Journal: Experimental hematology

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

doi: 10.1016/j.exphem.2020.09.187

Figure Lengend Snippet: (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 < 0.05

Article Snippet: Metformin and miRNAs – Metformin was purchased from SelleckChem and added to cells at indicated concentrations with a final DMSO concentration of 0.5%.

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

Journal: Experimental hematology

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

doi: 10.1016/j.exphem.2020.09.187

Figure Lengend Snippet: (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 < 0.05

Article Snippet: Metformin and miRNAs – Metformin was purchased from SelleckChem and added to cells at indicated concentrations with a final DMSO concentration of 0.5%.

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

Journal: Experimental hematology

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

doi: 10.1016/j.exphem.2020.09.187

Figure Lengend Snippet: (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 < 0.05

Article Snippet: Metformin and miRNAs – Metformin was purchased from SelleckChem and added to cells at indicated concentrations with a final DMSO concentration of 0.5%.

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

Journal: Experimental hematology

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

doi: 10.1016/j.exphem.2020.09.187

Figure Lengend Snippet: (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 < 0.05

Article Snippet: Metformin and miRNAs – Metformin was purchased from SelleckChem and added to cells at indicated concentrations with a final DMSO concentration of 0.5%.

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

Journal: Experimental hematology

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

doi: 10.1016/j.exphem.2020.09.187

Figure Lengend Snippet: (A) K562 cells stabling expressing luciferase alone (top panel), luciferase expressed behind a minimal promoter and the NLK proximal promoter (2nd panel), luciferase immediately upstream of the human NLK 3’UTR (3rd 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 < 0.05

Article Snippet: Metformin and miRNAs – Metformin was purchased from SelleckChem and added to cells at indicated concentrations with a final DMSO concentration of 0.5%.

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

Journal: Experimental hematology

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

doi: 10.1016/j.exphem.2020.09.187

Figure Lengend Snippet: (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 < 0.05

Article Snippet: Metformin and miRNAs – Metformin was purchased from SelleckChem and added to cells at indicated concentrations with a final DMSO concentration of 0.5%.

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

Journal: Experimental hematology

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

doi: 10.1016/j.exphem.2020.09.187

Figure Lengend Snippet: (A) Human CD34+ cells were transduced with shRNA against a control (shLuc) or RPS19 (shRPS19) and transfected with a miR-26a mimetic or a mock control. After differentiation in the absence or presence of metformin for 12 days, cells were counted and the percentage of CD235+ erythroblasts determined by flow cytometry. The calculated CD235+ population of each treatment goup is represented as a percentage of the untreated control group (upper). In parallel, qRT-PCR was performed to determine mRNA expression of NLK (middle) and miR-26a (lower). (B) Lin-Kit+ progenitors for RPL11+/+ and RPL+/lox mice were mock transfected or transfected with miR-26a mimetic and differentiated for 8 days. Cells were subjected to counting and the percentage of Ter119+ erythroblasts determined (upper) in conjunction with qRT-PCR examining NLK (middle) and miR-26a (lower) mRNA expression. Data are displayed as means +/− SD. Statistics: two-tailed Student’s t test, significant *P < 0.05

Article Snippet: Metformin and miRNAs – Metformin was purchased from SelleckChem and added to cells at indicated concentrations with a final DMSO concentration of 0.5%.

Techniques: Transduction, shRNA, Control, Transfection, Flow Cytometry, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Piperine Suppresses Pyroptosis and Interleukin-1β Release upon ATP Triggering and Bacterial Infection

doi: 10.3389/fphar.2016.00390

Figure Lengend Snippet: Metformin, an AMPK agonist, counteracted the effect of piperine on suppressing ATP-induced AMPK activation and inflammatory mediator release. J774A.1 cells (A–C) were pre-treated with 80 μM piperine and BMDMs (D) were pre-treated with 160 μM piperine for 4 h. Without being washed out of piperine, these cells were primed with LPS (500 ng/ml) for 4 h. Next, the cells were treated with metformin (1 mM) for 1 h (in the absence of piperine and LPS). Finally, in the presence of metformin, the BMDMs were stimulated with 3 mM ATP (final concentration) for 30 min while the J774A.1 cells were treated with 4 mM ATP (final concentration) for 1 h. After the cells were lysed with 1x SDS-PAGE loading buffer, protein levels were evaluated by western blotting (A,C,D) . β-Tubulin was used as a loading control for cell lysates. (B) The densitometry ratios of p-AMPK relative to AMPK in the blots of (A) were analyzed by FluorChem 8000 (Alpha Innotech). Data are presented as mean ± SD ( n = 3). Statistical significance was analyzed by one-way ANOVA with Tukey post hoc test. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Metformin was obtained from MedChem Express (Princeton, NJ, USA), dissolved in DMEM at 300 mM and stored at -20°C.

