Journal: Cell death discovery

Article Title: The inhibition of FKBP5 protects β-cell survival under inflammation stress via AKT/FOXO1 signaling.

doi: 10.1038/s41420-023-01506-x

Figure Lengend Snippet: Fig. 5 FOXO1 mediated the protective effect of FKBP5 inhibition on β cells. A, B Western blot analysis of Pdx1, p-Foxo1Ser256, Foxo1, p-Akt Ser473 and Akt in NIT-1 cells transfected with siFoxo1 or control siRNAs, and then treated with SAFit2 (A). Relative proteins expression levels were quantified by Image J (B). C, D Annexin-V/7-AAD staining and flow cytometry analysis in NIT-1 cells transfected with siFoxo1 or control siRNAs and then treated with cytokines, or cytokines + SAFit2 (C). Quantification of PE+7AAD+/PE+7AAD- cells (D). E, F Western blot analysis of FOXO1, BCL2, BAX in primary human islets transfected with siFOXO1 or control siRNAs and then treated with cytokines, or cytokines + SAFit2 (E). Relative proteins expression levels were quantified by Image J (F). Experiments were repeated for 3 times. Student’s t-test. Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The blotted membranes were blocked with 5% skim milk for 60min at room temperature and incubated with primary antibodies FKBP51 (1:1000, ABclonal, A3863, Woburn, MA, USA), p-FOXO1 (1:1000, NB100-81927, Novus Biological, Cambridge, UK), FOXO1 (1:1000, CST-2880, Cell signaling technology, Danvers, MA, USA), p-AKT(1:1000, CST4060S, Cell signaling technology, Danvers, MA, USA), AKT(1:1000, CST9272S, Cell signaling technology, Danvers, MA, USA), PDX1(1:1000, CST9679S, Cell signaling technology, Danvers, MA, USA), Sqstm1/p62 (1:1000, ab56416, Abcam, Cambridge, MA, USA), LC3I/II (1:1000, CST-12741S, Cell signaling technology, Danvers, MA, USA), BAX(1:1000, ab32503, Abcam, Cambridge, MA, USA), BCL2(1:1000, ab59348, Cambridge, MA, USA), β-actin (1:1000, CST-3700, Cell signaling technology, Danvers, MA, USA), Histone cluster 1, H3a (1:1000, 17168-1-AP, Proteintech, Rosemont, IL 60018, USA) at 4°C overnight.

Techniques: Inhibition, Western Blot, Transfection, Control, Expressing, Staining, Cytometry

Journal: American Journal of Cancer Research

Article Title: PF-429242 exhibits anticancer activity in hepatocellular carcinoma cells via FOXO1-dependent autophagic cell death and IGFBP1-dependent anti-survival signaling

doi:

Figure Lengend Snippet: Role of FOXO1 in PF-429242-induced autophagic cell death in hepatocellular carcinoma (HCC) cells. (A) PLC5 and HepG2 cells were treated with 20 μM PF-429242 with or without indicated doses of AS1842856 (AS) for 24 h. Protein expressions were determined by Western blotting. (B) PLC5 and HepG2 cells were treated with 20 μM PF-429242 with or without 120 μM carbenoxolone (CBX) for 24 h. Protein expressions were determined by Western blotting. (C, D) PLC5 and HepG2 cells were transfected with siRNAs specific for FOXO1 in (C) or IGFBP1 (BP1) in (D) for 24 h, and then treated with 20 μM PF-429242 for 24 h. Protein expressions were determined by Western blotting. (E) si-FOXO1-transfected PLC5 and HepG2 cells were seeded in a 96-well plate and then treated with the indicated doses of PF-429242 for 72 h. Cell viability was determined by Alamar Blue staining. Error bars are the mean ± SD (n = 5). *** indicates a significant difference (P < 0.01) between si-FOXO1- and si-Control-transfected cells. (F) PLC5 and HepG2 cells were treated with 10, 20, and 40 μM PF-429242 for 24 h. Protein expressions were determined by Western blotting. Ratios between indicated protein expressions are shown.

