Journal: Cell Reports Medicine

Article Title: Repression of LKB1 by miR-17∼92 Sensitizes MYC -Dependent Lymphoma to Biguanide Treatment

doi: 10.1016/j.xcrm.2020.100014

Figure Lengend Snippet: IM156 Is a Newly Developed Biguanide That Inhibits Mitochondrial Respiration (A) Chemical structure of the biguanides metformin, phenformin, and IM156. (B) Dose-dependent reduction of the OCR of Eμ- Myc + lymphoma cells by metformin, phenformin, and IM156. Data were derived from the maximal point of OCR reduction after 30 min of treatment with the indicated biguanide and expressed relative to untreated cells at the same time point. Data represent mean ± SEM for biological replicates (n = 4–6 per drug per concentration). (C) Mitochondrial ATP production rate of Eμ- Myc + lymphoma cells after incubation with 100 μM of either vehicle, phenformin (Phen), or IM156. Data represent mean ± SD for biological replicates (n = 8–9 per group). (D) Dose-dependent inhibition of substrate oxidation by electron transport chain complexes in IM156-treated purified bovine mitochondrial membranes. NADH oxidation was measured for complex I, III, and IV activity (dark blue), and succinate oxidation was measured for complex II, III, and IV activity (gray) over the indicated concentrations of IM156. Data represent mean ± SD for technical replicates (n = 3–4 per group). See also Figure S1 A. (E) Dose-dependent reduction of OCR by IM156 in HEK293T cells expressing empty vector (Ctrl) or yeast NDI1 (NDI1). Data were derived from the maximal point of OCR reduction after 30 min of treatment with IM156 and expressed relative to the maximal OCR at the same time point and group. Data represent mean ± SD for technical replicates (n = 10 per concentration per group). (F) Immunoblot for phosphorylated (pAMPK, T172) and total AMPKα following treatment with vehicle or equivalent doses of phenformin (100 μM) or IM156 (10 μM) for 2 h. Actin is shown as a loading control. (G) Viability of Eμ- Myc + lymphoma cells following 48 h treatment with phenformin or IM156, comparing 10 different doses. EC 50 for each treatment is indicated. Data represent mean ± SEM for biological replicates (n = 3 per drug per concentration). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: phenformin , Toronto Research Chemicals , Cat # P296900.

Techniques: Derivative Assay, Concentration Assay, Incubation, Inhibition, Purification, Activity Assay, Expressing, Plasmid Preparation, Western Blot, Control

Journal: Cell Reports Medicine

Article Title: Repression of LKB1 by miR-17∼92 Sensitizes MYC -Dependent Lymphoma to Biguanide Treatment

doi: 10.1016/j.xcrm.2020.100014

Figure Lengend Snippet: miR-17∼92 Sensitizes Lymphoma Cells to Apoptosis by Biguanides (A) Viability of Ctrl (fl/fl) and miR-17∼92 -deficient (Δ/Δ) Eμ- Myc + lymphoma cells untreated (Ctrl, gray) or treated with 100 μM phenformin (Phen, blue) for 48 h. Data represent mean ± SD for biological replicates (n = 3 per group). (B) Immunoblot for active (cleaved) caspase-3 in cells treated as in (A). Actin is shown as a loading control. (C) Dose of phenformin (left) or IM156 (right) required to achieve 50% decrease in cell viability (EC 50 ) in biguanide-sensitive Eμ- Myc + lymphoma cells expressing control (Ctrl) or miR-17∼92 (+17∼92) expression vectors. Cell viability was measured 48 h post-biguanide treatment. See also B and C. (D) Viability of control (Ctrl) or miR-17∼92 -expressing (+17∼92) Raji cells after 48 h treatment with 100 μM phenformin. Histogram shows representative staining for cell death using fluorescent viability dye (FVD), which is quantified at right. Data represent mean ± SD for biological replicates (n = 3 per group). (E) Dose of phenformin (left) or IM156 (right) required to achieve a 50% decrease in cell viability (EC 50 ) in biguanide-resistant Raji cells expressing control (Ctrl) or miR-17∼92 (+17∼92) expression vectors following 48 h of treatment with biguanide. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: phenformin , Toronto Research Chemicals , Cat # P296900.

