples are shown in the figure). The samples were blinded for analysis. Student’s t -test (unpaired), SD. c ABn treatment-induced “apoptotic ponds”. Representative images of tumor samples stained for cleaved caspase-3. d ABn treatment stimulates T-cell infiltration. WapMyc tumor samples were isolated from mice killed after the 21-day AB treatment period. Representative images are shown and the data below show average and SD of CD4+ or CD8+ T cells in the tumors. Immunohistochemical stainings were performed for at least 45 tumors per antibody and 3–6 field of view (fov) per tumor were analyzed. Student’s t -test (unpaired). e ABn treatment-induced changes in proportions of peripheral lymphocytes. N (vehicle) = 7 mice, N (ABn) = 4 mice. Blood was collected after 21 days treatment with either vehicle or ABn. Student’s t -test (unpaired), SD. f Tumor growth in ABn-treated mice. Student’s t -test (unpaired), SEM. g , h ABn treatment extends survival. P -values: Difference between the vehicle and ABn-treated cohorts. i Flow cytometry-based immunoprofiling of vehicle and ABn (navitoclax+metformin)-treated tumors. The heatmaps show fold-change compared to control. N = 6 (vehicle), N = 2 Navitoclax+Metformin. j Post-treatment ratios of tumor-infiltrating PD-1+CD8+ T cells. Student’s t -test (unpaired), SD
Figure Legend Snippet: ABn treatment inhibits tumor growth and extends survival of mice syngrafted with WapMyc tumors. a Combination treatment protocol for orthotopically syngrafted WapMyc tumors. The mice were treated with a vehicle, navitoclax, and two concentrations of metformin alone or in combination with navitoclax for 21 days. Immunoprofiling samples were collected right after the drug treatments or after a follow-up period. In the follow-up period the samples were collected when the tumors reached Ø 2 cm and the mice had to be killed. b ABn treatment-induces BIM activation in vivo. The level of BIM immunostaining was scored in IHC samples from treated tumors using a scale of 1–4 (representative examples are shown in the figure). The samples were blinded for analysis. Student’s t -test (unpaired), SD. c ABn treatment-induced “apoptotic ponds”. Representative images of tumor samples stained for cleaved caspase-3. d ABn treatment stimulates T-cell infiltration. WapMyc tumor samples were isolated from mice killed after the 21-day AB treatment period. Representative images are shown and the data below show average and SD of CD4+ or CD8+ T cells in the tumors. Immunohistochemical stainings were performed for at least 45 tumors per antibody and 3–6 field of view (fov) per tumor were analyzed. Student’s t -test (unpaired). e ABn treatment-induced changes in proportions of peripheral lymphocytes. N (vehicle) = 7 mice, N (ABn) = 4 mice. Blood was collected after 21 days treatment with either vehicle or ABn. Student’s t -test (unpaired), SD. f Tumor growth in ABn-treated mice. Student’s t -test (unpaired), SEM. g , h ABn treatment extends survival. P -values: Difference between the vehicle and ABn-treated cohorts. i Flow cytometry-based immunoprofiling of vehicle and ABn (navitoclax+metformin)-treated tumors. The heatmaps show fold-change compared to control. N = 6 (vehicle), N = 2 Navitoclax+Metformin. j Post-treatment ratios of tumor-infiltrating PD-1+CD8+ T cells. Student’s t -test (unpaired), SD

Techniques Used: Mouse Assay, Activation Assay, In Vivo, Immunostaining, Immunohistochemistry, Staining, Isolation, Flow Cytometry, Cytometry

ABv treatment with anti-PD-1 immunotherapy offers durable therapeutic response. a Treatment protocol for syngrafted WapMyc tumors. The mice received vehicle, venetoclax, metformin, or venetoclax+metformin (ABv) for 21 days. Tumors were surgically removed, and mice were given adjuvant treatments. ABv was given every day for 1 week, and control IgG or anti-PD-1 was given every third day, four times in total. The mice were followed up until the tumors reached Ø 2 cm. Flow cytometry-based immunoprofilings were done after the adjuvant treatment and after the follow-up. b Tumor growth during the neoadjuvant treatment. Student’s t -test (unpaired), SEM. N = 8 mice/treatment group. c Tumor volume during the adjuvant treatment (d7 from surgery). Student’s t -test (unpaired), SD. N = 4 mice/treatment group. d Tumor volume after the adjuvant treatment (d14 from surgery). Student’s t -test (unpaired), SD. e Box plot of tumor volumes in ABv+IgG- vs anti-PD-1-treated groups during and after the adjuvant treatment. After the adjuvant treatment, the only tumor in the ABv+anti-PD-1 group was surgically biopsied for immunoprofiling. f Tumor immunoprofiles after the adjuvant treatment. The heatmap shows fold-changes compared to vehicle+IgG. N = 4 mice/group, except N = 1 in the biopsy. Cell populations derived from parental populations with less than 90 cells are marked with asterisk (*). g Survival of the mice. P -value: Difference between vehicle+IgG and ABv+anti-PD-1-treated mice. h Total white blood cell (WBC) and red blood cell (RBC) counts in the treatment groups. Student’s t -test (unpaired), SD. i Liver injury measured by plasma ALAT levels. Student’s t -test (unpaired), SD. j Average tumor sizes during (d11) adjuvant treatment with vehicle+anti-PD-1, paclitaxel+anti-PD-1, or ABv+anti-PD-1, and after the drug withdrawal (d15). Length of one treatment cycle is 10 days. p -value denotes the difference between post-adjuvant vehicle+anti-PD-1 and ABv+anti-PD-1. N = 3 (vehicle+anti-PD-1), 7 (Paclitaxel+anti-PD-1), 10 (1 cycle of ABv+anti-PD-1). SD, student’s t -test. k Survival of the mice. N = 6 (Paclitaxel+anti-PD-1), 2 (1 cycle of ABv+anti-PD-1), 3 (3 cycles of ABv+anti-PD-1)
Figure Legend Snippet: ABv treatment with anti-PD-1 immunotherapy offers durable therapeutic response. a Treatment protocol for syngrafted WapMyc tumors. The mice received vehicle, venetoclax, metformin, or venetoclax+metformin (ABv) for 21 days. Tumors were surgically removed, and mice were given adjuvant treatments. ABv was given every day for 1 week, and control IgG or anti-PD-1 was given every third day, four times in total. The mice were followed up until the tumors reached Ø 2 cm. Flow cytometry-based immunoprofilings were done after the adjuvant treatment and after the follow-up. b Tumor growth during the neoadjuvant treatment. Student’s t -test (unpaired), SEM. N = 8 mice/treatment group. c Tumor volume during the adjuvant treatment (d7 from surgery). Student’s t -test (unpaired), SD. N = 4 mice/treatment group. d Tumor volume after the adjuvant treatment (d14 from surgery). Student’s t -test (unpaired), SD. e Box plot of tumor volumes in ABv+IgG- vs anti-PD-1-treated groups during and after the adjuvant treatment. After the adjuvant treatment, the only tumor in the ABv+anti-PD-1 group was surgically biopsied for immunoprofiling. f Tumor immunoprofiles after the adjuvant treatment. The heatmap shows fold-changes compared to vehicle+IgG. N = 4 mice/group, except N = 1 in the biopsy. Cell populations derived from parental populations with less than 90 cells are marked with asterisk (*). g Survival of the mice. P -value: Difference between vehicle+IgG and ABv+anti-PD-1-treated mice. h Total white blood cell (WBC) and red blood cell (RBC) counts in the treatment groups. Student’s t -test (unpaired), SD. i Liver injury measured by plasma ALAT levels. Student’s t -test (unpaired), SD. j Average tumor sizes during (d11) adjuvant treatment with vehicle+anti-PD-1, paclitaxel+anti-PD-1, or ABv+anti-PD-1, and after the drug withdrawal (d15). Length of one treatment cycle is 10 days. p -value denotes the difference between post-adjuvant vehicle+anti-PD-1 and ABv+anti-PD-1. N = 3 (vehicle+anti-PD-1), 7 (Paclitaxel+anti-PD-1), 10 (1 cycle of ABv+anti-PD-1). SD, student’s t -test. k Survival of the mice. N = 6 (Paclitaxel+anti-PD-1), 2 (1 cycle of ABv+anti-PD-1), 3 (3 cycles of ABv+anti-PD-1)

