Journal: Redox biology

Article Title: Mfn2-mediated mitochondrial fusion alleviates doxorubicin-induced cardiotoxicity with enhancing its anticancer activity through metabolic switch.

doi: 10.1016/j.redox.2022.102311

Figure Lengend Snippet: Fig. 7. Knockdown of FoxO1 restored Mfn2-mediated mitochondrial fusion and increased cell viability in the Dox-treated cardiomyocytes. (A) Heatmap showing the expression pattern of key transcription factors involved in the Gene Expression Omnibus (GEO) database (GSE157904). (B) Quantitative mRNA expression of FoxO1 in GSE157904. (C) Representative blots and quantitative data of total FoxO1 protein level. (D) Representative blots and quantitative data of nuclear FoxO1 protein level. Two-tailed unpaired Student’s t-test was used to compare the two groups in Figure B–D. (E-G) Representative blots and quantitative data of FoxO1 and Mfn2 protein levels. The blotting experiments were conducted 3 times independently per group in Figure C–G. (H) Quantitative mRNA expression of Mfn2. n = 3 independent experiments per group in Figure B and Figure H. (I) Representative MitoTracker Red-stained mitochondrial morphology images. (Quantitative data are presented in J–K). Original magnification × 600. (J) Quantitative data of mean mitochondrial size in the cardiomyocytes. (K) Quantitative data of mitochondrial number per cell. (L) Relative cell viability. n = 6 independent experiments per group in FigureI-L. One-way ANOVA with Turkey’s multiple comparison test was used to compare the groups in Figure F–L. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Since the protein expression of total FoxO1 was also upregulated in the Dox-treated hearts in vivo (Fig. 8A), the mice were then treated with FoxO1 inhibitor AS1842856 (HY-100596, MedChemExpress) at the dosage of 100 mg/kg by oral gavage twice daily for two days immediately following each Dox injection according to the previous study [26].

Techniques: Knockdown, Expressing, Gene Expression, Two Tailed Test, Staining, Comparison

Journal: Redox biology

Article Title: Mfn2-mediated mitochondrial fusion alleviates doxorubicin-induced cardiotoxicity with enhancing its anticancer activity through metabolic switch.

doi: 10.1016/j.redox.2022.102311

Figure Lengend Snippet: Fig. 8. FoxO1 inhibitor restored Mfn2-mediated mitochondrial fusion and enhanced cardiac function in the Dox-treated mice. (A) Representative blots and quantitative data of total FoxO1 protein level in the heart tissues. (B) Representative blots and quantitative data of Mfn2 protein level. The blotting experiments were conducted 6 times independently per group in Figure A–B. (C) Quantitative data of Mfn2 mRNA expression. (D) Representative M-mode echocardiography images. (Quantitative data are presented in E-F) (E) EF, ejection fraction. (F) LVESV, left ventricular end-systolic volume. (G) Quantitative data of mean mitochondrial size. (H) Representative mitochondrial images obtained by transmission electron microscope (Quantitative data are presented in G and I). Original magnification × 15000. Representative mitochondria on the bottom images (local magnifications of top images) were marked with light green pseudo-color to highlight the changes of the mitochondria. (I) The proportion of mitochondria in a specific area categorized into three size groups (< 0.6 μm2, within 0.6–1.0 μm2, >1.0 μm2) was counted. n = 6 independent experiments per group in Figure C–I. One-way ANOVA with Turkey’s multiple comparison test was used. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Since the protein expression of total FoxO1 was also upregulated in the Dox-treated hearts in vivo (Fig. 8A), the mice were then treated with FoxO1 inhibitor AS1842856 (HY-100596, MedChemExpress) at the dosage of 100 mg/kg by oral gavage twice daily for two days immediately following each Dox injection according to the previous study [26].

Techniques: Expressing, Transmission Assay, Microscopy, Comparison

Journal: Redox biology

Article Title: Mfn2-mediated mitochondrial fusion alleviates doxorubicin-induced cardiotoxicity with enhancing its anticancer activity through metabolic switch.