Techniques: Western Blot, Control, Expressing, Staining

Journal: Cell Reports Medicine

Article Title: Repression of LKB1 by miR-17∼92 Sensitizes MYC -Dependent Lymphoma to Biguanide Treatment

doi: 10.1016/j.xcrm.2020.100014

Figure Lengend Snippet: Biguanide Treatment Selectively Impairs the Growth of miR-17∼92 -Expressing Lymphoma Cells In Vivo (A and B) Kaplan-Meier curves for the viability of nude mice injected with 1 × 10 6 Myc + /Ctrl (A) or Myc + /+17∼92 (B) lymphoma cells. Mice were provided with untreated water (Ctrl, n = 10), 0.9 mg/mL phenformin (Phen, n = 8), or 0.8 mg/mL IM156 (IM156, n = 10) ad libitum following intravenous tumor cell injection. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: phenformin , Toronto Research Chemicals , Cat # P296900.

Techniques: Expressing, In Vivo, Injection

Journal: Cell Reports Medicine

Article Title: Repression of LKB1 by miR-17∼92 Sensitizes MYC -Dependent Lymphoma to Biguanide Treatment

doi: 10.1016/j.xcrm.2020.100014

Figure Lengend Snippet: miR-17∼92 Confers Sensitivity to Biguanides through the Suppression of LKB1 (A) Immunoblot of LKB1 and actin protein levels in Ctrl (fl/fl) and miR-17∼92-deficient (Δ/Δ) Eμ- Myc + lymphoma cells. (B and C) Immunoblot of AMPK pathway (B) and mTORC1 pathway (C) activation in control (Ctrl) or miR-17∼92-expressing (+17∼92) Eμ- Myc + lymphoma cells. AMPK activation was determined by measuring total and phosphorylated (p-AMPK, T172) AMPKα. mTORC1 activity was assessed by measuring levels of total and phosphorylated ribosomal S6 protein (rS6, S235/236), 4EBP (T37/46), and Raptor (S792). (D) Viability of the following Eμ- Myc + lymphoma cells: Ctrl (fl/fl), miR-17∼92-deficient (Δ/Δ), and miR-17∼92-deficient expressing shRNAs targeting LKB1 (Δ/Δ+shLKB1/2). Cells were treated with 10 μM IM156 for 48 h and viability assessed by flow cytometry. Data represent mean ± SD for biological replicates (n = 3 per group). (E) Viability of Eμ- Myc + lymphoma cells expressing miR-17∼92 (+17∼92) or miR-17∼92 lacking the miR-17 and -20 seed families (+17∼92Δ(17+20)). Cells were treated with vehicle (Ctrl) or 10 μM IM156 for 48 h and viability assessed by flow cytometry. Data represent mean ± SD for biological replicates (n = 3 per group). (F) Immunoblot of total and phosphorylated (p-AMPK, T172) AMPKα levels in control (Ctrl) and miR-17∼92 -expressing (+17∼92) Eμ- Myc + lymphoma cells following a 2-h treatment with vehicle (–), 100 μM phenformin (+Phen), or 10 μM IM156 (+IM156). (G) Schematic of transcriptional differences in miRNA between control (fl/fl) and miR-17∼92 expressing Eμ- Myc + lymphoma cells. miR-17 and -20 are responsible for the repression of LKB1, which increases biguanide sensitivity. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: phenformin , Toronto Research Chemicals , Cat # P296900.