Techniques: Activation Assay, Concentration Assay, SDS Page, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Piperine Suppresses Pyroptosis and Interleukin-1β Release upon ATP Triggering and Bacterial Infection

doi: 10.3389/fphar.2016.00390

Figure Lengend Snippet: Metformin reversed piperine’s effect on suppression of pyroptosis in macrophages treated with ATP. BMDMs (A,B) or J774A.1 cells (C,D) were pre-treated with indicated concentrations of piperine (BMDMs) or 80 μM piperine (J774A.1) for 4 h, and then primed with LPS (500 ng/ml) for 4 h in the presence of piperine. After washing out of piperine and LPS, the cells were then treated with metformin (1 mM) for 1 h followed by 3 mM ATP for 30 min (BMDMs) or 4 mM ATP for 1 h (J774A.1 cells) in the presence of metformin. The pyroptotic cell ratios in BMDMs (B) or J774A.1 cells (D) were evaluated as described in Figure . Data are presented as mean ± SD ( n = 5). Statistical significance was analyzed by one-way ANOVA with Tukey post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. PIP, piperine; Met, metformin.

Article Snippet: Metformin was obtained from MedChem Express (Princeton, NJ, USA), dissolved in DMEM at 300 mM and stored at -20°C.

Techniques:

Journal: Molecular Human Reproduction

Article Title: Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy

doi: 10.1093/molehr/gaaa084

Figure Lengend Snippet: The timeline of experiments and the number of ovarian follicles in different stages in each group . ( A ) The timeline of experiments was shown. Metformin (MET), sirolimus (SIRO) and everolimus (EVE) were administered by gavage on Days 1 to 5 for 4 weeks. Cyclophosphamide (CP) was provided after gavage on Day 1 by intraperitoneal injection weekly for 3 weeks. Other experimental details were described in Materials and methods section. ( B – F ) The numbers of ovarian follicles in different stages during folliculogenesis in C57BL/6 mice are shown. The mice were treated with CP-alone, MET-alone, SIRO-alone, or CP in combination with MET, SIRO or EVE. After 4 weeks of treatment, the ovaries were processed into paraffin blocks, sectioned, mounted and hematoxylin and eosin (H&E) stained for follicular counting. The number of primordial and tertiary follicles, and corpus luteum decreased in the CP-alone group compared with the control group ( P = 0.0014, 0.00003, 0.073, respectively). The deleterious effects of CP on follicular counts were diminished when oral MET was given to mice (Primordial follicle: P = 0.0274). The other two specific mTOR inhibitors, SIRO and EVE, also exhibited significant protective effects against CP damage (primary follicles: CP-alone versus CP + SIRO: P = 0.024; tertiary follicles: CP-alone versus CP + EVE: P = 0.0046). (B) and (C) The Y axis represented the average follicular counts per high-power field (HPF). N = 10 mice in the control and CP-alone group, while n = 5 mice in the other groups. Each value represents the average of 2–3 HPF per animal. (D)–(F) The Y axis represented the total follicular counts per ovarian section. N = 10 mice in the control and CP-alone group, while n = 5 mice in the other groups. Data are expressed as the mean ± standard deviation. Statistical analyses were performed by nonparametric Kruskal–Wallis test with Dunn's post-hoc for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001. Note: The double slash mark on the X axis separated the MET-alone and SIRO-alone group from other groups because these two control groups were run in a separate experiment.

Article Snippet: For in-vitro studies, metformin hydrochloride (dissolved in PBS) and CP (dissolved in DMSO) was purchased from Sigma (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK), respectively.