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Derivative Assay

AMPK activation potentiates MYC-dependent apoptosis by ABT-737. a Drug-targeted pathways. b Protocol for drug combination testing. MCF10A MycER cells were allowed to form mammospheres for 24 h. MYC was activated with 100 nM 4OHT for 24 h followed by 24 h incubation with drug combinations. c Combination drug testing to identify pharmacological triggers of MYC-dependent apoptosis. The drugs were administered as single agents or as ABT-737 with and without MYC activation. Each drug was tested in two concentrations. N = 3 biological repeats. Student’s t -test (unpaired), SD. d The relative level of apoptosis with and without MYC activity (+MYC: −MYC ratio). The ratio was calculated from fold-change in c . e Representative images of drug-treated mammospheres. f , g Sensitization to MYC-dependent apoptosis by 100 nM ABT-737 with either 1 μM A-769662 or 10 mM metformin. Mammospheres were treated as in b . Student’s t -test (unpaired), N = 3 biological replicates, SD. h Activation of AMPK alone does not sensitize to MYC-dependent apoptosis. N = 3 biological repeats, SD. i CRISPR/dead-Cas9-mediated transcriptional activation of MYC. HEK293 and MCF10A cells were transduced with vectors encoding dCas9-VP192 transcription-activating construct and MYC-promoter-targeted guide-RNAs. Western blot analysis shows MYC expression levels after 72 h treatment with doxycycline (DOX) and trimethoprim (TMP). Lamin B: Loading control. j CRISPR-mediated induction of endogenous MYC sensitizes cells to apoptosis by ABT-737+A-769662. Mammospheres with and without dCas9-VP192+MYC-gRNA were treated and analyzed as in b . Student’s t -test (unpaired), N = 3 biological replicates, SD
Figure Legend Snippet: AMPK activation potentiates MYC-dependent apoptosis by ABT-737. a Drug-targeted pathways. b Protocol for drug combination testing. MCF10A MycER cells were allowed to form mammospheres for 24 h. MYC was activated with 100 nM 4OHT for 24 h followed by 24 h incubation with drug combinations. c Combination drug testing to identify pharmacological triggers of MYC-dependent apoptosis. The drugs were administered as single agents or as ABT-737 with and without MYC activation. Each drug was tested in two concentrations. N = 3 biological repeats. Student’s t -test (unpaired), SD. d The relative level of apoptosis with and without MYC activity (+MYC: −MYC ratio). The ratio was calculated from fold-change in c . e Representative images of drug-treated mammospheres. f , g Sensitization to MYC-dependent apoptosis by 100 nM ABT-737 with either 1 μM A-769662 or 10 mM metformin. Mammospheres were treated as in b . Student’s t -test (unpaired), N = 3 biological replicates, SD. h Activation of AMPK alone does not sensitize to MYC-dependent apoptosis. N = 3 biological repeats, SD. i CRISPR/dead-Cas9-mediated transcriptional activation of MYC. HEK293 and MCF10A cells were transduced with vectors encoding dCas9-VP192 transcription-activating construct and MYC-promoter-targeted guide-RNAs. Western blot analysis shows MYC expression levels after 72 h treatment with doxycycline (DOX) and trimethoprim (TMP). Lamin B: Loading control. j CRISPR-mediated induction of endogenous MYC sensitizes cells to apoptosis by ABT-737+A-769662. Mammospheres with and without dCas9-VP192+MYC-gRNA were treated and analyzed as in b . Student’s t -test (unpaired), N = 3 biological replicates, SD

Techniques Used: Activation Assay, Incubation, Activity Assay, CRISPR, Transduction, Construct, Western Blot, Expressing

10) Product Images from "Pharmacological reactivation of MYC-dependent apoptosis induces susceptibility to anti-PD-1 immunotherapy"

Article Title: Pharmacological reactivation of MYC-dependent apoptosis induces susceptibility to anti-PD-1 immunotherapy