Article Snippet: Acetyl-Lys262/Lys265/Lys274-FOXO1 (#A17406) and Ki67 (#A11390) antibodies were purchased from ABclonal Technology (Woburn, MA, USA).

Techniques: Western Blot, Transfection, Staining, Control

Journal: Cells

Article Title: FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells

doi: 10.3390/cells11071122

Figure Lengend Snippet: FOXO1 increases in alveolar epithelial cells as a function of time in culture, and knockdown increases expression of the AT2 cell-specific marker SFTPC. Representative Western blot (WB) ( A ) and quantitation ( B – D ) show that the expression of FOXO1, but not FOXO3 and FOXO4, increases as rat AT2 cells (D0) differentiate into AT1-like cells at day eight (D8) in primary culture. EIF-4E, EIF-2α, and β -ACTIN were the loading controls. n = 4 for each group. One-way ANOVA, asterisk indicates p < 0.05 compared to D0. Representative WB ( E ) and quantitation ( F , G ) show that knockdown of FOXO1 increased pro-SFTPC expression in primary AEC six days following transduction with FoxO1 shRNA (+) or non-silencing shRNA (−) on Day two. EIF-2α was used as the loading control. The data were normalized to non-silencing shRNA. n = 4 for each group. Unpaired two-tailed t -test, asterisk indicates p < 0.05.

Article Snippet: The antibodies that were used for Western blotting analysis and immunostaining were as follows: FOXO1 (#2880 or #2880S, Cell Signaling, Danvers, MA, USA, 1:500); FOXO3 (#9467, Cell Signaling); FOXO4 (#9472, Cell Signaling, 1:500); p-FOXO1 (#ANTY011115, Antagene, Sunnyvale, CA, USA, 1:500); pro-SP-C (#AB3786, Millipore, Billerica, MA, USA, 1:500); AKT (#9272, Cell Signaling, 1:1000); p-AKT (ser473) (#3787, Cell Signaling, 1:1000); Lamin A/C (#SC20681, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000); NKX2.1 (#MS-699-P0, Thermo Scientific, Pittsburgh, PA, USA, 1:500); GAPDH (#AM4300, Applied Biosystems, Austin, TX, USA, 1:1000), EIF-4E (#610270, BD Biosciences, San Jose, CA, USA, 1:200); eIF2 α (#11386, Santa Cruz Biotechnology, 1:500); β -ACTIN (#ab6276, Abcam, Cambridge, MA, USA, 1:2000); and P180 lamellar body protein (#MMS-645R, Covance, Princeton, NJ, USA, 1:2000).

Techniques: Knockdown, Expressing, Marker, Western Blot, Quantitation Assay, Transduction, shRNA, Control, Two Tailed Test