Techniques: Injection, Staining, Control, Standard Deviation

Journal: Molecular Human Reproduction

Article Title: Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy

doi: 10.1093/molehr/gaaa084

Figure Lengend Snippet: The serum hormone concentration and the number of offspring in each group. C57BL/6 mice were treated with CP-alone, metformin (MET)-alone, sirolimus (SIRO)-alone, or CP in combination with MET, SIRO or everolimus (EVE). After 4 weeks of treatment, the mice were sacrificed and serum was collected for hormonal determination. ( A ) The serum levels of anti-Müllerian hormone (AMH) were significantly decreased in the CP-alone group (compared with control group: P < 0.0001). Although the AMH levels increased in CP + MET, CP + SIRO and CP + EVE group, the data did not reach statistical significance probably due to limited case number. ( B ) The serum levels of estradiol were significantly decreased in the CP-alone group ( P < 0.0001), and tended to increase in CP + MET group, CP + SIRO group and CP + EVE group. ( C ) The serum levels of progesterone were significantly decreased in the CP-alone group ( P < 0.0001) and tended to increase in CP + MET group and CP + EVE group. ( D ) A breeding test was conducted 1 week after the 4-week treatment. Only one round of timed mating was conducted per female mouse and the outcome of the first pregnancy in each mouse was evaluated. The number of the offspring was significantly decreased in the CP-alone group ( P < 0.0001) and tended to increase in the CP + MET group. In (A)–(C), n = 10 mice in the control and CP-alone group, while n = 5 mice in the other groups. In (D), n = 6 mice per group. Data are expressed as the mean ± standard deviation. Statistical analyses were performed by nonparametric Kruskal–Wallis test with Dunn's post-hoc for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Note: The double slash mark on the X axis separated the MET-alone and SIRO-alone group from other groups because these two control groups were run in a separate experiment.

Article Snippet: For in-vitro studies, metformin hydrochloride (dissolved in PBS) and CP (dissolved in DMSO) was purchased from Sigma (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK), respectively.

Techniques: Concentration Assay, Control, Standard Deviation

Journal: Molecular Human Reproduction

Article Title: Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy

doi: 10.1093/molehr/gaaa084

Figure Lengend Snippet: Immunohistochemical (IHC) staining of the proteins involved in mTOR signaling pathway. C57BL/6 mice were treated with CP-alone, metformin (MET)-alone, sirolimus (SIRO)-alone or CP in combination with MET or SIRO. After 4 weeks of treatment, the ovaries were processed into paraffin sections for the IHC detection of p-mTOR ( A , B ) and p-AMPK ( C , D ) proteins. The percentage of granulosa cells with positive staining was calculated by dividing the number of positive stained cells with the total number of granulosa cells in ovarian follicles under a microscopy at ×400 magnification. One largest tertiary follicle in each section was selected to calculate the number of positively stained cells. Five sections per mice were counted from a total of 5 mice in each group (n = 5 mice). Data are expressed as the mean (%) ± standard deviation. Statistical analyses were performed by nonparametric Kruskal–Wallis test with Dunn's post-hoc for multiple comparisons. * P < 0.05, ** P < 0.01. The scale bar is 50 μm. Note: The double slash mark on the X axis separated the MET-alone and SIRO-alone group from other groups because these two control groups were run in a separate experiment.

Article Snippet: For in-vitro studies, metformin hydrochloride (dissolved in PBS) and CP (dissolved in DMSO) was purchased from Sigma (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK), respectively.

Techniques: Immunohistochemical staining, Immunohistochemistry, Staining, Microscopy, Standard Deviation, Control

Journal: Molecular Human Reproduction

Article Title: Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy

doi: 10.1093/molehr/gaaa084

Figure Lengend Snippet: IHC staining of the cellular apoptotic and proliferative markers. C57BL/6 mice were treated with CP-alone, metformin (MET)-alone, sirolimus (SIRO)-alone or CP in combination with MET or SIRO. After 4 weeks of treatment, the ovaries were processed into paraffin sections for the TUNEL assay (A, B) and Ki67 staining (C, D) to separately evaluate the degree of cellular apoptosis and proliferation. The percentage of granulosa cells with positive staining was calculated by dividing the number of positive stained cells with the total number of granulosa cells in ovarian follicles under a microscopy at ×400 magnification. One largest tertiary follicle in each section was selected to calculate the number of positively stained cells. Five sections per mice were counted. N = 10 mice in the control and CP-alone group, while n = 5 mice in the other groups. Data are expressed as the mean (%) ± standard deviation. Statistical analyses were performed by nonparametric Kruskal–Wallis test with Dunn's post-hoc for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The scale bar is 50 μm. Note: The double slash mark on the X axis separated the MET-alone and SIRO-alone group from other groups because these two control groups were run in a separate experiment.