Journal: Nature Communications

doi: 10.1038/s41467-019-08541-2

ABn treatment reduces viability of MYC-high triple-negative breast cancer cell lines, inhibits tumor growth, and extends survival in patient-derived xenografts. a A summary of the results of BH3 mimetic screen in MCF10A MycER cells. The numbers refer to fold change as in Supplementary Figure 4A . The drugs were navitoclax, venetoclax, a BCL-X L -selective inhibitor A-1155463 and an MCL-1-selective inhibitor A-1210477. b AB treatment-induces apoptosis in MYC-high PDEc. The cultures were treated with DMSO, 1 μM navitoclax, 10 mM metformin or combination for 24 h. Blinded scoring of apoptosis was carried out for all samples. c MYC expression in 17 triple-negative breast cancer cell lines. MYC index: MYC intensity normalized to a blot-to-blot reference sample, highest intensity band (HCC1599) and loading control. N.B. HCC1599 excluded from the final analysis due to poor growth. Tubulin: Loading control. d The effect of AB treatment on the viability of MYC-low and MYC-high TNBC cell lines. The upper panel shows kill curves and the dashed line marks the EC 20 of navitoclax (20% reduction in survival); see also Fig.S 4E . The lower panel shows metformin effect at EC 20 of navitoclax. Student’s t -test (unpaired), SD. e Summary table of drug treatments in TNBC cell lines. The cell lines were categorized according to MYC index (0 = undetectable MYC expression; + to +++ = relative MYC expression level). Blue boxes indicate statistically non-significant and pink boxes statistically significant differences between each single-agent treatment and the corresponding combination. Student’s t -test (unpaired). f Representative images of tumors developing in TNBC-PDX mice. Black arrows: Primary tumors developing at the site of the tumor grafts; blue arrows: Metastases. g TNBC-PDX tumors retain MYC expression during in vivo passaging. Tumor generations G1–G3. h Effect of AB treatment on tumor growth and survival in cohorts of TNBC-PDX mice. The mice were treated with vehicle, 100 mg/kg/d navitoclax, 600 mg/kg/d metformin or the combination for 21 days, and followed up until day 60. Student’s t -test (unpaired), SEM. In the survival graph: P -value: Significant difference between vehicle and AB-treated cohorts
Figure Legend Snippet: ABn treatment reduces viability of MYC-high triple-negative breast cancer cell lines, inhibits tumor growth, and extends survival in patient-derived xenografts. a A summary of the results of BH3 mimetic screen in MCF10A MycER cells. The numbers refer to fold change as in Supplementary Figure 4A . The drugs were navitoclax, venetoclax, a BCL-X L -selective inhibitor A-1155463 and an MCL-1-selective inhibitor A-1210477. b AB treatment-induces apoptosis in MYC-high PDEc. The cultures were treated with DMSO, 1 μM navitoclax, 10 mM metformin or combination for 24 h. Blinded scoring of apoptosis was carried out for all samples. c MYC expression in 17 triple-negative breast cancer cell lines. MYC index: MYC intensity normalized to a blot-to-blot reference sample, highest intensity band (HCC1599) and loading control. N.B. HCC1599 excluded from the final analysis due to poor growth. Tubulin: Loading control. d The effect of AB treatment on the viability of MYC-low and MYC-high TNBC cell lines. The upper panel shows kill curves and the dashed line marks the EC 20 of navitoclax (20% reduction in survival); see also Fig.S 4E . The lower panel shows metformin effect at EC 20 of navitoclax. Student’s t -test (unpaired), SD. e Summary table of drug treatments in TNBC cell lines. The cell lines were categorized according to MYC index (0 = undetectable MYC expression; + to +++ = relative MYC expression level). Blue boxes indicate statistically non-significant and pink boxes statistically significant differences between each single-agent treatment and the corresponding combination. Student’s t -test (unpaired). f Representative images of tumors developing in TNBC-PDX mice. Black arrows: Primary tumors developing at the site of the tumor grafts; blue arrows: Metastases. g TNBC-PDX tumors retain MYC expression during in vivo passaging. Tumor generations G1–G3. h Effect of AB treatment on tumor growth and survival in cohorts of TNBC-PDX mice. The mice were treated with vehicle, 100 mg/kg/d navitoclax, 600 mg/kg/d metformin or the combination for 21 days, and followed up until day 60. Student’s t -test (unpaired), SEM. In the survival graph: P -value: Significant difference between vehicle and AB-treated cohorts

Techniques Used: Derivative Assay, Expressing, Mouse Assay, In Vivo, Passaging

ABn treatment inhibits tumor growth and extends survival of mice syngrafted with WapMyc tumors. a Combination treatment protocol for orthotopically syngrafted WapMyc tumors. The mice were treated with a vehicle, navitoclax, and two concentrations of metformin alone or in combination with navitoclax for 21 days. Immunoprofiling samples were collected right after the drug treatments or after a follow-up period. In the follow-up period the samples were collected when the tumors reached Ø 2 cm and the mice had to be killed. b ABn treatment-induces BIM activation in vivo. The level of BIM immunostaining was scored in IHC samples from treated tumors using a scale of 1–4 (representative examples are shown in the figure). The samples were blinded for analysis. Student’s t -test (unpaired), SD. c ABn treatment-induced “apoptotic ponds”. Representative images of tumor samples stained for cleaved caspase-3. d ABn treatment stimulates T-cell infiltration. WapMyc tumor samples were isolated from mice killed after the 21-day AB treatment period. Representative images are shown and the data below show average and SD of CD4+ or CD8+ T cells in the tumors. Immunohistochemical stainings were performed for at least 45 tumors per antibody and 3–6 field of view (fov) per tumor were analyzed. Student’s t -test (unpaired). e ABn treatment-induced changes in proportions of peripheral lymphocytes. N (vehicle) = 7 mice, N (ABn) = 4 mice. Blood was collected after 21 days treatment with either vehicle or ABn. Student’s t -test (unpaired), SD. f Tumor growth in ABn-treated mice. Student’s t -test (unpaired), SEM. g , h ABn treatment extends survival. P -values: Difference between the vehicle and ABn-treated cohorts. i Flow cytometry-based immunoprofiling of vehicle and ABn (navitoclax+metformin)-treated tumors. The heatmaps show fold-change compared to control. N = 6 (vehicle), N = 2 Navitoclax+Metformin. j Post-treatment ratios of tumor-infiltrating PD-1+CD8+ T cells. Student’s t -test (unpaired), SD
Figure Legend Snippet: ABn treatment inhibits tumor growth and extends survival of mice syngrafted with WapMyc tumors. a Combination treatment protocol for orthotopically syngrafted WapMyc tumors. The mice were treated with a vehicle, navitoclax, and two concentrations of metformin alone or in combination with navitoclax for 21 days. Immunoprofiling samples were collected right after the drug treatments or after a follow-up period. In the follow-up period the samples were collected when the tumors reached Ø 2 cm and the mice had to be killed. b ABn treatment-induces BIM activation in vivo. The level of BIM immunostaining was scored in IHC samples from treated tumors using a scale of 1–4 (representative examples are shown in the figure). The samples were blinded for analysis. Student’s t -test (unpaired), SD. c ABn treatment-induced “apoptotic ponds”. Representative images of tumor samples stained for cleaved caspase-3. d ABn treatment stimulates T-cell infiltration. WapMyc tumor samples were isolated from mice killed after the 21-day AB treatment period. Representative images are shown and the data below show average and SD of CD4+ or CD8+ T cells in the tumors. Immunohistochemical stainings were performed for at least 45 tumors per antibody and 3–6 field of view (fov) per tumor were analyzed. Student’s t -test (unpaired). e ABn treatment-induced changes in proportions of peripheral lymphocytes. N (vehicle) = 7 mice, N (ABn) = 4 mice. Blood was collected after 21 days treatment with either vehicle or ABn. Student’s t -test (unpaired), SD. f Tumor growth in ABn-treated mice. Student’s t -test (unpaired), SEM. g , h ABn treatment extends survival. P -values: Difference between the vehicle and ABn-treated cohorts. i Flow cytometry-based immunoprofiling of vehicle and ABn (navitoclax+metformin)-treated tumors. The heatmaps show fold-change compared to control. N = 6 (vehicle), N = 2 Navitoclax+Metformin. j Post-treatment ratios of tumor-infiltrating PD-1+CD8+ T cells. Student’s t -test (unpaired), SD

Techniques Used: Mouse Assay, Activation Assay, In Vivo, Immunostaining, Immunohistochemistry, Staining, Isolation, Flow Cytometry, Cytometry

