foxo1 antagonist as1842856  (MedChemExpress)

Journal: Frontiers in Physiology

Article Title: Protective Properties of FOXO1 Inhibition in a Murine Model of Non-alcoholic Fatty Liver Disease Are Associated With Attenuation of ER Stress and Necroptosis

doi: 10.3389/fphys.2020.00177

Figure Lengend Snippet: Effects of PA on FOXO1, ER stress, and necroptosis in AML12 cells. (A) Immunoblot analysis of FoxO1, p-FoxO1, PERK, GRP78, CHOP, RIP1, RIP3, p-MLKL in CD and HFD feeding mice. (B) Immunoblot analysis of FoxO1, p-FoxO1, PERK, GRP78, CHOP, RIP1, RIP3, p-MLKL in cells treated with PA. (C) Quantitative analysis of (A) . (D) Quantitative analysis of (B) ∗ P < 0.05 vs. CD controls. t -Test, data are shown as mean ± standard deviation.

Article Snippet: To determine whether ER stress and necroptosis could be attenuated via inhibition of FOXO1, we treated AML12 cells with a FOXO1 antagonist (AS1842856, 1 μM, Selleck Chemicals) for 2 h before stimulation with PA (500 μM, Sigma-Aldrich).

Techniques: Western Blot, Standard Deviation

Journal: Frontiers in Physiology

Article Title: Protective Properties of FOXO1 Inhibition in a Murine Model of Non-alcoholic Fatty Liver Disease Are Associated With Attenuation of ER Stress and Necroptosis

doi: 10.3389/fphys.2020.00177

Figure Lengend Snippet: Effects of inhibition of FOXO1 in AML12 cells treated with PA. (A) Immunoblot analysis of PERK, GRP78, CHOP, RIP1, RIP3, p-MLKL. (B) Quantitative analysis of (A) . (C) Representative immunofluorescence staining of RIP3 was performed in Ctr, PA, PA + AS. (D) Representative immunofluorescence staining of PI. # P < 0.05 vs. PA controls. t -Test, data are shown as mean ± standard deviation.

Article Snippet: To determine whether ER stress and necroptosis could be attenuated via inhibition of FOXO1, we treated AML12 cells with a FOXO1 antagonist (AS1842856, 1 μM, Selleck Chemicals) for 2 h before stimulation with PA (500 μM, Sigma-Aldrich).

Techniques: Inhibition, Western Blot, Immunofluorescence, Staining, Standard Deviation

Journal: Frontiers in Physiology

Article Title: Protective Properties of FOXO1 Inhibition in a Murine Model of Non-alcoholic Fatty Liver Disease Are Associated With Attenuation of ER Stress and Necroptosis

doi: 10.3389/fphys.2020.00177

Figure Lengend Snippet: Effects of inhibition of FOXO1 in mice fed with a high fat diet. (A) Representative H&E staining, Oil red staining and Transmission electron microscopy of liver sections after CD or HFD or HFD + AS. (B) Weight, TG, AST, ALT, and CHO in CD, HFD, HFD + AS. (C) Graphical abstract. # P < 0.05 vs. HFD controls, ∗ P < 0.05 vs. CD controls. t -Test, data are shown as mean ± standard deviation.

Article Snippet: To determine whether ER stress and necroptosis could be attenuated via inhibition of FOXO1, we treated AML12 cells with a FOXO1 antagonist (AS1842856, 1 μM, Selleck Chemicals) for 2 h before stimulation with PA (500 μM, Sigma-Aldrich).

Techniques: Inhibition, Staining, Transmission Assay, Electron Microscopy, Standard Deviation

Journal: Scientific Reports

Article Title: Oral Pathobiont Activates Anti-Apoptotic Pathway, Promoting both Immune Suppression and Oncogenic Cell Proliferation

doi: 10.1038/s41598-018-35126-8

Figure Lengend Snippet: DPG3 activated DC-SIGN, AKT, regulated FOXO1 phosphorylation/expression was required for the survival effect of DPG3 in MDDSCs. Immature monocytes isolated from human peripheral blood mononuclear cells (PBMCs) were infected with Pg 381, DPG3 and MFI for 6 hours at 1 MOI. ( A ) Immunoblot analysis of DC-SIGN, p/tAKT, p/tFOXO1 and BIM in MDDSCs and MoDCs with GAPDH as loading control. Immunoblot detected increased expression of DC-SIGN, p/tAKT, p/tFOXO1 and decreased expression of BIM in DPG3- infected MDDSCs compared with uninfected control MoDCs. ( B ) DC-SIGN was blocked with HIV-gp120 (6ug/ml) for 30 minutes before infection. Notably, DC-SIGN expression and internalization was abolished by its antagonist HIV-gp120 and consequently the downstream signaling cascade also blocked as evident by dephosphorylated or decreased expression of pAKT ( B , D ), pFOXO1 ( B , E ) and increased expression of BIM ( B , F ). ( C – F ) Quantification analysis of DC-SIGN ( C ), and relative ratio of p/tAKT ( D ), p/tFOXO1 ( E ) and BIM ( F ) after normalized with the internal control GAPDH. Results are from one representative of three independent experiments. Data were analyzed by performing one-way ANOVA with Tukey’s post hoc using Prism GraphPad. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: The antagonists for AKT1 (MK-2206 2HCl) and FOXO1 (AS1842856) were from Selleckchem and Calbiochem, respectively.