Article Snippet: For in-vitro studies, metformin hydrochloride (dissolved in PBS) and CP (dissolved in DMSO) was purchased from Sigma (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK), respectively.

Techniques: Immunohistochemistry, TUNEL Assay, Staining, Microscopy, Control, Standard Deviation

Journal: Molecular Human Reproduction

Article Title: Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy

doi: 10.1093/molehr/gaaa084

Figure Lengend Snippet: Cell cycle phase analysis by flow cytometry using cultured mouse granulosa cells. Mouse granulosa cells were treated with CP-alone (1ug/ml) or with CP + metformin (MET) (10 mM) or with MET-alone for 72 h. Fresh granulosa cells were collected from five mice, and then mixed and cultured together. ( A ) The cell cycles, including subG1 (apoptotic phase), G1, S and G2/M of the assayed cells were determined by quantifying their DNA contents with propidium iodide (PI) staining and flow cytometry. The M1, M2, M3 and M4 bars represent the subG1, G1, S and G2/M phases, respectively. ( B ) The quantitative results of the cell cycle phases in each group were shown. The percentage of cells in the subG1 phase was significantly increased in the CP group ( P = 0.0009) and tended to decrease when cells were cotreated with MET. There was no significant difference between control group and MET-alone group. All the experiments were repeated five times and therefore N = 5 per group. Statistical analyses were performed by nonparametric Kruskal–Wallis test with Dunn's post-hoc for multiple comparisons. * P < 0.05, *** P < 0.001.

Article Snippet: For in-vitro studies, metformin hydrochloride (dissolved in PBS) and CP (dissolved in DMSO) was purchased from Sigma (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK), respectively.

Techniques: Flow Cytometry, Cell Culture, Staining, Control

Journal: Molecular Human Reproduction

Article Title: Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy

doi: 10.1093/molehr/gaaa084

Figure Lengend Snippet: Metformin exerted AMPK/p53/p21-mediated anti-apoptotic effect on cultured mouse granulosa cells. ( A ) Mouse granulosa cells were treated with p53 siRNA (25 nM) or control siRNA for 24 h prior CP (1ug/ml) or metformin (MET) (10 mM) treatment, after 72 h. Apoptotic cells were determined by flow cytometry with propidium iodide staining and quantified by the subG1 ratio. The experiments were repeated five times and therefore N = 5 per group. The apoptosis rate significantly increased in the CP-alone group ( P = 0.0029) and tended to decrease after cotreating the cells with MET. The anti-apoptosis effect of MET was diminished after blocking the p53 activity with siRNA. ( B ) The experimental conditions were the same as A, except that p21 siRNA (25 nM) was applied instead of p53 siRNA. ( C ) Mouse granulosa cells were treated with AMPK inhibitor BML275(10 μM) for 30 min prior MET (10 mM) treatment. At indicated time periods, the expression of p53 mRNA was determined by qRT-PCR. The experiments were repeated four times and therefore N = 4 per group. Sequential measurement of p53 mRNA expression in culture granulosa cells significantly increased 8 h after the addition of MET ( P = 0.0098). The expression of p53 mRNA was blocked with the addition of cell-permeable AMPK inhibitor BML. ( D ) The expression of p21 mRNA was determined by qRT-PCR and sequential measurement of p21 mRNA expression in cultured granulosa cells significantly increased 24 h after the addition of MET ( P = 0.0095). The experiments were repeated five times and therefore N = 5 per group. ( E ) C57BL/6 mice were treated with CP-alone or in combination with MET or MET-alone. After 4 weeks of treatment, the ovaries were processed into paraffin sections for the p53 and p21 staining. Both p53 and p21 activity were generally low in normal untreated ovarian tissue but was elevated after CP treatment (p53: P = 0.6529; p21: P = 0.0452) as determined with IHC staining under microscopic examination at ×400 magnification. The coadministration of MET with CP increased p53 and p21 protein expression in the ovarian tissue even higher ( P < 0.0004 in both). Statistical analyses were performed by nonparametric Kruskal–Wallis test with Dunn's post-hoc for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001. ( F ) Representative p53 and p21 IHC images of each group were shown. The scale bar is 50 μm.