ABv treatment with anti-PD-1 immunotherapy offers durable therapeutic response. a Treatment protocol for syngrafted WapMyc tumors. The mice received vehicle, venetoclax, metformin, or venetoclax+metformin (ABv) for 21 days. Tumors were surgically removed, and mice were given adjuvant treatments. ABv was given every day for 1 week, and control IgG or anti-PD-1 was given every third day, four times in total. The mice were followed up until the tumors reached Ø 2 cm. Flow cytometry-based immunoprofilings were done after the adjuvant treatment and after the follow-up. b Tumor growth during the neoadjuvant treatment. Student’s t -test (unpaired), SEM. N = 8 mice/treatment group. c Tumor volume during the adjuvant treatment (d7 from surgery). Student’s t -test (unpaired), SD. N = 4 mice/treatment group. d Tumor volume after the adjuvant treatment (d14 from surgery). Student’s t -test (unpaired), SD. e Box plot of tumor volumes in ABv+IgG- vs anti-PD-1-treated groups during and after the adjuvant treatment. After the adjuvant treatment, the only tumor in the ABv+anti-PD-1 group was surgically biopsied for immunoprofiling. f Tumor immunoprofiles after the adjuvant treatment. The heatmap shows fold-changes compared to vehicle+IgG. N = 4 mice/group, except N = 1 in the biopsy. Cell populations derived from parental populations with less than 90 cells are marked with asterisk (*). g Survival of the mice. P -value: Difference between vehicle+IgG and ABv+anti-PD-1-treated mice. h Total white blood cell (WBC) and red blood cell (RBC) counts in the treatment groups. Student’s t -test (unpaired), SD. i Liver injury measured by plasma ALAT levels. Student’s t -test (unpaired), SD. j Average tumor sizes during (d11) adjuvant treatment with vehicle+anti-PD-1, paclitaxel+anti-PD-1, or ABv+anti-PD-1, and after the drug withdrawal (d15). Length of one treatment cycle is 10 days. p -value denotes the difference between post-adjuvant vehicle+anti-PD-1 and ABv+anti-PD-1. N = 3 (vehicle+anti-PD-1), 7 (Paclitaxel+anti-PD-1), 10 (1 cycle of ABv+anti-PD-1). SD, student’s t -test. k Survival of the mice. N = 6 (Paclitaxel+anti-PD-1), 2 (1 cycle of ABv+anti-PD-1), 3 (3 cycles of ABv+anti-PD-1)
Figure Legend Snippet: ABv treatment with anti-PD-1 immunotherapy offers durable therapeutic response. a Treatment protocol for syngrafted WapMyc tumors. The mice received vehicle, venetoclax, metformin, or venetoclax+metformin (ABv) for 21 days. Tumors were surgically removed, and mice were given adjuvant treatments. ABv was given every day for 1 week, and control IgG or anti-PD-1 was given every third day, four times in total. The mice were followed up until the tumors reached Ø 2 cm. Flow cytometry-based immunoprofilings were done after the adjuvant treatment and after the follow-up. b Tumor growth during the neoadjuvant treatment. Student’s t -test (unpaired), SEM. N = 8 mice/treatment group. c Tumor volume during the adjuvant treatment (d7 from surgery). Student’s t -test (unpaired), SD. N = 4 mice/treatment group. d Tumor volume after the adjuvant treatment (d14 from surgery). Student’s t -test (unpaired), SD. e Box plot of tumor volumes in ABv+IgG- vs anti-PD-1-treated groups during and after the adjuvant treatment. After the adjuvant treatment, the only tumor in the ABv+anti-PD-1 group was surgically biopsied for immunoprofiling. f Tumor immunoprofiles after the adjuvant treatment. The heatmap shows fold-changes compared to vehicle+IgG. N = 4 mice/group, except N = 1 in the biopsy. Cell populations derived from parental populations with less than 90 cells are marked with asterisk (*). g Survival of the mice. P -value: Difference between vehicle+IgG and ABv+anti-PD-1-treated mice. h Total white blood cell (WBC) and red blood cell (RBC) counts in the treatment groups. Student’s t -test (unpaired), SD. i Liver injury measured by plasma ALAT levels. Student’s t -test (unpaired), SD. j Average tumor sizes during (d11) adjuvant treatment with vehicle+anti-PD-1, paclitaxel+anti-PD-1, or ABv+anti-PD-1, and after the drug withdrawal (d15). Length of one treatment cycle is 10 days. p -value denotes the difference between post-adjuvant vehicle+anti-PD-1 and ABv+anti-PD-1. N = 3 (vehicle+anti-PD-1), 7 (Paclitaxel+anti-PD-1), 10 (1 cycle of ABv+anti-PD-1). SD, student’s t -test. k Survival of the mice. N = 6 (Paclitaxel+anti-PD-1), 2 (1 cycle of ABv+anti-PD-1), 3 (3 cycles of ABv+anti-PD-1)

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Derivative Assay

AMPK activation potentiates MYC-dependent apoptosis by ABT-737. a Drug-targeted pathways. b Protocol for drug combination testing. MCF10A MycER cells were allowed to form mammospheres for 24 h. MYC was activated with 100 nM 4OHT for 24 h followed by 24 h incubation with drug combinations. c Combination drug testing to identify pharmacological triggers of MYC-dependent apoptosis. The drugs were administered as single agents or as ABT-737 with and without MYC activation. Each drug was tested in two concentrations. N = 3 biological repeats. Student’s t -test (unpaired), SD. d The relative level of apoptosis with and without MYC activity (+MYC: −MYC ratio). The ratio was calculated from fold-change in c . e Representative images of drug-treated mammospheres. f , g Sensitization to MYC-dependent apoptosis by 100 nM ABT-737 with either 1 μM A-769662 or 10 mM metformin. Mammospheres were treated as in b . Student’s t -test (unpaired), N = 3 biological replicates, SD. h Activation of AMPK alone does not sensitize to MYC-dependent apoptosis. N = 3 biological repeats, SD. i CRISPR/dead-Cas9-mediated transcriptional activation of MYC. HEK293 and MCF10A cells were transduced with vectors encoding dCas9-VP192 transcription-activating construct and MYC-promoter-targeted guide-RNAs. Western blot analysis shows MYC expression levels after 72 h treatment with doxycycline (DOX) and trimethoprim (TMP). Lamin B: Loading control. j CRISPR-mediated induction of endogenous MYC sensitizes cells to apoptosis by ABT-737+A-769662. Mammospheres with and without dCas9-VP192+MYC-gRNA were treated and analyzed as in b . Student’s t -test (unpaired), N = 3 biological replicates, SD
Figure Legend Snippet: AMPK activation potentiates MYC-dependent apoptosis by ABT-737. a Drug-targeted pathways. b Protocol for drug combination testing. MCF10A MycER cells were allowed to form mammospheres for 24 h. MYC was activated with 100 nM 4OHT for 24 h followed by 24 h incubation with drug combinations. c Combination drug testing to identify pharmacological triggers of MYC-dependent apoptosis. The drugs were administered as single agents or as ABT-737 with and without MYC activation. Each drug was tested in two concentrations. N = 3 biological repeats. Student’s t -test (unpaired), SD. d The relative level of apoptosis with and without MYC activity (+MYC: −MYC ratio). The ratio was calculated from fold-change in c . e Representative images of drug-treated mammospheres. f , g Sensitization to MYC-dependent apoptosis by 100 nM ABT-737 with either 1 μM A-769662 or 10 mM metformin. Mammospheres were treated as in b . Student’s t -test (unpaired), N = 3 biological replicates, SD. h Activation of AMPK alone does not sensitize to MYC-dependent apoptosis. N = 3 biological repeats, SD. i CRISPR/dead-Cas9-mediated transcriptional activation of MYC. HEK293 and MCF10A cells were transduced with vectors encoding dCas9-VP192 transcription-activating construct and MYC-promoter-targeted guide-RNAs. Western blot analysis shows MYC expression levels after 72 h treatment with doxycycline (DOX) and trimethoprim (TMP). Lamin B: Loading control. j CRISPR-mediated induction of endogenous MYC sensitizes cells to apoptosis by ABT-737+A-769662. Mammospheres with and without dCas9-VP192+MYC-gRNA were treated and analyzed as in b . Student’s t -test (unpaired), N = 3 biological replicates, SD