Techniques: Phospho-proteomics, Expressing, Isolation, Infection, Western Blot, Control

Journal: Scientific Reports

Article Title: Oral Pathobiont Activates Anti-Apoptotic Pathway, Promoting both Immune Suppression and Oncogenic Cell Proliferation

doi: 10.1038/s41598-018-35126-8

Figure Lengend Snippet: Oral infection of mice with P. gingivalis induces pAkt/pFoxo1/Foxp3 mediated immunosuppression. The gene expression profile shows differential response of blood ( A ) and splenocytes ( B ) isolated from DPG3-infected mice (n = 3) at 12 hours compared with negative control group (n = 3; 2% CMC without bacteria). Total of 36 mice were sacrificed at 1, 12 and 24 hours (from groups:- 1. CMC; 2. Pg 381; 3. DPG3 and 4. MFI), and blood (refer Fig. ) and splenocytes were collected and genes quantified by TaqMan® array. ( C ) P. gingivalis DPG3-fimbriae mutants induce lower IgG antibody titer after 4-weeks oral infection. Sera from infected mice (n = 12) was collected after 4 weeks of infection with Pg 381, DPG3, MFI and CMC control to determine total IgG titers against respective formalin-killed P. gingivalis strains, measured by ELISA. DPG3 was shown to induce reduced antibody titer compared to Pg 381, whereas MFI induced minimal IgG. ***P < 0.001, ANOVA. ( D ) Immunofluorescence analysis (IFA) of pAkt1 protein expression in gingival interproximal papilla area between M1&M2, pFoxo1 and Foxp3 in spleen tissue from DPG3, Pg 381 infected mice, compared with uninfected CMC group (refer Fig. ). Arrow shows Foxo1 phosphorylation and nuclear exclusion of pFoxo1 (middle panel). For independent channels refer Fig. . Scale bar: D 20 µm. Images are representative of three independent experiments. ( E – G ) Quantification analysis of pAkt, pFoxo1 and Foxp3 expression in DPG3 and Pg 381 compared to CMC infected mice gingiva ( E ) and splenocytes ( F and G ). ( H ) Immunoblot shows increased expression of Foxp3 in Pg 381 and DPG3 infected mice splenocytes, compared with CMC. * P ≤ 0.05, ** P ≤ 0.01.

Article Snippet: The antagonists for AKT1 (MK-2206 2HCl) and FOXO1 (AS1842856) were from Selleckchem and Calbiochem, respectively.

Techniques: Infection, Gene Expression, Isolation, Negative Control, Bacteria, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Expressing, Phospho-proteomics, Western Blot

Journal: Scientific Reports

Article Title: Oral Pathobiont Activates Anti-Apoptotic Pathway, Promoting both Immune Suppression and Oncogenic Cell Proliferation

doi: 10.1038/s41598-018-35126-8

Figure Lengend Snippet: FimA+Mfa1 − Pg strain MFI stimulates proliferation of Human oral squamous carcinoma cells via CXCR4. ( A ) Pg-MFI (Mfa1 − /FimA + ) stimulates proliferation of human head and neck OSC cell lines HN6 and HN12, compared to uninfected cells or those incubated with E. coli -LPS, but not the noncancerous ( B ) oral keratinocytes (hTERT HAK Clone 41). For other noncancerous ARPE and MEF cells refer Fig. . ( C ) Wild-type Pg381 , DPG3 and MFI induces expression of CXCR4, AKT1, FOXO1 and SDF1 in HN6 cells at 24 hours, whereas decreases the expression of BIM and FOXO3, as determined by qPCR. ( D ) Suppression of MFI-stimulated cell growth by inhibiting CXCR4. MFI-stimulated HN6 growth was inhibited by the CXCR4-antagonist AMD3100, but weakly or not by the DC-SIGN inhibitor gp120 after infected with DPG3. ( E ) MFI activates CXCR4-mediated Cellular Signaling in the direction of oncogenesis. Immunoblot analysis was used to detect the following proteins SDF-1/CXCR4, p/tAKT1, and p/tFOXO1 in E. coli -LPS, FadA + , Pg 381, DPG3 and MIF treated HN6 cells compared with uninfected control, but pAKT1, pFOXO1 and SDF-1/CXCR4, were inhibited by their respective inhibitors such as MK-2206 (MK), AS1842856 (AS) and AMD3100. ( F ) DC-SIGN was not detectable by immunoblot in HN6 cells, thus confirming role of CXCR4 in FimA + MFI stimulation of proliferation. The results are presented (as the mean ± SD) from one representative of four independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: The antagonists for AKT1 (MK-2206 2HCl) and FOXO1 (AS1842856) were from Selleckchem and Calbiochem, respectively.