Article Snippet: For in-vitro studies, metformin hydrochloride (dissolved in PBS) and CP (dissolved in DMSO) was purchased from Sigma (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK), respectively.

Techniques: Cell Culture, Control, Flow Cytometry, Staining, Blocking Assay, Activity Assay, Expressing, Quantitative RT-PCR, Immunohistochemistry

Journal: Current Issues in Molecular Biology

Article Title: Astragalus Polysaccharides and Metformin May Have Synergistic Effects on the Apoptosis and Ferroptosis of Lung Adenocarcinoma A549 Cells

doi: 10.3390/cimb46080461

Figure Lengend Snippet: Synergistic effect of APS and metformin on A549 cell viability. A549 cell viability under the treatment of ( a , b ) APS/metformin alone for ( a ) 24 h and ( b ) for 48 h. ( c , d ) APS in combination with different concentrations of metformin after treatment of ( c ) 24 h and ( d ) 48 h. The combination group significantly decreased compared to using either APS or metformin alone. All the results are representative of at least three independent experiments. (Error bars = mean ± S.E.M. Asterisks mark samples significantly different from control group with (*) p < 0.05; (**) with p < 0.01; (***) with p < 0.001).

Article Snippet: Co., Limited, Compton, UK (product code: FA41820, from Mongolian Astragalus, using a low concentration of ethanol for precipitation and gel chromatography for purification; the approximate molecular weight was 301 kDa), while metformin HCl was obtained from Toronto Research Chemicals, CA, USA (product number: TRC-M258815, molecular weight: 165.62 g/mol).

Techniques: Control

Journal: Current Issues in Molecular Biology

Article Title: Astragalus Polysaccharides and Metformin May Have Synergistic Effects on the Apoptosis and Ferroptosis of Lung Adenocarcinoma A549 Cells

doi: 10.3390/cimb46080461

Figure Lengend Snippet: The apoptotic effect on A549 cells of APS, metformin detected by flow cytometry with annexin V-FITC/PI dual staining: ( a , c ) A549 cells treated for 24 h and ( b , d ) for 48 h. (Error bars = mean ± S.E.M. Asterisks mark samples significantly different from control group with (**) with p < 0.01; (***) with p < 0.001).

Article Snippet: Co., Limited, Compton, UK (product code: FA41820, from Mongolian Astragalus, using a low concentration of ethanol for precipitation and gel chromatography for purification; the approximate molecular weight was 301 kDa), while metformin HCl was obtained from Toronto Research Chemicals, CA, USA (product number: TRC-M258815, molecular weight: 165.62 g/mol).

Techniques: Flow Cytometry, Staining, Control

Journal: Current Issues in Molecular Biology

Article Title: Astragalus Polysaccharides and Metformin May Have Synergistic Effects on the Apoptosis and Ferroptosis of Lung Adenocarcinoma A549 Cells

doi: 10.3390/cimb46080461

Figure Lengend Snippet: Effect of APS, metformin, and their combination on ferroptosis of A549 cells in vitro. The A549 cells were treated with either APS, metformin, or their combination under different concentrations for 24 h and then collected for the following test: ( a ) ROS assay; ( b ) MAD assay; ( c ) GSH levels; ( d ) Western blot for GPx4 protein. B-actin was used as the internal control. (Error bars = mean ± S.E.M. Asterisks mark samples significantly different from control group with (*) p < 0.05; (**) with p < 0.01; (***) with p < 0.001).

Article Snippet: Co., Limited, Compton, UK (product code: FA41820, from Mongolian Astragalus, using a low concentration of ethanol for precipitation and gel chromatography for purification; the approximate molecular weight was 301 kDa), while metformin HCl was obtained from Toronto Research Chemicals, CA, USA (product number: TRC-M258815, molecular weight: 165.62 g/mol).