Techniques: Western Blot, Activation Assay, Control, Expressing, Activity Assay, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Repression of LKB1 by miR-17∼92 Sensitizes MYC -Dependent Lymphoma to Biguanide Treatment

doi: 10.1016/j.xcrm.2020.100014

Figure Lengend Snippet: Biguanide Treatment Affects Central Carbon Metabolism in miR-17∼92 -Expressing Lymphoma Cells (A) ECAR and OCR of control (Ctrl) or miR-17∼92 -expressing (+17∼92) Eμ- Myc + lymphoma cells. Data represent mean± SD for biological replicates (n = 12 per group). (B) ATP production rates (J ATP ) for Eμ- Myc + lymphoma cells expressing control (Ctrl) or miR-17∼92 (+17∼92) expression vectors. Seahorse experiments were performed under standard cell culture media conditions (Glc, 25 mM; Gln, 2 mM). J ATP total is the sum of the glycolytic (J ATP gly) and OXPHOS (J ATP ox) ATP production rates. Data represent mean ± SDs for biological replicates (n = 12 per group) (Ctrl, n = 8; Ctrl+IM156, n = 8; +17∼92, n = 11; +17∼92+IM156, n = 12). (C) Bioenergetic capacity plot of Eμ- Myc + lymphoma cells expressing control (Ctrl) or miR-17∼92 overexpression (+17∼92) vectors following a 2-h treatment with vehicle (gray) or IM156 (purple). Rectangles define the maximum bioenergetic space of each cell type. (D) Heatmap showing relative metabolite abundances for control (Ctrl) or miR-17∼92 -expressing (+17∼92) Eμ- Myc + lymphoma cells following a 2-h treatment with vehicle (Ctrl), phenformin (Phen), or IM156 (n = 3 per group). (E) Fractional enrichment of U-[ 13 C]-glucose-derived lactate in miR-17∼92 -expressing Eμ- Myc + lymphoma cells following a 2-h treatment with vehicle (Ctrl), phenformin (Phen), or IM156. Data represent mean ± SD for biological replicates (n = 3 per group). (F) Fractional enrichment of U-[ 13 C]-labeled glutamine in miR-17∼92 -expressing Eμ- Myc + lymphoma cells following a 2-h treatment with vehicle (Ctrl), phenformin (Phen), or IM156. Data represent mean ± SD for biological replicates (n = 3 per group). (G) Fractional enrichment of U-[ 13 C]-glucose-derived citrate and U-[ 13 C]-glutamine-derived citrate and aspartate in miR-17∼92 -expressing Eμ- Myc + lymphoma cells, following a 2-h treatment with vehicle (Ctrl), phenformin (Phen), or IM156. Data represent mean ± SD for biological replicates (n = 3 per group). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: phenformin , Toronto Research Chemicals , Cat # P296900.

Techniques: Expressing, Control, Cell Culture, Over Expression, Derivative Assay, Labeling

Journal: Cell Reports Medicine

Article Title: Repression of LKB1 by miR-17∼92 Sensitizes MYC -Dependent Lymphoma to Biguanide Treatment

doi: 10.1016/j.xcrm.2020.100014

Figure Lengend Snippet: miR-17 and - 20 Expression Correlates with Biguanide Sensitivity in Human Lymphoma Cells (A and B) EC 50 for phenformin (Phen, left) and IM156 (right) correlated to the relative levels of pri-miR-17 (A) and mature miR-17 (B) transcripts for 10 human lymphoma cell lines. Linear regression (dotted line) is shown for both drugs with shaded 95% confidence interval. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: phenformin , Toronto Research Chemicals , Cat # P296900.

Techniques: Expressing

Journal: Cell Reports Medicine

Article Title: Repression of LKB1 by miR-17∼92 Sensitizes MYC -Dependent Lymphoma to Biguanide Treatment

doi: 10.1016/j.xcrm.2020.100014

Figure Lengend Snippet:

Article Snippet: phenformin , Toronto Research Chemicals , Cat # P296900.