Journal: Cells

Article Title: FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells

doi: 10.3390/cells11071122

Figure Lengend Snippet: FOXO1 attenuates NKX2.1 activation of SFTPC and SFTPB . ( A ) MLE-15 cells were co-transfected with a 3.7 kb SFTPC reporter construct (3.7- SFTPC -Luc) and NKX2.1 or FOXO1 expression constructs alone or in combination. Dual luciferase assays 48 h following transduction showed that FOXO1 inhibited NKX2.1 activation of the SFTPC reporter. Firefly luciferase activity was normalized to Renilla luciferase. n = 4 for each group. One-way ANOVA, asterisk indicates p < 0.05. ( B ) MLE-15 cells were co-transfected with 3.7- SFTPC -Luc, pRC/CMV/ NKX2.1 and increasing concentrations of FOXO1 expression construct. Dual luciferase assays 48 h post-transduction showed that FOXO1 inhibited NKX2.1-mediated induction of the SFTPC reporter in a dose-dependent manner. The data are shown as normalized to the absence of FOXO1. n = 4 for each group. One-way ANOVA, asterisk indicates p < 0.05 compared to absence of FOXO1 (empty vector). ( C ) MLE-15 cells were co-transfected with a 1.4 kb SFTPB reporter construct (1.4- SFTPB -Luc), NKX2.1 and FOXO1 expression constructs alone, or in combination. Dual luciferase assays 48 h post-transduction showed that FOXO1 inhibited NKX2.1 activation of the SFTPB reporter. Firefly luciferase activity was normalized to Renilla luciferase. The data are shown as normalized to the absence of FOXO1 and NKX2.1. n = 3 for each group. One-way ANOVA, asterisk indicates p < 0.05. ( D ) Schematic showing the putative FOXO1 and NKX2.1 binding sites on human SFTPC promoter. ( E ) MLE-15 cells were co-transfected with a 3.7 kb SFTPC reporter construct (3.7- SFTPC C-Luc), NKX2.1 and FOXO1 wild-type (WT), or FOXO1H215R (DNA binding mutant) expression constructs alone or in combination. Dual luciferase assays 48 h post-transduction showed that FOXO1-H215R maintained the ability to repress NKX2.1 activation of the SFTPC reporter. Firefly luciferase activity was normalized to Renilla luciferase. The data are shown as normalized to the absence of FOXO1 and NKX2.1. n = 3 for each group. One-way ANOVA, asterisk indicates p < 0.05. ( F ) MLE-15 cells were co-transfected with a 318 bp Sftpc reporter p318mu sftpc -Luc which lacks a FOXO1 binding site and a NKX2.1 expression construct, together with increasing amounts of FOXO1 expression construct. Dual luciferase assays 48 h post-transduction showed that FOXO1 inhibited NKX2.1-induced activation of the 318 bp Sftpc reporter in a dose-dependent manner. The data are shown as normalized to the absence of FOXO1 (empty vector). n = 3 for each group. One-way ANOVA, asterisk indicates p < 0.05 compared to absence of FOXO1.

Article Snippet: The antibodies that were used for Western blotting analysis and immunostaining were as follows: FOXO1 (#2880 or #2880S, Cell Signaling, Danvers, MA, USA, 1:500); FOXO3 (#9467, Cell Signaling); FOXO4 (#9472, Cell Signaling, 1:500); p-FOXO1 (#ANTY011115, Antagene, Sunnyvale, CA, USA, 1:500); pro-SP-C (#AB3786, Millipore, Billerica, MA, USA, 1:500); AKT (#9272, Cell Signaling, 1:1000); p-AKT (ser473) (#3787, Cell Signaling, 1:1000); Lamin A/C (#SC20681, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000); NKX2.1 (#MS-699-P0, Thermo Scientific, Pittsburgh, PA, USA, 1:500); GAPDH (#AM4300, Applied Biosystems, Austin, TX, USA, 1:1000), EIF-4E (#610270, BD Biosciences, San Jose, CA, USA, 1:200); eIF2 α (#11386, Santa Cruz Biotechnology, 1:500); β -ACTIN (#ab6276, Abcam, Cambridge, MA, USA, 1:2000); and P180 lamellar body protein (#MMS-645R, Covance, Princeton, NJ, USA, 1:2000).

Techniques: Activation Assay, Transfection, Construct, Expressing, Luciferase, Transduction, Activity Assay, Plasmid Preparation, Binding Assay, Mutagenesis