Techniques: In Vitro, ROS Assay, Western Blot, Control

Journal: Physiological Reports

Article Title: Metformin does not slow cyst growth in the PCK rat model of polycystic kidney disease

doi: 10.14814/phy2.15776

Figure Lengend Snippet: Metformin (MET; 300 mg/kg) resulted in weight loss, had no effect on cyst indices, worsened kidney function, and increased l ‐lactate levels. The effect of vehicle (VEH) vs. MET 300 mg/kg on body weight (a), two kidney weights (b), kidney weight corrected for body weight (c), cyst index (the cross‐sectional area of the kidney that was cystic) (d), cyst number per cross‐sectional area (e), cyst size (f) in the medulla, cyst index (g), cyst number per the cross‐sectional area (h), cyst size (i) in the cortex, BUN (j), serum creatinine (k), and serum lactate levels (l). Representative images of hematoxylin–eosin sections from the mid‐pole of the kidney in vehicle‐ (m) and MET‐treated kidneys (n). Representative images from the upper pole of the kidney in vehicle‐ (o) and MET‐treated kidneys (p). Scale bar = 1000 μm. Comparisons between two groups were made using Student's t test. A p value of <0.05 was considered statistically significant. Values are expressed as the mean ± SEM. BW, total body weight; 2KW, two kidney weight; 2KW/BW (%), 2 kidney weight to body weight ratio expressed as a percentage; creat = serum creatinine.

Article Snippet: Metformin and d6‐metformin were purchased from Toronto Research Chemicals.

Techniques:

Journal: Physiological Reports

Article Title: Metformin does not slow cyst growth in the PCK rat model of polycystic kidney disease

doi: 10.14814/phy2.15776

Figure Lengend Snippet: Metformin (MET; 300 mg/kg) had no effect on heart weight or liver cysts. Heart weight (a), heart weight corrected for body weight (b), liver weight (c), and liver weight corrected for body weight (d) in vehicle (VEH) and MET. Liver cyst index (% of the liver) (e), liver cyst count (number of cysts) (f), average (g), and median liver cyst size (h) are shown. Cyst index, count, and size data for two separate sections of each liver are shown. Representative images of liver cysts are shown in (i and j). Scale bar = 2 mm. Comparisons between the two groups were made using Student's t test. A p value of <0.05 was considered statistically significant. Values are expressed as the mean ± SEM. wt, weight; BW, body weight.

Article Snippet: Metformin and d6‐metformin were purchased from Toronto Research Chemicals.

Techniques:

Journal: Physiological Reports

Article Title: Metformin does not slow cyst growth in the PCK rat model of polycystic kidney disease

doi: 10.14814/phy2.15776

Figure Lengend Snippet: Metformin (MET) has no effect on p‐AMPK T172 or p‐ACC S79 , increases p‐S6 (mTORC1), but has no consistent effect on LC3‐II (autophagy), p‐Akt T308 (upstream of mTORC1), or p‐Akt S473 (mTORC2). Quantitative immunoblot analysis for mTOR proteins in vehicle‐ (VEH) and MET‐treated kidneys. Representative densitometry of immunoblot analysis of proteins in the kidney is demonstrated: p‐AMPK (a), p‐ACC (b), p‐S6 (c). LC3‐II (d), the conversion of LC3‐I to LC3‐II (e), p62 (f). p‐Akt T308 (g), p‐Akt S473 (h). Immunohistochemistry staining for p‐S6 (brown) mostly in tubules in vehicle‐ (i) and metformin (j)‐treated PCK rats. Scale bar = 100 μm. Quantitation of p‐S6 staining is shown in (k). Comparisons between the two groups were made using Student's t test. A p value of <0.05 was considered statistically significant. Values are expressed as the mean ± SEM. NS, not significant; RDU, relative densitometry units.

Article Snippet: Metformin and d6‐metformin were purchased from Toronto Research Chemicals.

Techniques: Western Blot, Immunohistochemistry, Staining, Quantitation Assay

Journal: Physiological Reports

Article Title: Metformin does not slow cyst growth in the PCK rat model of polycystic kidney disease

doi: 10.14814/phy2.15776

Figure Lengend Snippet: Metformin (MET; 300 mg/kg) had no effect on proliferation. The effect of vehicle (VEH) versus MET on PCNA staining (arrows) in the noncystic tubules or cystic tubular cells in the PCK rats. Scale bar = 100 μm. Comparisons between the two groups were made using Student's t test. A p value of <0.05 was considered statistically significant. Values are expressed as the mean ± SEM.

Article Snippet: Metformin and d6‐metformin were purchased from Toronto Research Chemicals.