doi: 10.1016/j.redox.2022.102311

Figure Lengend Snippet: Fig. 9. FoxO1 inhibited the transcription of Mfn2 by binding to its promoter sites. Ad-FoxO1 reduced Mfn2 mRNA and protein expression in a dose-dependent manner. (A-C) Representative blots and quantitative data of FoxO1 and Mfn2 protein levels. The blotting experiments were conducted 3 times independently per group in Figure A–C. (D) Quantitative data of Mfn2 mRNA expression. n = 3 independent experiments per group in Figure D. One-way ANOVA with Bonferroni’s multiple comparison test was used to compare the groups in Figure B–D. (E) Representative MitoTracker Red-stained mitochondrial morphology images (Quanti tative data are presented in F-G). Original magnification × 600. (F) Quantitative data of mean mitochondrial size in the cardiomyocytes. (G) Quantitative data of mitochondrial number per cell. n = 6 independent experiments per group in Figure E–G. Two-tailed unpaired Student’s t-test was used to compare the groups in Figure F–G. (H) ChIP analysis of FoxO1 at five positions of Mfn2 promoter in the cardiomyocytes. One-way ANOVA with Turkey’s multiple comparison test was used. (I) Allocation of five primers of Mfn2 promoter. (J-K) ChIP analysis for FoxO1 binding to Mfn2 promoter using prime 3 in the cardiomyocytes subjected to different treatments. (L) FoxO1 inhibits the luciferase activity of the Mfn2 promoter in HEK-293T cells. n = 3 independent experiments per group in Figure H–L. Two-way ANOVA with Bonferroni’s multiple comparison test was used to compare the groups in Figure K–L. *P < 0.05, **P < 0.01, ***P < 0.001; ###P < 0.001 vs. other primers. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Since the protein expression of total FoxO1 was also upregulated in the Dox-treated hearts in vivo (Fig. 8A), the mice were then treated with FoxO1 inhibitor AS1842856 (HY-100596, MedChemExpress) at the dosage of 100 mg/kg by oral gavage twice daily for two days immediately following each Dox injection according to the previous study [26].

Techniques: Binding Assay, Expressing, Comparison, Staining, Two Tailed Test, Luciferase, Activity Assay

Journal: Redox biology

Article Title: Mfn2-mediated mitochondrial fusion alleviates doxorubicin-induced cardiotoxicity with enhancing its anticancer activity through metabolic switch.

doi: 10.1016/j.redox.2022.102311

Figure Lengend Snippet: Fig. 12. Schematic figure illustrating that Mfn2- mediated mitochondrial fusion alleviates Dox- induced cardiotoxicity and enhances its anti cancer efficacy via metabolic shift from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). In the cardiomyocytes, Dox-elicited upregulation of FoxO1 negatively inhibits the tran scriptional expression of Mfn2 directly by binding to its promoter. Reconstitution of mitochondrial fusion through upregulation of Mfn2 alleviates Dox-induced myocardial injury by enhancing OXPHOS. In the tumor cells, Mfn2-mediated mitochondrial fusion delays tumor growth and strengthens antitumor therapy of Dox mainly by inhibiting aerobic glycol ysis metabolism. OCR/ECAR, the ratio of oxygen consumption rate to extracellular acidification rate. Δψm, mitochondrial membrane potential.

Article Snippet: Since the protein expression of total FoxO1 was also upregulated in the Dox-treated hearts in vivo (Fig. 8A), the mice were then treated with FoxO1 inhibitor AS1842856 (HY-100596, MedChemExpress) at the dosage of 100 mg/kg by oral gavage twice daily for two days immediately following each Dox injection according to the previous study [26].

Techniques: Phospho-proteomics, Expressing, Binding Assay, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Rapid Renal Regulation of Peroxisome Proliferator-activated Receptor γ Coactivator-1α by Extracellular Signal-Regulated Kinase 1/2 in Physiological and Pathological Conditions *

doi: 10.1074/jbc.M116.754762

Figure Lengend Snippet: FOXO1 phosphorylation in the cortex and nuclear phosphorylation decrease after ERK1/2 inhibition. A, representative immunoblot of phosphorylated FOXO1 and ERK1/2, AKT, and p38 kinases following trametinib administration. p, phosphorylated; MW, molecular weight. B, densitometry analysis of phosphorylated FOXO1 when compared with total FOXO1. C, densitometry analysis of phosphorylated AKT to total AKT, and phosphorylated p38 to total p38 after trametinib treatment. D, representative immunoblot of nuclear phosphorylated FOXO1 and ERK1/2 after trametinib treatment in mouse kidney cortex. E, densitometry analysis of phosphorylated FOXO1 when compared with total FOXO1 after inhibition of ERK1/2 in the nucleus. F, mRNA expression of FOXO1, SOD2, and catalase at 4 h following trametinib administration. Data are represented as mean ± S.E., n ≥ 4. Different superscripts indicate statistically significant differences (p < 0.05).

Article Snippet: Primary antibodies used in these studies included phospho-ERK1/2 (1:1000) (#4370), total ERK1/2 (1:1000) (#4695), AKT (1:1000) (#9272), phospho-AKT (1:1000) (#4060), phospho-p38 (1:1000) (#4511), p38 MAPK (1:1000) (#8690), EGF receptor (1:1000) (#4267), phospho-EGFR Tyr-1173 (1:750) (#4407s), phospho-EGFR Tyr-1068 (1:1000) (#3777s), total FOXO3a (1:1000), phospho-Ser-294-FOXO3a (1:1000) (#5538), total FOXO1 (1:1000) (#2880), and histone H3 (1:2000) (#9715), all from Cell Signaling Technology, Danvers, MA; phospho-Ser-329-FOXO1 (1:1000) (ab192201), TFAM (1:1000) (ab131607), lamin B1 (1:1000) (ab16048), and PGC-1α (1:1000) (ab54481), all from Abcam; kidney injury molecule-1 (KIM-1) (1:1000; AF1817), from R&D Systems, Minneapolis, MN; and β-actin (1:2000) (sc-47778), from Santa Cruz Biotechnology, Dallas, TX.

