Journal: Cell metabolism

Article Title: Fructose-1,6-bisphosphatase is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness

doi: 10.1016/j.cmet.2023.03.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-P-AMPK(Thr172) , Cell Signaling , Cat# 2535; RRID:AB_331250.

Techniques: Immunoprecipitation, Transfection, Enzyme-linked Immunosorbent Assay, ATP Assay, Colorimetric Assay, Activity Assay, Glucose Assay, In Situ, Recombinant, Sequencing, Construct, Software

Journal: Redox Biology

Article Title: Energy stress modulation of AMPK/FoxO3 signaling inhibits mitochondria-associated ferroptosis

doi: 10.1016/j.redox.2023.102760

Figure Lengend Snippet: Energy stress inhibits ferroptosis partly through AMPK/FoxO3a signaling pathway. Cells treated with or without erastin(16 μM) and cultured in normal or sugar-free medium for 24 h Then the protein levels of FoxO3a, p -FoxO3a(Ser413), AMPK and p -AMPK(Thr172) in ( A/C )MEFs and ( B/D )MCF-7 cells were determined by Western blot. * p < 0.05, ** p < 0.01, *** p < 0.0001 versus the Glc group. # p < 0.05, ## p < 0.01, ### p < 0.001 versus the -Glc group. ( E) MCF-7 cells were treated with compound C (2 μM) or erastin (16 μM) in normal and sugar-free medium for 36 h, then the cell viability was measured by CCK-8 kit. * p < 0.05, ** p < 0.01, *** p < 0.001. (F) MCF-7 cells were transfected with FoxO3 specific siRNA for 24 h, the efficiency of knockdown was detected by western blotting analysis. 24 h after transfection with FoxO3a shRNA or Negative control (NC), MCF-7 cells were treated with 16 μM erastin (G) or 5 μM RSL3 (H) and cultured in normal or sugar-free medium for 24 h. Cell viability was measured by CCK-8 kit. ** p < 0.01 versus NC group cultured in sugar-free medium, ## p < 0.01 versus the group cultured in sugar-free medium for (G) while ## p < 0.01 versus the erastin group cultured in normal medium for (H) ， aa p < 0.01, aaa p < 0.001 versus the control group. (I/J) 24 h after transfection with lentivirus containing FoxO3a shRNA, MCF-7 cells were treated with erastin and cultured in normal or sugar-free medium for 24 h. Oxidized C11-BODIPY581/591 (Green) indicating lipid ROS were imaged by fluorescent microscope.* p ＜0.05,** p ＜0.01 versus NC; aa p ＜0.01, aaa p ＜0.001 versus the Glc group; # p ＜0.05， ## p ＜0.01 versus the erastin group; @@ p ＜0.01 versus the -Glc group. Data are expressed as mean ± SEM, n = 3.

Article Snippet: The membranes were blocked in 5% skim-milk and then incubated overnight at 4 °C with the primary antibodies for mouse AMPK (1:1000,Proteintech,#66536-1-Ig), rabbit p -AMPK(Thr172) (1:1500,Cell Signaling technology,#2535), rabbit FoxO3a (1:1000,proteintech,#66428-1-Ig), rabbit p -FoxO3a(Ser413)(1:1000,Cell Signaling technology,#8174), rabbit CYCs (1:1000,Cell Signaling technology,#11940S), rabbit HMOX1 (1:1000,ABclonal,#A19062), rabbit SOD2 (1:1000,proteintech,#6647-1-Ig),mouse PGC1α(1:1000,proteintech,#66369-1-Ig),mouse TOM20(1:1000,proteintech,#66777-1-Ig),rabbit GPX4 (1:1000,ABclonal,#A11243),rabbit SLC7A11 (1:1000,ABclonal,#A2413),rabbit DRP1 (1:1500,proteintech,#12957-1-AP),mouse Hsp60 (1:1000,proteintech,#66041-1-Ig), rabbit p -MFF (ser172/ser146)(1:1500,Cell Signaling technology,#AF2365),mouse Total OXPHOS(1:2500,Abcam,#ab110413),mouse β-actin (1:1000,TRANS,#HC201).

Techniques: Cell Culture, Western Blot, CCK-8 Assay, Transfection, shRNA, Negative Control, Microscopy

Journal: Redox Biology

Article Title: Energy stress modulation of AMPK/FoxO3 signaling inhibits mitochondria-associated ferroptosis

doi: 10.1016/j.redox.2023.102760

Figure Lengend Snippet: TFP plays a protective effect on CIR injury in rats through AMPK/FoxO3a/Hif1α pathway (A) Experimental design of this study. For the sham group rats, only their blood vessels were exposed, and no embolization coils were introduced. Rats in the trifluoperazine groups were treated medically after MCAO surgery for 5 min and repeated 24 h after first injection. Neurological score and Collection of brain were evaluated at 48 h of reperfusion, and tissue samples were subsequently tested. (B) The representative TTC-stained brain slices from each group, ( C) Quantitative analysis of brain infarct volume. (D) Effect of TFP on neurological scores. (E/F) Immunofluorescence staining results of the penumbral brain tissue in each group. DAPI (blue), FoxO3a (green), Hif1α and SLC7A11 (red). (G/H/I) The protein expression of SLC7A11, FoxO3a, p -FoxO3a(ser413), AMPK and p -AMPK(Thr172) in the penumbral brain tissue were detected by western blotting analysis (J) The mRNA expression of SLC7A11 in the penumbral brain tissue were detected by qRT-PCR analysis. (K) BV-2 cells were treated with different concentration of erastin for 24 h then the expression of FoxO3a/SLC7A11 were measured by western blotting analysis. (L) BV-2 cells were transfected with FoxO3a siRNA for 36 h, then the expression of SLC7A11 were measured by western blotting analysis. * p < 0.05, *** p < 0.001 versus control group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus MCAO group .(M) Statistics of distribution of FoxO3a-binding sites in the mouse genome. (N) FoxO3a binding profile containing the gene of SLC7A11 in BV-2 cells. Data are expressed as mean ± SEM, n = 5.

Article Snippet: The membranes were blocked in 5% skim-milk and then incubated overnight at 4 °C with the primary antibodies for mouse AMPK (1:1000,Proteintech,#66536-1-Ig), rabbit p -AMPK(Thr172) (1:1500,Cell Signaling technology,#2535), rabbit FoxO3a (1:1000,proteintech,#66428-1-Ig), rabbit p -FoxO3a(Ser413)(1:1000,Cell Signaling technology,#8174), rabbit CYCs (1:1000,Cell Signaling technology,#11940S), rabbit HMOX1 (1:1000,ABclonal,#A19062), rabbit SOD2 (1:1000,proteintech,#6647-1-Ig),mouse PGC1α(1:1000,proteintech,#66369-1-Ig),mouse TOM20(1:1000,proteintech,#66777-1-Ig),rabbit GPX4 (1:1000,ABclonal,#A11243),rabbit SLC7A11 (1:1000,ABclonal,#A2413),rabbit DRP1 (1:1500,proteintech,#12957-1-AP),mouse Hsp60 (1:1000,proteintech,#66041-1-Ig), rabbit p -MFF (ser172/ser146)(1:1500,Cell Signaling technology,#AF2365),mouse Total OXPHOS(1:2500,Abcam,#ab110413),mouse β-actin (1:1000,TRANS,#HC201).

Techniques: Injection, Staining, Immunofluorescence, Expressing, Western Blot, Quantitative RT-PCR, Concentration Assay, Transfection, Binding Assay