Journal: Cancers

Article Title: P2Y 2 R-Mediated PAK1 Activation Is Involved in ESM-1 Overexpression in RT-R-MDA-MB-231 through FoxO1 Regulation

doi: 10.3390/cancers14174124

Figure Lengend Snippet: ESM-1 overexpression in RT-R-MDA-MB-231 cells compared to MDA-MB-231 cells was downregulated by an inhibitor of FoxO1. ( A ) MDA-MB-231 and RT-R-MDA-MB-231 cells were treated with the indicated inhibitors (digoxin, a HIF-1α inhibitor; Bay11-7085, an NF-κB inhibitor; SR11302, an AP-1 inhibitor; AS1842856, a FoxO1 inhibitor; and AG490, a STAT-3 inhibitor) for 8 h, and then ESM-1 and GAPDH mRNA expression levels were analyzed with total RNA extracted from the cells by RT–PCR. The results are presented as the mean ± SD of five independent experiments. ** p < 0.01 compared to the MDA-MB-231 cells; # p < 0.05 compared to the untreated RT-R-MDA-MB-231 cells. ( B – E ) MDA-MB-231 and RT-R-MDA-MB-231 cells were treated with AS1842856, and ESM-1 mRNA expression levels ( B , D ) and protein expression levels ( C , E ) were analyzed by RT-PCR and Western blotting, respectively. GAPDH and β-actin were used as loading controls. The results are presented as the mean ± SD of five independent experiments. * p < 0.05, ** p < 0.01 compared to the MDA-MB-231 cells; # p < 0.05, ## p < 0.01 compared to the untreated RT-R-MDA-MB-231 cells. ( F ) RT-R-MDA-MB-231 cells were treated with the indicated doses of AS1842856 for 24 h, and cell viability was measured by cell counting kit (CCK) viability assay. The results are presented as the mean ± SD of five independent experiments.

Article Snippet: Digoxin (4683), Bay11-7085 (1743), SR11302 (2476), AS1842856 (4265), AG490 (0414), SP600125 (1496), SB203580 (1202), GF109203X (0741) and OSU03012 (5682) were purchased from Tocris (Bristol, UK).

Techniques: Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Counting, Viability Assay

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Insulin regulation of solute carrier family 2 member 1 (glucose transporter 1) expression and glucose uptake in decidualizing human endometrial stromal cells: an in vitro study

doi: 10.1186/s12958-020-00674-0

Figure Lengend Snippet: Relative gene expression levels of SLC2A1 in decidualizing human endometrial stromal cells in response to treatment with a FOXO1 inhibitor. The results are presented as mean values with 95% confidence intervals (CI) for each culture condition. All groups were compared to the control group (decidual cells) and differences considered statistically significant if 95% CI did not include the numerical value of the control group, in which case the significance was marked with one asterisk (*) corresponding to the significance level of p < 0.05

Article Snippet: In the experiment with FOXO1 inhibitor, cells were pre-decidualized for 3 days before further treatment with decidualizing agents in the presence of 100 nM and 500 nM AS1842856 (FOXO1-inhibitor by Merck Millipore, Germany, dissolved in DMSO) for 2 more days.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Participation of PLK1 and FOXM1 in the hyperplastic proliferation of pulmonary artery smooth muscle cells in pulmonary arterial hypertension

doi: 10.1371/journal.pone.0221728

Figure Lengend Snippet: Cells were grown in medium containing 5% FBS for 24 h in the presence or absence of 1uM AS1842856 (FOXO1 inhibitor). Western blots were probed for FOXM1 and beta actin. Shown is a representative blot of a non-PAH and PAH HPASMC cropped from the same blot (uncropped image in ). This experiment was done in duplicate. Box whisker plot displays the sum results of n = 4 HPASMC (two non-PAH and two PAH) treated with or without (CON) FOXO1 inhibitor. * = p < 0.05.

Article Snippet: Pharmacological inhibitor, thiostrepton was purchased from Santa Cruz Biotechnology, Inc (Dallas, TX), volasertib (BI 6727) from Selleckchem (Houston, TX) and AS1842856 from Cayman Chemical (Ann Arbor, Michigan).