Techniques: Inhibition, Western Blot, Molecular Weight, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Rapid Renal Regulation of Peroxisome Proliferator-activated Receptor γ Coactivator-1α by Extracellular Signal-Regulated Kinase 1/2 in Physiological and Pathological Conditions *

doi: 10.1074/jbc.M116.754762

Figure Lengend Snippet: Erlotinib blocks ERK1/2 phosphorylation in naïve mice and following IR, preventing decreases in PGC-1α and NRF1 expression. A, representative immunoblot of phosphorylated ERK1/2 after 4 h of treatment with erlotinib in mouse kidney cortex. p, phosphorylated; MW, molecular weight. B, densitometry analysis of phosphorylated ERK1/2 when compared with total ERK1/2 following 4 h of erlotinib treatment. C, mRNA expression of PGC-1α and FOXO1 at 4 h in mouse cortex following erlotinib treatment. D, representative immunoblot of phosphorylated ERK1/2 and total ERK1/2, as well as PGC-1α following IR AKI. E, densitometry analysis of phosphorylated ERK1/2 when compared with total ERK1/2 following 3 h of IR AKI. F, densitometry analysis of PGC-1α protein when compared with β-actin following 3 h of IR AKI. G, mRNA expression of PGC-1α and NRF1 following 3 h of IR AKI. H, serum creatinine was assessed 3 h after IR AKI. Data are represented as mean ± S.E., n ≥ 4. Different superscripts indicate statistically significant differences (p < 0.05).

Article Snippet: Primary antibodies used in these studies included phospho-ERK1/2 (1:1000) (#4370), total ERK1/2 (1:1000) (#4695), AKT (1:1000) (#9272), phospho-AKT (1:1000) (#4060), phospho-p38 (1:1000) (#4511), p38 MAPK (1:1000) (#8690), EGF receptor (1:1000) (#4267), phospho-EGFR Tyr-1173 (1:750) (#4407s), phospho-EGFR Tyr-1068 (1:1000) (#3777s), total FOXO3a (1:1000), phospho-Ser-294-FOXO3a (1:1000) (#5538), total FOXO1 (1:1000) (#2880), and histone H3 (1:2000) (#9715), all from Cell Signaling Technology, Danvers, MA; phospho-Ser-329-FOXO1 (1:1000) (ab192201), TFAM (1:1000) (ab131607), lamin B1 (1:1000) (ab16048), and PGC-1α (1:1000) (ab54481), all from Abcam; kidney injury molecule-1 (KIM-1) (1:1000; AF1817), from R&D Systems, Minneapolis, MN; and β-actin (1:2000) (sc-47778), from Santa Cruz Biotechnology, Dallas, TX.

Techniques: Expressing, Western Blot, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Rapid Renal Regulation of Peroxisome Proliferator-activated Receptor γ Coactivator-1α by Extracellular Signal-Regulated Kinase 1/2 in Physiological and Pathological Conditions *

doi: 10.1074/jbc.M116.754762

Figure Lengend Snippet: Mouse primer sequence pairs with forward (F) and reverse (R) primers identified

Article Snippet: Primary antibodies used in these studies included phospho-ERK1/2 (1:1000) (#4370), total ERK1/2 (1:1000) (#4695), AKT (1:1000) (#9272), phospho-AKT (1:1000) (#4060), phospho-p38 (1:1000) (#4511), p38 MAPK (1:1000) (#8690), EGF receptor (1:1000) (#4267), phospho-EGFR Tyr-1173 (1:750) (#4407s), phospho-EGFR Tyr-1068 (1:1000) (#3777s), total FOXO3a (1:1000), phospho-Ser-294-FOXO3a (1:1000) (#5538), total FOXO1 (1:1000) (#2880), and histone H3 (1:2000) (#9715), all from Cell Signaling Technology, Danvers, MA; phospho-Ser-329-FOXO1 (1:1000) (ab192201), TFAM (1:1000) (ab131607), lamin B1 (1:1000) (ab16048), and PGC-1α (1:1000) (ab54481), all from Abcam; kidney injury molecule-1 (KIM-1) (1:1000; AF1817), from R&D Systems, Minneapolis, MN; and β-actin (1:2000) (sc-47778), from Santa Cruz Biotechnology, Dallas, TX.