Journal: Cells

Article Title: FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells

doi: 10.3390/cells11071122

Figure Lengend Snippet: FOXO1 physically interacts with the homeodomain of NKX2.1. Co-immunoprecipitation of nuclear extracts that were harvested from primary AT2 cells on day three in culture with anti-NKX2.1 ( A ) or anti-FOXO1 ( B ) Abs shows the association of endogenous FOXO1 and NKX2.1 ( n = 4). IgG was used as a negative control. ( C ) Schematic of GST-tagged FOXO1 fusion proteins (left panel) and GST pull-down assay (right panel). In vitro translated NKX2.1 was incubated with GST-FOXO1 fusion proteins (GST-FOXO1 full-length (FL), GST-FOXO1-N, GST-FOXO1-FK, GST-FOXO1-N + FK, GST-FOXO1-M, GST-FOXO1-C) coupled to glutathione sepharose. Bound NKX2.1 was visualized by Western blotting using an anti-NKX2.1 antibody. The FOXO1 FL and FOXO1 FK domains interact with NKX2.1 (right panel). n = 3. ( D ) Schematic of GST-tagged NKX2.1 fusion proteins (left panel) and GST pull-down assay (right panel). In vitro translated FOXO1 was incubated with GST- NKX2.1 fusion proteins (GST-NKX2.1 FL, GST-NKX2.1-N, GST-NKX2.1-HD, and GST-NKX2.1-C) that were coupled to glutathione sepharose. The bound FOXO1 was visualized by Western blotting using an anti-FOXO1 Ab. FOXO1 interacts with the NKX2.1 homeodomain (right panel). n = 3. ( E , F ) EMSA was performed with nuclear extracts from MLE-15 cells and biotin-labeled oligonucleotides encompassing the NKX2.1 DNA-binding site (−186 to −163 bp) of the SFTPC promoter. The arrow points to the inhibition of the NKX2.1 protein/DNA complexes (lane 2) with increasing amounts of GST-FOXO1 fusion protein (lane 8–10) ( E ) or GST-FOXO1 FK domain fusion protein (lane 8–10) ( F ) but not by GST alone (lane 4–6). Lane 1 is probe only. GST (lane 4) and GST-FOXO1 or GST-FOXO1 FK (lane 7) proteins do not form a complex with the oligonucleotide probe. n = 3. ( G ) Publicly available ChIPseq analysis of NKX2-1 binding surrounding the Sftpc and Sftpb loci in Sftpc + AT2 and Wnt3A + AT1 cells. Peaks were called using MACS2.0 as outlined by Little DR et al. . 0–150 indicates number of Chip-Seq reads overlapping at a given base.

Article Snippet: The antibodies that were used for Western blotting analysis and immunostaining were as follows: FOXO1 (#2880 or #2880S, Cell Signaling, Danvers, MA, USA, 1:500); FOXO3 (#9467, Cell Signaling); FOXO4 (#9472, Cell Signaling, 1:500); p-FOXO1 (#ANTY011115, Antagene, Sunnyvale, CA, USA, 1:500); pro-SP-C (#AB3786, Millipore, Billerica, MA, USA, 1:500); AKT (#9272, Cell Signaling, 1:1000); p-AKT (ser473) (#3787, Cell Signaling, 1:1000); Lamin A/C (#SC20681, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000); NKX2.1 (#MS-699-P0, Thermo Scientific, Pittsburgh, PA, USA, 1:500); GAPDH (#AM4300, Applied Biosystems, Austin, TX, USA, 1:1000), EIF-4E (#610270, BD Biosciences, San Jose, CA, USA, 1:200); eIF2 α (#11386, Santa Cruz Biotechnology, 1:500); β -ACTIN (#ab6276, Abcam, Cambridge, MA, USA, 1:2000); and P180 lamellar body protein (#MMS-645R, Covance, Princeton, NJ, USA, 1:2000).

Techniques: Immunoprecipitation, Negative Control, Pull Down Assay, In Vitro, Incubation, Western Blot, Labeling, Binding Assay, Inhibition, ChIP-sequencing