Techniques: Recombinant, Lysis, Luciferase, shRNA, Software

Journal: eLife

Article Title: ANGPTL4 mediates shuttling of lipid fuel to brown adipose tissue during sustained cold exposure

doi: 10.7554/eLife.08428

Figure Lengend Snippet: ( A ) Angptl4 mRNA in differentiated T37i adipocytes treated for 6 hr with 1 mM AICAR, 100 μM A769662, 1 mM metformin or 250 μM phenformin hydrochloride. ( B ) Angptl4 mRNA in differentiated primary brown adipocytes, BA adipocytes, or T37i adipocytes treated for indicated times with 1 mM AICAR. ( C ) Immunoblot for ANGPTL4, LPL, AMPKα1,2 and phospho-AMPK Thr172 in differentiated T37i cells treated with control (CTRL) or 1 mM AICAR for 3 hr. ( D ) Angptl4 mRNA in differentiated T37i adipocytes treated with CTRL siRNA or siRNA against AMPKα1 and AMPKα2 for 48 hr, followed by incubation with control (CTRL) or 1 mM AICAR for 3 hr. ( E ) Angptl4 mRNA in differentiated T37i adipocytes treated with CTRL siRNA or siRNA against AMPKα1 and AMPKα2 for 48 hr, followed by incubation with H 2 O control medium (CTRL) or 100 μM A769662 for 6 hr. ( E ) Ampkα1 and Ampkα2 mRNA in differentiated T37i adipocytes treated with CTRL siRNA or siRNA against AMPKα1 and AMPKα2 for 48 hr. ( F ) Angptl4 mRNA levels in BAT and WAT explants from C57BL/6J wild-type mice (∼50 μg) treated with H 2 O control medium (CTRL) or 1 mM AICAR for 3 hr. ( G ) Immunoblot for ANGPTL4 in BAT and WAT explants from C57BL/6J wild-type mice (∼50 mg) treated with H 2 O control medium (CTRL) or 1 mM AICAR for 3 hr. *Statistically significant compared to control samples or between indicated treatments, according to Student’s t-test (p<0.05). Error bars represent ± SEM. DOI: http://dx.doi.org/10.7554/eLife.08428.013

Article Snippet: A769662 was purchased from Abcam (Cambridge, United Kingdom), phenformin hydrochloride was purchased from Cayman Chemicals (via SanBio, Uden, the Netherlands).

Techniques: Western Blot, Control, Incubation

Journal: Marine Drugs

Article Title: Antidiabetic Activity of Differently Regioselective Chitosan Sulfates in Alloxan-Induced Diabetic Rats

doi: 10.3390/md13053072

Figure Lengend Snippet: The effects of differently regioselective chitosan sulfates on the body weights of alloxan-induced diabetic rats.

Article Snippet: Phenformin hydrochloride tablets were purchased from Zhejiang Yatai Pharmaceutical Co. Ltd. (Shaoxing, Zhejiang, China).

Techniques: Control

Journal: Marine Drugs

Article Title: Antidiabetic Activity of Differently Regioselective Chitosan Sulfates in Alloxan-Induced Diabetic Rats

doi: 10.3390/md13053072

Figure Lengend Snippet: The effects of differently regioselective chitosan sulfates on the fasting blood glucose level of alloxan-induced diabetic rats.

Article Snippet: Phenformin hydrochloride tablets were purchased from Zhejiang Yatai Pharmaceutical Co. Ltd. (Shaoxing, Zhejiang, China).

Techniques: Control

Journal: Marine Drugs

Article Title: Antidiabetic Activity of Differently Regioselective Chitosan Sulfates in Alloxan-Induced Diabetic Rats

doi: 10.3390/md13053072

Figure Lengend Snippet: Glucose tolerance tests in normal and experimental groups.

Article Snippet: Phenformin hydrochloride tablets were purchased from Zhejiang Yatai Pharmaceutical Co. Ltd. (Shaoxing, Zhejiang, China).

Techniques: Control