Techniques: Sequencing

Journal: Cell Stress & Chaperones

Article Title: The regulation of Akt and FoxO transcription factors during dehydration in the African clawed frog ( Xenopus laevis )

doi: 10.1007/s12192-020-01123-y

Figure Lengend Snippet: Changes in the protein levels of total and phosphorylated FoxO1 over medium and high dehydration in the skeletal muscle of X. laevis. Total FoxO1, p-FoxO1 Thr-24, p-FoxO1 Ser-319, and p-FoxO1 Ser-256 protein levels were visualized at control, medium dehydration (15%), and high dehydration (30%) experimental conditions. Representative immunoblots are shown for all sampling points for all targets. Histograms show mean band densitometries (± SEM, n = 4 independent protein isolations from different animals). Data were analyzed using one-way ANOVAwith post hoc Tukey’s test (p < 0.05); for each parameter measured, values that are not statistically different from each other share the same letter notation

Article Snippet: Primary antibodies were purchased from Cell Signaling Technology for p-Akt Ser-473 (CS 9271), p-Akt Thr-308 (CS 4056), p-FoxO1 Ser-319 (CS 9471), p-FoxO3 Ser-318/321 (CS 9465), and p-FoxO1/3 Thr-24/32 (CS 9464P); Genscript for total Akt (Cat#: A00275) and p-FoxO1 Ser-256 (Cat#: A00395); Genetex for total FoxO1 (GTX110724), total FoxO3 (GTX100277), and MuRF1 (GTX110475); and Santa Cruz Biotechnology for MAFbx (SC27845).

Techniques: Control, Western Blot, Sampling

Journal: Hepatobiliary Surgery and Nutrition

Article Title: Xanthophyll β-cryptoxanthin treatment inhibits hepatic steatosis without altering vitamin A status in β-carotene 9',10'-oxygenase knockout mice

doi: 10.21037/hbsn-20-404

Figure Lengend Snippet: Hepatic SRT1 protein expression (A), acetylation of FOXO1 (B), PGC1α protein expression (C), and (D) PPARα protein expression in BCO2-KO mice fed control or BCX diet for 3 months. Values are mean ± SEM, n=5. *, different from –BCX, P≤0.05. BCO2, β-carotene-9',10'-oxygenase; KO, knock out; BCX, β-cryptoxanthin; SEM, standard error of the mean.

Article Snippet: The following antibodies were used for Western blotting: SIRT1 (sc-74465), acetylated and total FOXO1 (sc-374427), PGC1α (sc-518025), and PPARα (sc-398394) (Santa Cruz Biotechnology, Inc.), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (MAB374) (Millipore).

Techniques: Expressing, Knock-Out

Journal: Kidney Research and Clinical Practice

Article Title: Pan-Nox inhibitor treatment improves renal function in aging murine diabetic kidneys

doi: 10.23876/j.krcp.23.004

Figure Lengend Snippet: (A) Representative immunoblots for plasminogen activator inhibitor 1 (PAI-1) and type IV collagen (Col4); (B) densitometric analysis of immunoblots for PAI-1 and Col4; (C) representative immunoblot for Klotho, p-FoxO1, t-FoxO1 and Nrf2; and (D) densitometric analysis of immunoblot for Klotho, p-FoxO1/t-FoxO1 ratio, Nrf2. Data are shown as the mean ± standard error of mean. * p < 0.05, p < 0.001 vs. control or APX-115, # p < 0.05, ### p < 0.001 vs. streptozotocin (STZ).

Article Snippet: Proteins were transferred onto a polyvinylidene difluoride membrane, and the membrane was hybridized in blocking buffer overnight at 4 °C with rabbit polyclonal anti-Nox1 antibody (1:1,000; Abcam Plc), rabbit polyclonal anti-Nox2 antibody (1:1,000; Bioworld Technology), rabbit polyclonal anti-Nox4 antibody (1:1,000; Bioworld Technology), rabbit polyclonal anti-PAI-1 antibody (1:1,000; Santa Cruz Biotechnology), mouse monoclonal anti-monocyte chemoattractant protein (MCP)-1 antibody (1:1000; Lsbio), rabbit polyclonal anti-type IV collagen antibody (1:1,000; Abcam Plc), rabbit polyclonal anti-TGF-β1 antibody (1:1,000; Abbkine), goat polyclonal anti-Klotho antibody (1:1,000; R&D Systems Inc.), rabbit polyclonal anti-phospho FoxO1 antibody (1:1,000; Lsbio), rabbit polyclonal anti-total-FoxO1 antibody (1:1,000; Cell Signaling Technology), rabbit polyclonal anti-NRF2 antibody (1:1,000; Cell Signaling Technology), or mouse monoclonal anti-β actin antibody (1:5,000; Sigma-Aldrich).

Techniques: Western Blot, Control