Techniques: Staining

Journal: Physiological Reports

Article Title: Metformin does not slow cyst growth in the PCK rat model of polycystic kidney disease

doi: 10.14814/phy2.15776

Figure Lengend Snippet: Metformin (MET; 150 mg/kg) had no effect on cyst indices, kidney function, or serum l ‐lactate levels. The effect of vehicle (VEH) versus MET 150 mg/kg on body weight (a), kidney weight (b), kidney weight corrected for body weight (c), cyst index (the cross‐sectional area of the kidney that was cystic) (d), cyst number per cross‐sectional area (e), cyst size (f) in the medulla, cyst index (g), cyst number per cross‐sectional area (h), cyst size (i) in the cortex, BUN (j), serum creatinine (k), and serum lactate levels (l). Representative images of hematoxylin–eosin sections from the mid‐pole of the kidney in vehicle‐ (m) and MET‐treated kidneys (n). Representative images from the upper pole of the kidney in vehicle (o) and MET‐treated kidneys (p). Scale bar = 1000 μm. Comparisons between the two groups were made using Student's t test. A p value of <0.05 was considered statistically significant. Values are expressed as the mean ± SEM. BW = total body weight, 2KW = two kidney weight, 2 K/BW (%) = 2 kidney weight to body weight ratio expressed as a percentage, creat = serum creatinine.

Article Snippet: Metformin and d6‐metformin were purchased from Toronto Research Chemicals.

Techniques:

Journal: Physiological Reports

Article Title: Metformin does not slow cyst growth in the PCK rat model of polycystic kidney disease

doi: 10.14814/phy2.15776

Figure Lengend Snippet: Metformin (MET; 150 mg/kg) had no effect on liver or heart weight. Heart weight (a), heart weight corrected for body weight (b), liver weight (c), and liver weight corrected for body weight (d) in vehicle (VEH) and MET (Met). Liver cyst index (% of the liver) (e), cyst count (number of cysts) (f), average (g), and median cyst size (h) is shown. Representative images of liver cysts in vehicle‐ (i) and metformin‐treated (j) rats. Scale bar = 2 mm. Cyst index, count, and size data for two separate sections of each liver are shown. Comparisons between the two groups were made using Student's t test. A p value of <0.05 was considered statistically significant. Values are expressed as the mean ± SEM. wt, weight; BW, body weight.

Article Snippet: Metformin and d6‐metformin were purchased from Toronto Research Chemicals.

Techniques:

Journal: Physiological Reports

Article Title: Metformin does not slow cyst growth in the PCK rat model of polycystic kidney disease

doi: 10.14814/phy2.15776

Figure Lengend Snippet: Metformin (MET; 150 mg/kg) has no effect on p‐AMPK, p‐ACC, p‐S6 (mTORC1), autophagy (LC‐3, p62), p‐Akt T308 , or p‐Akt s473 (mTORC2). Quantitative immunoblot analysis for mTOR proteins in vehicle‐ (VEH) and MET‐treated kidneys. Representative densitometry of immunoblot analysis of proteins in the kidney is demonstrated. p‐AMPK (a), p‐ACC (b), p‐S6 (c), LC3‐II (d), the conversion of LC3‐I to LC3‐II (e), p62 (f), p‐Akt S473 (g), and p‐Akt T308 (h). Comparisons between the two groups were made using Student's t test. A p value of <0.05 was considered statistically significant. Values are expressed as the mean ± SEM. NS, not significant; RDU, relative densitometry units.

Article Snippet: Metformin and d6‐metformin were purchased from Toronto Research Chemicals.

Techniques: Western Blot

Journal: Physiological Reports

Article Title: Metformin does not slow cyst growth in the PCK rat model of polycystic kidney disease

doi: 10.14814/phy2.15776

Figure Lengend Snippet: Metformin (MET; 150 mg/kg) had no effect on proliferation. The effect of vehicle (VEH) versus MET on PCNA staining (arrows) in the noncystic tubules or cystic tubular cells in the PCK rats. Scale bar = 100 μm. Comparisons between two groups were made using Student's t test. A p value of <0.05 was considered statistically significant. Values are expressed as the mean ± SEM.

Article Snippet: Metformin and d6‐metformin were purchased from Toronto Research Chemicals.

Techniques: Staining