Techniques Used: Activation Assay, Incubation, Activity Assay, CRISPR, Transduction, Construct, Western Blot, Expressing

11) Product Images from "Body Weight Considerations in the Management of Type 2 Diabetes"

Article Title: Body Weight Considerations in the Management of Type 2 Diabetes

Journal: Advances in Therapy

doi: 10.1007/s12325-018-0824-8

Profiles of antidiabetic medications. AGI alpha-glucosidase inhibitor, ASCVD atherosclerotic cardiovascular disease, BCR-QR bromocriptine qui release, CHF congestive heart failure, COLSVL colesevelam, DPP-4i dipeptidyl peptidase 4 inhibitor, FDA US Food and Drug Administration, GI Sx gastrointestinal side effects, GLN glinides, GLP-1 RA glucagon-like peptide-1 receptor agonist, MET metformin, PRAML pramlintide, SGLT-2i sodium glucose co-transporter 2 inhibitor, SU sulfonylurea, TZD thiazolidinedione Reprinted with permission from American Association of Clinical Endocrinologists © 2018 AACE. Garber AJ, Abrahamson MJ, Barzilay JI, et al. AACE/ACE comprehensive type 2 diabetes management algorithm 2018. Endocr Pract. 2018;24:91–120
Figure Legend Snippet: Profiles of antidiabetic medications. AGI alpha-glucosidase inhibitor, ASCVD atherosclerotic cardiovascular disease, BCR-QR bromocriptine qui release, CHF congestive heart failure, COLSVL colesevelam, DPP-4i dipeptidyl peptidase 4 inhibitor, FDA US Food and Drug Administration, GI Sx gastrointestinal side effects, GLN glinides, GLP-1 RA glucagon-like peptide-1 receptor agonist, MET metformin, PRAML pramlintide, SGLT-2i sodium glucose co-transporter 2 inhibitor, SU sulfonylurea, TZD thiazolidinedione Reprinted with permission from American Association of Clinical Endocrinologists © 2018 AACE. Garber AJ, Abrahamson MJ, Barzilay JI, et al. AACE/ACE comprehensive type 2 diabetes management algorithm 2018. Endocr Pract. 2018;24:91–120

Techniques Used:

12) Product Images from "AMPK Re-Activation Suppresses Hepatic Steatosis but its Downregulation Does Not Promote Fatty Liver Development"

Article Title: AMPK Re-Activation Suppresses Hepatic Steatosis but its Downregulation Does Not Promote Fatty Liver Development

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2018.01.008

Indirect and small-molecule AMPK activators inhibit lipid synthesis in primary human hepatocytes. (A) Assessment of AMPK subunit levels in primary human and mouse hepatocytes by western blotting with the indicated antibodies. (B-J) Human primary hepatocytes were cultured in the absence or presence of various concentrations of A-769662, C13, AICAR or metformin, alone or in combination, at the indicated concentrations for 3 h. (B) Immunoblots were performed with the indicated antibodies. (C-J) Fatty acid and sterol synthesis were assessed from the incorporation of [1- 14 C]-acetate into saponifiable and non-saponifiable lipids, respectively. Data are means ± SEM from 3 independent experiments. ⁎ P
Figure Legend Snippet: Indirect and small-molecule AMPK activators inhibit lipid synthesis in primary human hepatocytes. (A) Assessment of AMPK subunit levels in primary human and mouse hepatocytes by western blotting with the indicated antibodies. (B-J) Human primary hepatocytes were cultured in the absence or presence of various concentrations of A-769662, C13, AICAR or metformin, alone or in combination, at the indicated concentrations for 3 h. (B) Immunoblots were performed with the indicated antibodies. (C-J) Fatty acid and sterol synthesis were assessed from the incorporation of [1- 14 C]-acetate into saponifiable and non-saponifiable lipids, respectively. Data are means ± SEM from 3 independent experiments. ⁎ P

Techniques Used: Western Blot, Cell Culture

Small-molecule AMPK activators enhance the action of metformin and AICAR on AMPK activation, lipid synthesis and fatty acid oxidation in primary hepatocytes. Control AMPKα1α2 floxed (WT) and AMPKα1α2 KO (AMPK KO) primary hepatocytes were incubated with or without various concentrations of AICAR or metformin in the absence or presence of A-769662 (1 or 10 μM) or C13 (1 μM) for 3 h. (A) Immunoblots were performed with the antibodies indicated. (B) P-AMPKα/AMPKα and P-ACC/ACC ratios from the quantification of immunoblot images. Data are means ± SEM. ⁎ P
Figure Legend Snippet: Small-molecule AMPK activators enhance the action of metformin and AICAR on AMPK activation, lipid synthesis and fatty acid oxidation in primary hepatocytes. Control AMPKα1α2 floxed (WT) and AMPKα1α2 KO (AMPK KO) primary hepatocytes were incubated with or without various concentrations of AICAR or metformin in the absence or presence of A-769662 (1 or 10 μM) or C13 (1 μM) for 3 h. (A) Immunoblots were performed with the antibodies indicated. (B) P-AMPKα/AMPKα and P-ACC/ACC ratios from the quantification of immunoblot images. Data are means ± SEM. ⁎ P

Techniques Used: Activation Assay, Incubation, Western Blot

Effect of various AMPK activators on triglyceride content and lipogenic gene expression in control and AMPK-deficient hepatocytes. control AMPKα1α2 floxed (WT) and AMPKα1α2 KO (AMPK KO) primary hepatocytes were cultured for 16 h in medium containing 5 or 25 mM glucose (G5 or G25), with or without 100 nM insulin, in the presence or absence of various concentrations of AICAR, metformin or A-769662. (A) Intracellular triglyceride content. (B) Immunoblots were performed with the antibodies indicated. (C) Lipogenic gene expression. Data are means ± SEM from 5 independent experiments. ⁎ P
Figure Legend Snippet: Effect of various AMPK activators on triglyceride content and lipogenic gene expression in control and AMPK-deficient hepatocytes. control AMPKα1α2 floxed (WT) and AMPKα1α2 KO (AMPK KO) primary hepatocytes were cultured for 16 h in medium containing 5 or 25 mM glucose (G5 or G25), with or without 100 nM insulin, in the presence or absence of various concentrations of AICAR, metformin or A-769662. (A) Intracellular triglyceride content. (B) Immunoblots were performed with the antibodies indicated. (C) Lipogenic gene expression. Data are means ± SEM from 5 independent experiments. ⁎ P