Journal: Cells

Article Title: FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells

doi: 10.3390/cells11071122

Figure Lengend Snippet: FOXO1 repressor function is negatively regulated by PI-3K-dependent phosphorylation. ( A ) MLE-15 cells were co-transfected with the 3.7-SFTPC-Luc reporter construct, NKX2.1 expression construct and FOXO1 or FOXO1-AAA, a dephosphorylated constitutively active form of FOXO1. Dual luciferase assays were performed 48 h after transfection. Firefly luciferase activity was normalized to Renilla luciferase. The data are shown as normalized to the absence of FOXO1 and NKX2.1. n = 3 for each group. One-way ANOVA, asterisk indicates p < 0.05. ( B ) Western blotting for p -AKT (ser473) and p-FOXO1 in MLE-15 cells shows that Ly294002 treatment (7 h) decreases p-AKT and p-FOXO1. n = 2. ( C ) MLE-15 cells were co-transfected with a 3.7-SFTPC-Luc reporter construct and NKX2.1 expression construct for 24 h, followed by treatment with Ly294002 (1 µM) for an additional 24 h. Dual luciferase assay showed that Ly294002 inhibited SFTPC reporter activity. The data are shown as normalized to the absence of both NKX2 and Ly294002. n = 3 for each group. One-way ANOVA, asterisk indicates p < 0.05. ( D ) Co-IP was performed with cell lysates that were harvested from MLE-15 cells that were cultured in the presence or absence of PI-3K inhibitor Ly294002 (6 μM) for 48 h. Increased association of NKX2.1 with FOXO1 was detected in the Ly294002-treated samples. n = 3.

Article Snippet: The antibodies that were used for Western blotting analysis and immunostaining were as follows: FOXO1 (#2880 or #2880S, Cell Signaling, Danvers, MA, USA, 1:500); FOXO3 (#9467, Cell Signaling); FOXO4 (#9472, Cell Signaling, 1:500); p-FOXO1 (#ANTY011115, Antagene, Sunnyvale, CA, USA, 1:500); pro-SP-C (#AB3786, Millipore, Billerica, MA, USA, 1:500); AKT (#9272, Cell Signaling, 1:1000); p-AKT (ser473) (#3787, Cell Signaling, 1:1000); Lamin A/C (#SC20681, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000); NKX2.1 (#MS-699-P0, Thermo Scientific, Pittsburgh, PA, USA, 1:500); GAPDH (#AM4300, Applied Biosystems, Austin, TX, USA, 1:1000), EIF-4E (#610270, BD Biosciences, San Jose, CA, USA, 1:200); eIF2 α (#11386, Santa Cruz Biotechnology, 1:500); β -ACTIN (#ab6276, Abcam, Cambridge, MA, USA, 1:2000); and P180 lamellar body protein (#MMS-645R, Covance, Princeton, NJ, USA, 1:2000).

Techniques: Phospho-proteomics, Transfection, Construct, Expressing, Luciferase, Activity Assay, Western Blot, Co-Immunoprecipitation Assay, Cell Culture

Journal: Cells

Article Title: FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells

doi: 10.3390/cells11071122

Figure Lengend Snippet: FOXO1 phosphorylation is regulated as a function of AEC differentiation by KGF in an AKT-dependent manner. ( A ) Double labeling immunofluorescence was performed on freshly isolated rat lung epithelial cells with p-FOXO1 (FITC, green) and either lamellar membrane protein P180 (LBM-180, an AT2 cell-specific marker) (red), (i) or VIIIB2 (an AT1 cell-specific marker) (red) (ii). The nuclei were stained with DAPI (blue). Species-specific IgGs were used as controls (iii and iv). p-FOXO1 co-localized with LBM-180, but not VIIIB2. n = 4. Representative Western blot ( B ) and quantitation of p-FOXO1 ( C ) and pro-SFTPC ( D ) in AT2 cells that were grown in primary culture from day zero (D0) to day eight (D8) and that were treated with KGF (10 ng/mL) from either D0–8 or D4–8 shows increased phosphorylation of FOXO1 and preserved expression of pro-SFTPC. β -ACTIN was the loading control. The data are shown as normalized to the absence of KGF. n = 3 for each group. One-way ANOVA, asterisk indicates p < 0.05. Representative Western blot ( E ) and quantitation of p-FOXO1 ( F ) and pro-SFTPC ( G ) in primary rat AEC that were treated with KGF ± Ly294002 from D4–D8. Ly294002 reduced p-FOXO1 and pro-SFTPC. β -ACTIN was a loading control. The data are shown as normalized to the absence of KGF and Ly294002. n = 3 for each group. One-way ANOVA, asterisk indicates p < 0.05. ( H ) Representative immunofluorescence analysis of p-FOXO1 (FITC, green) in AEC that were treated with KGF ± LY294002 from D4–8 shows that Ly294002 reduces p-FOXO1 expression. The untreated AEC (DMSO) were used as controls. The nuclei were counterstained with propidium iodide (PI). n = 4. Representative Western blot ( I ) and quantitation ( J ) of p-AKT (Ser473) in primary AT2 cells that were treated with KGF ± Ly294002. Ly294002 inhibits KGF-induced p-AKT expression. Total AKT was a loading control. n = 4 for each group. One-way ANOVA, asterisk indicates p < 0.05.

