Journal: Molecular Medicine

Article Title: TOP2A deficiency leads to human recurrent spontaneous abortion and growth retardation of mouse pre-implantation embryos

doi: 10.1186/s10020-022-00592-4

Figure Lengend Snippet: Downregulation of TOP2A affects the FOXO signaling pathway. A Western blotting showed changes in the levels of phosphorylated- FoxO1, phosphorylated-FoxO3a and phosphorylated-Rb after TOP2A downregulation. B Western blotting was used to assess changes in the level of phosphorylated-FoxO1 in the inhibitor-treated group. C q-PCR assay was used to verify changes in the mRNA expression of several downstream signaling molecules. D mRNA expression of cycle-associated genes was assessed after TOP2A knock down in JEG-3 cells and after treatment with chemical inhibitors that reduce FoxO1 activity. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Equal amounts of protein (30 μg/lane) were separated using SDS–PAGE and transferred on to PVDF membranes (Millipore Sigma, Burlington, MA, USA), which was subsequently blocked with quick blocking buffer (NCM Biotech, Suzhou, China) for 15 min. Membranes were washed with phosphate-buffered saline (PBS) and then incubated with the following primary antibodies overnight at 4 °C: TOP2A (1:10,000; Abcam, Cambridge, UK), matrix metalloproteinase 9 (MMP9) (1:1000; Abcam), MMP2 (1:1000; Abcam), cyclin-dependent kinase 2 (CDK2) (1:1000, Cell Signaling Technology, Danvers, MA, USA), CDK4 (1:1000, Cell Signaling Technology), phosphor-FoxO1 (1:1000, Cell Signaling Technology), P21 (1:1000; Cell Signaling Technology), P27 (1:1000; Cell Signaling Technology), cleaved caspase 3 (1:1000; Abcam), caspase 3 (1:1000; Abcam), and β-actin (1:2000; Proteintech, Wuhan, China).

Techniques: Western Blot, Expressing, Activity Assay

Journal: PLoS ONE

Article Title: Regulation of AKT Phosphorylation at Ser473 and Thr308 by Endoplasmic Reticulum Stress Modulates Substrate Specificity in a Severity Dependent Manner

doi: 10.1371/journal.pone.0017894

Figure Lengend Snippet: Cells were transfected with either siCon or siGRP78 RNA duplexes for 24 hour before treatment with tunicamycin for 24 hour. Proteins were extracted for immunoblot analysis with GRP78, P-PDK1(Ser241), PDK1, P-AKT(Thr308), P-AKT(Ser473), AKT, P-AKT substrate (RXRXXS/T), P-mTOR(Ser2448), P-HDM2(Ser166), P-FOXO1(Ser319) and P-GSK-3α/β(Ser21/9). Densitometry of band intensity is expressed relative to siCon untreated control (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are mean±SEM for 3 independent experiments. ** indicates P≤ 0.01; n.s indicates non-significant change. A & B ) Down-regulation of GRP78 elevates Ser473 phosphorylation but not at Thr308. C ) Knock-down of GRP78 alters AKT downstream substrates recognition. Arrows indicate the substrates changed their phosphorylation pattern in tunicamycin-treated siGRP78 cells.

Article Snippet: Anti-phospho-AKT (Ser473), anti-AKT, anti-AKT1, anti-phospho-mTOR (Ser2448), anti-phospho-HDM2 (Ser166), anti-phospho-FOXO1 (Ser319), anti-phospho-GSK3α/β (Ser21/9), immobilized AKT (1G1) antibodies (bead conjugated), immobilized phospho-AKT (Ser473) (D9E) antibodies (bead conjugated) and immobilized IgG mouse (bead conjugated) were from Cell Signaling Technology (New England Biolabs Ltd, UK).

Techniques: Transfection, Western Blot

Journal: Frontiers in Pharmacology

Article Title: RSK2 promotes melanoma cell proliferation and vemurafenib resistance via upregulating cyclin D1

doi: 10.3389/fphar.2022.950571

Figure Lengend Snippet: RSK2 upregulates cyclin D1 via promoting FOXO1 degradation. (A) Western blot was used to measure FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (B) RT-qPCR was used to measure FOXO1 mRNA expression in A375 cells transfected with RSK2 siRNA. (C) The influence of RSK2 on FOXO1 degradation was estimated in A375 cells under MG132 treatment. (D) The effect of RSK2 on FOXO1 protein stability was evaluated in A375 cells under CHX treatment for the indicated time. (E) HEK293T cells were co-transfected with Flag-FOXO1 and HA-RSK2 plasmid as indicated, immunoprecipitation with anti-Flag antibody was performed. (F) Western blot was used to measure p-FOXO1 (S319) and FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (G) Western blot was used to measure cyclin D1 expression in A375 cells co-transfected with Flag-FOXO1 and HA-RSK2 as indicated. (H) A schematic model of RSK2–FOXO1–cyclin D1 axis leading to melanoma proliferation and vemurafenib resistance.

Article Snippet: Antibodies used in immunoblotting were purchased from Cell Signaling Technologies: RSK2 (No.5528, 1:1000), FOXO1 (No.2880,1:1000), p-FOXO1/Ser319(No.2486,1:1000) and cyclin D1 (No.55506, 1:1000).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Immunoprecipitation

Journal: Scientific Reports

Article Title: FOXO1 forkhead domain mutants in B-cell lymphoma lack transcriptional activity

doi: 10.1038/s41598-022-05334-4

Figure Lengend Snippet: Expression of FOXO1 mutants, phosphorylation and interaction with ATG7. ( A ) HEK293T cells were transfected with wild-type or mutated pCMV-FOXO1 before protein extraction. Total protein lysates were analyzed by western blot with anti-phospho-FOXO1 (phospho-Thr24, phospho-Ser256 and phospho-Ser319), anti-FOXO1 and anti-β-actin antibodies. ( B ) HEK293T cells were transfected with wild-type or mutated pCMV-FOXO1 before protein extraction. FOXO1 was immunoprecipitated with anti-FOXO1 antibody and analyzed by western blot with anti-FOXO1 and anti-ATG7 antibodies. Original unprocessed images are available in Supplementary Fig. . ( C ) Schematic conclusion of cellular and molecular mechanisms of FOXO1 DBD versus Nt mutations.

Article Snippet: Anti-phospho-FOXO1 (anti-Thr24 (#9464), anti-Ser256 (#9461), anti-Ser319 (#2486)), anti-FOXO1 (#2880) and anti-ATG7 (#2631) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and the anti-β-actin antibody (#A5441) from Sigma (Saint-Louis, MO, USA).

Techniques: Expressing, Transfection, Protein Extraction, Western Blot, Immunoprecipitation