Techniques: Incubation, Expressing, Infection, Western Blot, Control

Journal: Scientific Reports

Article Title: Oral Pathobiont Activates Anti-Apoptotic Pathway, Promoting both Immune Suppression and Oncogenic Cell Proliferation

doi: 10.1038/s41598-018-35126-8

Figure Lengend Snippet: Enriched blood panDCs and gingival tissues of CP patients reveal immunosuppressive state. ( A ) 29 genes associated with transcriptional regulation, inflammation, immunosuppression, cell proliferation, anti-apoptosis and other homeostatic functions were identified from the RNAseq data. Bar graphs show both up- (red) and down- (blue) regulation of genes (≥±2 fold) in ex-vivo isolated and panDC-enriched blood samples from CP patients compared to healthy controls confirmed by TaqManPCR. The overall validation data shows the same trend as in RNAseq analysis. ( B ) H&E of healthy control and CP patient’s gingival tissue. The arrows indicate the infiltrating cells of inflammatory responses; (20X), CT- Connective Tissue, ET- Epithelial Tissue. Results are representative of two experiments (n = 9/group). ( C ) Co-localized expression of pAKT1/pFOXO1, pAKT1/DC-SIGN (receptor for Mfa1) and DC-SIGN/FOXP3 (arrowhead) in gingival connective tissue (marked in b) from CP, compared with healthy control. Images are representative of three independent experiments (Scale bar- 100 µm; pls refer Fig. for independent channel and their quantification measurement of co-localization. ( D ) Immunoblot analysis of p/tAKT1, p/tFOXO1, FOXP3, IDO1, BIM, CXCR4 and SDF1 in gingival tissue of CP compared with healthy control. ( E ) Mfa1 and FimA mRNA in ex-vivo isolated panDCs from CP patients and Healthy control and normalized with the internal control. ( F–H ) Regulation of FOXO1, FOXP3, BIM transcription by FOXO1A or pAKT1. Immunoprecipitation of chromatin from the human FOXO1 ( F ), FOXP3 ( G ) and BIM ( H ) locus in mixed immune cell population from gingival tissues of CP and healthy control with anti-pAKT1 or anti-FOXO1A, followed by qPCR analysis of immunoprecipitants; results are presented relative to input by immunoprecipitation with isotype-matched control antibody. Data are representative of six independent experiments. * P ≤ 0.05, ** P ≤ 0.01.

Article Snippet: The antagonists for AKT1 (MK-2206 2HCl) and FOXO1 (AS1842856) were from Selleckchem and Calbiochem, respectively.

Techniques: Ex Vivo, Isolation, Biomarker Discovery, Control, Expressing, Western Blot, Immunoprecipitation

Journal: Scientific Reports

Article Title: Oral Pathobiont Activates Anti-Apoptotic Pathway, Promoting both Immune Suppression and Oncogenic Cell Proliferation

doi: 10.1038/s41598-018-35126-8

Figure Lengend Snippet: Model of immune homeostasis disruption and dysbiosis by P. gingivalis . DC-SIGN - TLR2/CXCR4 crosstalk activated by ligation via P. gingivalis Mfa1 - FimA, respectively, activates STAT3, resulting in downstream AKT1 activation that phosphorylates and inactivates FOXO1. Direct suppression of apoptotic activity through BIM also occurs. DC-SIGN routes P. gingivalis into non-autophagosomal compartments where they survive. Upon phosphorylation by AKT1 the FOXO proteins are excluded from the nucleus and are sequestered or degraded. FOXO inactivation contributes to extended cell survival as FOXO members activate genes encoding pro-apoptotic molecules including Bcl-2-member BIM. The resultant MDDSCs are highly resistant to apoptosis and IDO-1 competent, traveling to local and distant sites bearing the bacteria and promoting immunosuppression through induced-T regs (FOXP3). In addition to the immunosuppressive route through DC-SIGN by DPG3 (Mfa1), \FimA (MFI) leads the direct oncogenesis route through CXCR4.

Article Snippet: The antagonists for AKT1 (MK-2206 2HCl) and FOXO1 (AS1842856) were from Selleckchem and Calbiochem, respectively.

Techniques: Disruption, Ligation, Activation Assay, Activity Assay, Phospho-proteomics, Bacteria