Article Snippet: The antibodies that were used for Western blotting analysis and immunostaining were as follows: FOXO1 (#2880 or #2880S, Cell Signaling, Danvers, MA, USA, 1:500); FOXO3 (#9467, Cell Signaling); FOXO4 (#9472, Cell Signaling, 1:500); p-FOXO1 (#ANTY011115, Antagene, Sunnyvale, CA, USA, 1:500); pro-SP-C (#AB3786, Millipore, Billerica, MA, USA, 1:500); AKT (#9272, Cell Signaling, 1:1000); p-AKT (ser473) (#3787, Cell Signaling, 1:1000); Lamin A/C (#SC20681, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000); NKX2.1 (#MS-699-P0, Thermo Scientific, Pittsburgh, PA, USA, 1:500); GAPDH (#AM4300, Applied Biosystems, Austin, TX, USA, 1:1000), EIF-4E (#610270, BD Biosciences, San Jose, CA, USA, 1:200); eIF2 α (#11386, Santa Cruz Biotechnology, 1:500); β -ACTIN (#ab6276, Abcam, Cambridge, MA, USA, 1:2000); and P180 lamellar body protein (#MMS-645R, Covance, Princeton, NJ, USA, 1:2000).

Techniques: Phospho-proteomics, Labeling, Immunofluorescence, Isolation, Membrane, Marker, Staining, Western Blot, Quantitation Assay, Expressing, Control

Journal: Cells

Article Title: FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells

doi: 10.3390/cells11071122

Figure Lengend Snippet: Model of mechanisms underlying FOXO1 regulation of lung-specific NKX2.1-activated genes via PI-3K/AKT-dependent phosphorylation. In AT2 and AT2-like cells in the presence of KGF, the PI-3K/AKT pathway is activated, leading to downstream phosphorylation of FOXO1 and subsequent cytoplasmic extrusion and degradation. The absence of nuclear FOXO1 allows active NKX2.1 to remain at the SFTPC and SFTPB promoters, increasing the expression of these AT2 cell-specific target genes. During the transition from AT2 to AT1-like cells (AT1.5 intermediate cells), or in the absence of KGF, the PI-3K/AKT pathway becomes inactivated. This in turn prevents the phosphorylation of FOXO1, allowing it to remain in the nucleus and interact with and pull NKX2.1 from its target promoters. This process is completed in differentiated AT1-like cells, where NKX2.1 removal from target genes by FOXO1 leads to the inhibition of gene expression. The figure was created with BioRender (BioRender Academic License https://biorender.com/ (accessed on 14 March 2022)).

Article Snippet: The antibodies that were used for Western blotting analysis and immunostaining were as follows: FOXO1 (#2880 or #2880S, Cell Signaling, Danvers, MA, USA, 1:500); FOXO3 (#9467, Cell Signaling); FOXO4 (#9472, Cell Signaling, 1:500); p-FOXO1 (#ANTY011115, Antagene, Sunnyvale, CA, USA, 1:500); pro-SP-C (#AB3786, Millipore, Billerica, MA, USA, 1:500); AKT (#9272, Cell Signaling, 1:1000); p-AKT (ser473) (#3787, Cell Signaling, 1:1000); Lamin A/C (#SC20681, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000); NKX2.1 (#MS-699-P0, Thermo Scientific, Pittsburgh, PA, USA, 1:500); GAPDH (#AM4300, Applied Biosystems, Austin, TX, USA, 1:1000), EIF-4E (#610270, BD Biosciences, San Jose, CA, USA, 1:200); eIF2 α (#11386, Santa Cruz Biotechnology, 1:500); β -ACTIN (#ab6276, Abcam, Cambridge, MA, USA, 1:2000); and P180 lamellar body protein (#MMS-645R, Covance, Princeton, NJ, USA, 1:2000).