Techniques: Western Blot, Whisker Assay

Journal: Journal of Inflammation Research

Article Title: Upregulation of COX-2 and PGE 2 Induced by TNF-α Mediated Through TNFR1/MitoROS/PKCα/P38 MAPK, JNK1/2/FoxO1 Cascade in Human Cardiac Fibroblasts

doi: 10.2147/JIR.S313665

Figure Lengend Snippet: Involvement of FoxO1 in TNF-α-induced COX-2 expression and PGE 2 production in HCFs. (A) The cells were pretreated with various concentrations of AS1842856 (10, 30, and 100 nM) for 1 h and then incubated with 15 ng/mL TNF-α for 16 h. The levels of COX-2 and GAPDH protein were determined by Western blot. (B) The cells were pretreated with 1 μM AS1842856 for 1 h and then incubated with 15 ng/mL TNF-α for 4 h. The levels of cox-2 and gapdh mRNA were analyzed by real-time PCR (open bars). Cells were co-transfected with COX-2-luci-plasmid along with a β-galactosidase plasmid, sequentially pretreated with 1 μM AS1842856 for 1 h, and then incubated with 15 ng/mL TNF-α for 2 h. Promoter activity was determined in the cell lysates using a promoter assay kit (solid bars). (C) The cells were transfected with scrambled or FoxO1 siRNA and then incubated with 15 ng/mL TNF-α for 16 h. The levels of COX-2, FoxO1, and GAPDH proteins were analyzed by Western blot. (D) The cells were pretreated with 0.1 μM AS1842856, 1 μg/mL TNFR1 nAb, 3 μM Gö6976, 1 μM MitoTEMPO, 1 μM p38i VIII, or 10 μM SP600125 for 1 h and then incubated with 15 ng/mL TNF-α for the indicated time intervals (15, 30, and 60 min). The levels of phospho-FoxO1 and total FoxO1 proteins were analyzed by Western blot. (E) The cells were pretreated with 0.1 μM AS1842856 and then incubated with 15 ng/mL TNF-α for 60 min. The DNA binding ability of FoxO1 was detected by chromatin immunoprecipitation assay. (F) The media saved from (A) were used to determine the levels of PGE 2 . Data are expressed as the mean ± S.E.M. of three independent experiments. # p<0.05, as compared with the control.

Article Snippet: SP600125, p38 inhibitor (p38i) VIII, Gö6976, MitoTEMPO, and AS1842856 were purchased from Enzo Life Science (Farmingdale, NY).

Techniques: Expressing, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Activity Assay, Promoter Assay, Binding Assay, Chromatin Immunoprecipitation

Journal: Science Advances

Article Title: Atf3-mediated metabolic reprogramming in hepatic macrophage orchestrates metabolic dysfunction–associated steatohepatitis

doi: 10.1126/sciadv.ado3141

Figure Lengend Snippet: ( A ) Co-immunoprecipitation (Co-IP) was performed using hepatic macrophages isolated from C57BL/6J mice fed a chow diet. ( B ) Representative image of basal FoxO1/Atf3 dimerization in hepatic macrophages. ( C and D ) Hepatic macrophages were pretreated with 1 μM AS1842856 for 24 hours and infected with Lv-Gfp or Lv-Atf3 for 24 hours. The Pck2 activity (C) and glucose levels (D) were determined. ( E and L ) C57BL/6J mice were fed chow diet (CD) or WD for 8 or 16 weeks. The levels of acetyl-FoxO1 and Cd36 palmitoylation in hepatic macrophages were measured. ( F and M ) Lv-Atf3 , Atf3 M−/− , or control mice were fed WD for 20 weeks. The level of acetyl-FoxO1 and CD36 palmitoylation in liver macrophages was determined. ( G ) Hepatic macrophages were infected with Lv-Gfp or Lv-Atf3 for 36 hours. Protein samples were subjected to immunoprecipitation and Western blotting and developed with appropriate antibodies, as shown in the figures. ( H ) Co-IP was performed using hepatic macrophages isolated from C57BL/6J mice. ( I ) Hepatic macrophages were pretreated with 10 μM MS-275 for 24 hours and infected with Lv-Gfp or Lv-Atf3 for 24 hours. Protein samples were subjected to immunoprecipitation and Western blotting and developed with appropriate antibodies, as shown in the figures. ( J ) The FoxO1 has three putative Hdac1 deacetylating sites. ( K ) Human embryonic kidney (HEK) 293T cells cotransfected pCMV-Hdac1 or pCMV-Atf3 with pCMV-flag-FoxO1-Wt or pCMV-flag-FoxO1-Mut for 36 hours. Levels of acetylated FoxO1 were determined. ( N ) The levels of CD36 palmitoylation in Atf3 fl/fl or Atf3 M−/− macrophages were measured. ( O ) Hepatic macrophages were isolated from C57BL/6J mice, and CUT&Tag assay was performed. ( P ) The heatmap shows that palmitoylation-related genes were regulated by Atf3. ( Q and R ) Cd36 WT or Cd36 Mut macrophages were infected with Lv-Gfp or Lv-Atf3 for 24 hours. Fatty acid uptake and oxidation were measured. ( S ) Simplified model depicting the role of Atf3 in glucolipid metabolism. * P < 0.05; ** P < 0.01. NS, not significant.

Article Snippet: AS1842856 (catalog no. GC19040) was purchased from GLPBIO.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Isolation, Infection, Activity Assay, Control, Western Blot