Techniques Used: Expressing, Cell Culture, Western Blot

Effects of various AMPK activators on lipid synthesis and fatty acid oxidation rates in control and AMPK-deficient hepatocytes. control AMPKα1α2 floxed (WT) and AMPKα1α2 KO (AMPK KO) primary hepatocytes were incubated in the presence or absence of various concentrations of AICAR, metformin, A-769662, C13, 991 or TOFA for 3 h. (A) Immunoblots were performed with the indicated antibodies. (B, C) Fatty acid and sterol synthesis were assessed from the incorporation of [1- 14 C]-acetate into saponifiable and non-saponifiable lipids, respectively. (D) Palmitate oxidation rates were determined by measuring the production of 14 C–labeled acid-soluble metabolites from [1- 14 C]-palmitic acid. Data are means ± SEM from 3 to 5 independent experiments. ⁎ P
Figure Legend Snippet: Effects of various AMPK activators on lipid synthesis and fatty acid oxidation rates in control and AMPK-deficient hepatocytes. control AMPKα1α2 floxed (WT) and AMPKα1α2 KO (AMPK KO) primary hepatocytes were incubated in the presence or absence of various concentrations of AICAR, metformin, A-769662, C13, 991 or TOFA for 3 h. (A) Immunoblots were performed with the indicated antibodies. (B, C) Fatty acid and sterol synthesis were assessed from the incorporation of [1- 14 C]-acetate into saponifiable and non-saponifiable lipids, respectively. (D) Palmitate oxidation rates were determined by measuring the production of 14 C–labeled acid-soluble metabolites from [1- 14 C]-palmitic acid. Data are means ± SEM from 3 to 5 independent experiments. ⁎ P

Techniques Used: Incubation, Western Blot, Labeling

13) Product Images from "AMPK Re-Activation Suppresses Hepatic Steatosis but its Downregulation Does Not Promote Fatty Liver Development"

Article Title: AMPK Re-Activation Suppresses Hepatic Steatosis but its Downregulation Does Not Promote Fatty Liver Development

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2018.01.008

Indirect and small-molecule AMPK activators inhibit lipid synthesis in primary human hepatocytes. (A) Assessment of AMPK subunit levels in primary human and mouse hepatocytes by western blotting with the indicated antibodies. (B-J) Human primary hepatocytes were cultured in the absence or presence of various concentrations of A-769662, C13, AICAR or metformin, alone or in combination, at the indicated concentrations for 3 h. (B) Immunoblots were performed with the indicated antibodies. (C-J) Fatty acid and sterol synthesis were assessed from the incorporation of [1- 14 C]-acetate into saponifiable and non-saponifiable lipids, respectively. Data are means ± SEM from 3 independent experiments. ⁎ P
Figure Legend Snippet: Indirect and small-molecule AMPK activators inhibit lipid synthesis in primary human hepatocytes. (A) Assessment of AMPK subunit levels in primary human and mouse hepatocytes by western blotting with the indicated antibodies. (B-J) Human primary hepatocytes were cultured in the absence or presence of various concentrations of A-769662, C13, AICAR or metformin, alone or in combination, at the indicated concentrations for 3 h. (B) Immunoblots were performed with the indicated antibodies. (C-J) Fatty acid and sterol synthesis were assessed from the incorporation of [1- 14 C]-acetate into saponifiable and non-saponifiable lipids, respectively. Data are means ± SEM from 3 independent experiments. ⁎ P

Techniques Used: Western Blot, Cell Culture

Small-molecule AMPK activators enhance the action of metformin and AICAR on AMPK activation, lipid synthesis and fatty acid oxidation in primary hepatocytes. Control AMPKα1α2 floxed (WT) and AMPKα1α2 KO (AMPK KO) primary hepatocytes were incubated with or without various concentrations of AICAR or metformin in the absence or presence of A-769662 (1 or 10 μM) or C13 (1 μM) for 3 h. (A) Immunoblots were performed with the antibodies indicated. (B) P-AMPKα/AMPKα and P-ACC/ACC ratios from the quantification of immunoblot images. Data are means ± SEM. ⁎ P
Figure Legend Snippet: Small-molecule AMPK activators enhance the action of metformin and AICAR on AMPK activation, lipid synthesis and fatty acid oxidation in primary hepatocytes. Control AMPKα1α2 floxed (WT) and AMPKα1α2 KO (AMPK KO) primary hepatocytes were incubated with or without various concentrations of AICAR or metformin in the absence or presence of A-769662 (1 or 10 μM) or C13 (1 μM) for 3 h. (A) Immunoblots were performed with the antibodies indicated. (B) P-AMPKα/AMPKα and P-ACC/ACC ratios from the quantification of immunoblot images. Data are means ± SEM. ⁎ P

Techniques Used: Activation Assay, Incubation, Western Blot

Effect of various AMPK activators on triglyceride content and lipogenic gene expression in control and AMPK-deficient hepatocytes. control AMPKα1α2 floxed (WT) and AMPKα1α2 KO (AMPK KO) primary hepatocytes were cultured for 16 h in medium containing 5 or 25 mM glucose (G5 or G25), with or without 100 nM insulin, in the presence or absence of various concentrations of AICAR, metformin or A-769662. (A) Intracellular triglyceride content. (B) Immunoblots were performed with the antibodies indicated. (C) Lipogenic gene expression. Data are means ± SEM from 5 independent experiments. ⁎ P
Figure Legend Snippet: Effect of various AMPK activators on triglyceride content and lipogenic gene expression in control and AMPK-deficient hepatocytes. control AMPKα1α2 floxed (WT) and AMPKα1α2 KO (AMPK KO) primary hepatocytes were cultured for 16 h in medium containing 5 or 25 mM glucose (G5 or G25), with or without 100 nM insulin, in the presence or absence of various concentrations of AICAR, metformin or A-769662. (A) Intracellular triglyceride content. (B) Immunoblots were performed with the antibodies indicated. (C) Lipogenic gene expression. Data are means ± SEM from 5 independent experiments. ⁎ P