Techniques: Phospho-proteomics, Expressing, Inhibition, Gene Expression

Journal: Frontiers in Pharmacology

Article Title: Akkermansia muciniphila ameliorates olanzapine-induced metabolic dysfunction-associated steatotic liver disease via PGRMC1/SIRT1/FOXO1 signaling pathway

doi: 10.3389/fphar.2025.1550015

Figure Lengend Snippet: Effects of AKK and OLZ on the hepatic PGRMC1/SIRT1/FOXO1 signaling pathway. Relative mRNA expression of PGRMC1 (a) , SIRT1 (b) , FOXO1 (c) . Relative protein expression of PGRMC1 (d) , SIRT1 (e) , FOXO1 (f) , p-FOXO1 (g) . (h) The ratio of p-FOXO1/FOXO1. Differences were considered statistically significant when p < 0.05. * p < 0.05, ** p < 0.01, *** p < 0.001, HFD vs. LFD. # p < 0.05, ## p < 0.01, ### p < 0.001, OLZ vs. HFD. $ p < 0.05, $$ p < 0.01, $$$ p < 0.001, OLZ-AKK vs. OLZ-PBS.

Article Snippet: The antibodies used included PGRMC1 (1:5000; Proteintech; 12990-1-AP), SIRT1 (1:2000; HUABIO; ER130811), FOXO1 (1:1000; Proteintech; 18592-1-AP), p-FOXO1 (s256) (1:500; ProMab Biotechnologies Inc.; P40793), and β-actin (1:10000; Proteintech; 66009-1-Ig).

Techniques: Expressing

Journal: Foods

Article Title: “Biqi” Bayberry Extract Promotes Skeletal Muscle Fiber Type Remodeling by Increasing Fast Myofiber Formation via the Akt/FoxO1 Pathway in Mice

doi: 10.3390/foods12132471

Figure Lengend Snippet: BBE activates the Akt/FoxO1 pathway in skeletal muscles of mice. ( A ) The binding modes of myricitrin, hyperoside, myricetin, quercitrin, cyanidin-3-O-glucoside, and quercetin in the Akt protein. ( B , C ) Protein expression and quantification of p-Akt/Akt and p-FoxO1/FoxO1 in the gastrocnemius muscles of each group mice ( n = 4). ( D , E ) Protein expression and quantification of p-Akt/Akt and p-FoxO1/FoxO1 in soleus muscles of each group mice ( n = 4). Results are presented as mean ± SD, (*) p < 0.05, (**) p < 0.01.

Article Snippet: The membranes were blocked with 10% fat-free milk for 2 h and then placed in primary antibodies specific for MyHC-I (Proteintech, Wuhan, China, 22280-1-AP), MyHC-IIA (Abcam, Cambridge, UK, ab124937), MyHC-IIB (Proteintech, Wuhan, China, 20140-1-AP), Akt (Cell Signaling Technology, 4685S), P-Akt (ser473) (Proteintech, Wuhan, China, 66444-1-Ig), FoxO1 (Proteintech, Wuhan, China, 18592-1-AP), P-FoxO1 (ser256) (Signalway Antibody, Greenbelt, MD, USA, 12198), and GAPDH (Proteintech, Wuhan, China, 60004-1-Ig) overnight at 4 °C and finally incubated in secondary antibodies (Proteintech, Wuhan, China, SA00001-1 or SA00001-2) for 1 h at room temperature.

Techniques: Muscles, Binding Assay, Expressing