Techniques Used: Expressing, Cell Culture, Western Blot

Effects of various AMPK activators on lipid synthesis and fatty acid oxidation rates in control and AMPK-deficient hepatocytes. control AMPKα1α2 floxed (WT) and AMPKα1α2 KO (AMPK KO) primary hepatocytes were incubated in the presence or absence of various concentrations of AICAR, metformin, A-769662, C13, 991 or TOFA for 3 h. (A) Immunoblots were performed with the indicated antibodies. (B, C) Fatty acid and sterol synthesis were assessed from the incorporation of [1- 14 C]-acetate into saponifiable and non-saponifiable lipids, respectively. (D) Palmitate oxidation rates were determined by measuring the production of 14 C–labeled acid-soluble metabolites from [1- 14 C]-palmitic acid. Data are means ± SEM from 3 to 5 independent experiments. ⁎ P
Figure Legend Snippet: Effects of various AMPK activators on lipid synthesis and fatty acid oxidation rates in control and AMPK-deficient hepatocytes. control AMPKα1α2 floxed (WT) and AMPKα1α2 KO (AMPK KO) primary hepatocytes were incubated in the presence or absence of various concentrations of AICAR, metformin, A-769662, C13, 991 or TOFA for 3 h. (A) Immunoblots were performed with the indicated antibodies. (B, C) Fatty acid and sterol synthesis were assessed from the incorporation of [1- 14 C]-acetate into saponifiable and non-saponifiable lipids, respectively. (D) Palmitate oxidation rates were determined by measuring the production of 14 C–labeled acid-soluble metabolites from [1- 14 C]-palmitic acid. Data are means ± SEM from 3 to 5 independent experiments. ⁎ P

Techniques Used: Incubation, Western Blot, Labeling

14) Product Images from "Towards natural mimetics of metformin and rapamycin"

Article Title: Towards natural mimetics of metformin and rapamycin

Journal: Aging (Albany NY)

doi: 10.18632/aging.101319

DL‐based similarity to metformin ( A ) and rapamycin ( B ). Significance of natural compound was determined as the ‐log10(p‐value) and odds ratio for compound according to Fisher's exact test performed on the DNN output for each perturbed sample. Only compounds with ‐log10(p‐value) > 4 and odds ratio > 1 are shown.
Figure Legend Snippet: DL‐based similarity to metformin ( A ) and rapamycin ( B ). Significance of natural compound was determined as the ‐log10(p‐value) and odds ratio for compound according to Fisher's exact test performed on the DNN output for each perturbed sample. Only compounds with ‐log10(p‐value) > 4 and odds ratio > 1 are shown.

Techniques Used:

Workflow diagram showing multi‐level analysis for screening and ranking nutraceuticals that mimic metformin and rapamycin in transcriptional and pathway activation response. A subset of 871 LINCS compounds were selected from the UNPD and KEGG BRITE databases. Perturbations with those compounds in cancer cell lines were compared with perturbations with metformin and rapamycin to estimate similarity at the gene and pathway level and deep learning techniques were employed to recognize the transcriptional signature of metformin and rapamycin and screen for matches amongst the LINCS compounds.
Figure Legend Snippet: Workflow diagram showing multi‐level analysis for screening and ranking nutraceuticals that mimic metformin and rapamycin in transcriptional and pathway activation response. A subset of 871 LINCS compounds were selected from the UNPD and KEGG BRITE databases. Perturbations with those compounds in cancer cell lines were compared with perturbations with metformin and rapamycin to estimate similarity at the gene and pathway level and deep learning techniques were employed to recognize the transcriptional signature of metformin and rapamycin and screen for matches amongst the LINCS compounds.

Techniques Used: Activation Assay

Gene‐ and pathway‐level similarity to metformin ( A ) and rapamycin ( B ). Significance of natural compound was determined as the ‐log10(p‐value) of the most significant perturbation of compound according to Fisher's exact test. Percentage of common pathways designates the amount of pathways that have the same direction of the change as Metformin. Only compounds with ‐log10(p‐value) > 4 and over 50% of common pathways are shown.
Figure Legend Snippet: Gene‐ and pathway‐level similarity to metformin ( A ) and rapamycin ( B ). Significance of natural compound was determined as the ‐log10(p‐value) of the most significant perturbation of compound according to Fisher's exact test. Percentage of common pathways designates the amount of pathways that have the same direction of the change as Metformin. Only compounds with ‐log10(p‐value) > 4 and over 50% of common pathways are shown.

Techniques Used:

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    Mimetics metformin
    Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM <t>metformin</t> treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value
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    Mimetics metformin ipc mimetics
    Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM <t>metformin</t> treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value
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    Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value

    Journal: Scientific Reports

    Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

    doi: 10.1038/s41598-019-41839-1

    Figure Lengend Snippet: Gene Ontology (GO) terms for genes changing ≥2-fold and ≥5-fold in response to 0.5 mM and 1.0 mM metformin treatments in 2DD fibroblasts. ( A ) Pie charts visualizing GO Biological Processes in response to genes up and down-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( B ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥2-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( C ) Pie charts visualizing GO Biological Processes in response to genes up regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. ( D ) Pie charts visualizing GO Molecular Function in response to genes up-regulated ≥5-fold in response to 0.5 mM (left) and 1.0 mM (right) metformin. All data presented had an FDR and P-value

    Article Snippet: The closer relationship between glucose depletion and metformin indicates that metformin is having a similar impact on energy sensing and that rapamycin and metformin, although both considered to be mimetics of caloric restriction, have different impacts on gene expression profiles in primary fibroblasts.

    Techniques:

    Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.

    Journal: Scientific Reports

    Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

    doi: 10.1038/s41598-019-41839-1

    Figure Lengend Snippet: Chromosome territories re-locate following 0.5 mM/1.0 mM metformin treatment. Chromosomes ( A ) 10, ( B ) 18, ( C ) X were identified in proliferative (Proliferative, first column), 0.5 mM metformin-treated (0.5 mM Met, second column) and 1.0 mM metformin-treated (1.0 mM Met, third column) 2DD fibroblasts by chromosome painting. Red signal represents the identified chromosomes; chromatin was counter stained with H33342 (blue). Scale bar = 10 μm. Cell Nucleus Analyser (CNA) software broke nuclei into five concentric shells of area, shell 1 being the most exterior and 5 the most interior (X-axis). Y-axes of graphs for each chromosome (X, 10, 18) represent the measured ratio of % chromosome signal/% H333432 signal in each shell. This ratio normalizes for DNA content in each shell. Error bars = S.E.M. *p-value ≤ 0.05 between treatment and proliferative. # Significant difference (p-value ≤ 0.05) between 0.5 mM and 1.0 mM metformin.

    Article Snippet: The closer relationship between glucose depletion and metformin indicates that metformin is having a similar impact on energy sensing and that rapamycin and metformin, although both considered to be mimetics of caloric restriction, have different impacts on gene expression profiles in primary fibroblasts.

    Techniques: Staining, Software, Significance Assay

    Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.

    Journal: Scientific Reports

    Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

    doi: 10.1038/s41598-019-41839-1

    Figure Lengend Snippet: Proposed model for the impact of Metformin on FOXO3a and gene transcription in primary human fibroblasts. A model of the proposed impact of metformin on FOXO3a. In response to metformin treatment, it is likely that AMPK is phosphorylated and FOXO3a is translocated into the nucleus from the cytoplasm, resulting in increased transcription of cytokine genes and gens in the activator protein-1 transcription factor pathway. Simultaneously, as a result of AMPK activation, mTORC1 likely becomes inhibited, resulting in previously described changes in autophagy, protein translation, metabolism and other down-stream pathways.

    Article Snippet: The closer relationship between glucose depletion and metformin indicates that metformin is having a similar impact on energy sensing and that rapamycin and metformin, although both considered to be mimetics of caloric restriction, have different impacts on gene expression profiles in primary fibroblasts.

    Techniques: Activation Assay

    Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.

    Journal: Scientific Reports

    Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

    doi: 10.1038/s41598-019-41839-1

    Figure Lengend Snippet: Scatter plots demonstrating transcriptome profile in 0.5 mM/1.0 mM metformin treatments. ( A ) Scatter plot comparing the transcript abundance in 0.5 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. ( B ) Scatter plot comparing the transcript abundance in 1.0 mM metformin (Y-axis) and proliferating (X-axis) fibroblasts. Counts identified for each transcript by RNA-seq for proliferative were log-base-2 transformed. Each square represents a single transcript. Transcripts exhibiting ≥2-fold change in 0.5 mM metformin-treated fibroblasts when compared to proliferative are marked in blue. Transcripts exhibiting ≥2-fold change in 1.0 mM metformin-treated fibroblasts when compared to proliferative are marked in red. Gray squares represent transcripts that did not change abundance ≥2-fold. Green squares represent transcripts that had a ≥2-fold change in response to both 0.5 mM and 1.0 mM metformin when compared to proliferative fibroblasts. Black text highlights individual transcripts within each scatter plot. ( C ) Venn diagrams demonstrating the number of genes up-regulated (left) or down-regulated (right) shared between 0.5 mM (red) and 1.0 mM (blue) metformin treatments. Numbers in each segment represent genes that are not shared, and in overlapping segments, shared genes between 0.5 mM and 1.0 mM metformin-treated fibroblasts.

    Article Snippet: The closer relationship between glucose depletion and metformin indicates that metformin is having a similar impact on energy sensing and that rapamycin and metformin, although both considered to be mimetics of caloric restriction, have different impacts on gene expression profiles in primary fibroblasts.

    Techniques: RNA Sequencing Assay, Transformation Assay

    Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).

    Journal: Scientific Reports

    Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

    doi: 10.1038/s41598-019-41839-1

    Figure Lengend Snippet: Genes changing ≥2-fold in response to 0.5 mM/1.0 mM metformin are divergent to those changing in response to 500 nM rapamycin treated fibroblasts. ( A ) Venn diagrams demonstrating the number of genes up-regulated (top) or down-regulated (bottom) between 0.5 mM (red), 1.0 mM (blue) metformin and 500 nM rapamycin (green). Numbers in each segment represent genes that are not shared. Genes in overlapping segments represent genes shared either between samples. ( B ) Principal Component Analysis (PCA) demonstrating the divergence between sample-sets. Two proliferative sets are included (Pro Set 1: Black; Pro Set 2: Grey) to represent controls in RNAseq assays for rapamycin-treated samples and for metformin or glucose deprived samples. Each circle represents an RNAseq replicate, with each sample having two identically coloured circles. A key is given in the bottom left corner corresponding circle colour to condition (0.5 mM Metformin (Met): Red; 1.0 mM Met (Blue); 500 nM Rap (Green); 1.0 g/L Glucose (Orange)). Y-axis represents PC2 (22% explained var.) and X-axis represents PC1 (35.1% explained var).

    Article Snippet: The closer relationship between glucose depletion and metformin indicates that metformin is having a similar impact on energy sensing and that rapamycin and metformin, although both considered to be mimetics of caloric restriction, have different impacts on gene expression profiles in primary fibroblasts.

    Techniques:

    Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.

    Journal: Scientific Reports

    Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

    doi: 10.1038/s41598-019-41839-1

    Figure Lengend Snippet: Metformin decreases the rate of fibroblast proliferation. ( A ) 2DD fibroblasts were grown under normal culture conditions or in the presence of 0.5/1.0 mM metformin. Cell numbers were monitored and population doubling times (Y axis) calculated at 120 h. ( B ) The total population doublings (Y axis) for 0.5 mM and 1.0 mM metformin treatments (X axis) were calculated. 2DD were immuno-labelled for Ki67 following 120 h of treatment. ( C ) Percent Ki67 positive (Y axis) for 0.5 mM/1.0 mM metformin (X-axis) is plotted. Ki67 positive and negative cells are shown at the bottom of the panel. Ki67 is false coloured green and chromatin counterstained with H33342 (blue). ( D ) Percent EdU positive (Y axis) 2DD fibroblasts at 120 h following 0.5 mM/1.0 mM metformin treatment. Below the panel, EdU positive and negative fibroblasts are shown. EdU is false coloured red and chromatin counterstained with H33342 (blue). Data represent three biological replicates. Error bars represent S.E.M. Scale bars = 10 μm.

    Article Snippet: The closer relationship between glucose depletion and metformin indicates that metformin is having a similar impact on energy sensing and that rapamycin and metformin, although both considered to be mimetics of caloric restriction, have different impacts on gene expression profiles in primary fibroblasts.

    Techniques:

    FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P

    Journal: Scientific Reports

    Article Title: Metformin induces the AP-1 transcription factor network in normal dermal fibroblasts

    doi: 10.1038/s41598-019-41839-1

    Figure Lengend Snippet: FOXO3a promoter occupancy is increased in genes up-regulated by 0.5 mM metformin treatments in primary human fibroblasts. ( A ) Using CLOVER, promoters of genes changing expression following metformin treatments had overrepresentation of FOXO3a and SRF transcription factor binding sites. Position weight matrices/sequence logos of these binding sites are shown. Log-base-2 of the information content of each nucleotide (Y-axis) and position of these nucleotides (X-axis) are given. ( B ) Immunofluorescence for FOXO3a (green) in proliferative, 0.5 mM and 1.0 mM metformin treated fibroblasts. Chromatin is counterstained with H33342 (blue). Scale bar = 10 μm. Western blot assays for FOXO3a (top), SRF (centre) and beta actin (bottom) are given for proliferative (pro) 0.5 mM and 1.0 mM metformin (met) treated whole protein lysates. ( C ) ChIP assays were used to compare promoter occupancy of FOXO3a (top) and SRF (bottom) in proliferative (dark grey) and 0.5 mM metformin treated (light grey) samples. Promoters of genes analysed are given (X-axis) and percent (%) enrichment over input reported (Y-axis). Error bars = S.E.M. *P

    Article Snippet: The closer relationship between glucose depletion and metformin indicates that metformin is having a similar impact on energy sensing and that rapamycin and metformin, although both considered to be mimetics of caloric restriction, have different impacts on gene expression profiles in primary fibroblasts.

    Techniques: Expressing, Binding Assay, Sequencing, Immunofluorescence, Western Blot, Chromatin Immunoprecipitation