Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 1. FoxO1 was predominantly expressed in ME cells. (A–D) ME cells were isolated from 8-week-old male Myh11- CreERT2/tdTomato (tdT)fl/fl mice (n = 4). Cre recombination was induced by tamoxifen injection 1 day before the assay. (A) Flow cytometry histograms. The Myh11-positive ME cells (tdT+) represented 10.6% (right) of salivary gland tissue cells without endothelial cells, hematocytes, and erythroid cells (CD31−C45−TER119−; 96.06%, left). (B,C) tdT fluorescence and FoxO1 immunofluorescence (IF) in FACS-sorted cells [crude, Myh(+), Myh(−)] (B). Bar = 20 μm. The cell population expressing FoxO1 and tdT double positive (%) in each FACS-sorted cells [crude, Myh(+), Myh(−)] (C). Threshold intensity was 30. *P < 0.05. (D) Expression of αSMA and FoxO1 in CD31−C45−TER119− cells (crude), tdT-positive [Myh(+)] and -negative [Myh(−)] cells. *P < 0.05. (E) tdT fluorescence and IF of FoxO1 and E-cadherin (E-cad) in submandibular glands (SMG) of Myh11-CreERT2/tdTfl/fl on embryonic day 16 (E16, n = 4) and at 8 weeks (8w, n = 3). The arrow head showed αSMA and FoxO1 double positive cells. Bar = 20 μm. All data were representative of three independent experiments. See also Supplementary Fig. S1.

Article Snippet: Cells (2 × 105) were transfected with FoxO1 Mouse siRNA Oligo Duplex (#SR427332/SR418715, OriGene) and control siRNA duplex (#SR30002, OriGene) at a final concentration of 100 nmol/L using Lipofectamine RNAiMax reagent (Invitrogen, #13778-100) without antibiotics according to the manufacturer’s instructions.

Techniques: Isolation, Injection, Flow Cytometry, Fluorescence, Immunofluorescence, Expressing

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 2. Overexpression of FoxO1 in ME cells. (A) Scatter plot of CD49f (x-axis) and EpCAM (y-axis). The cells were isolated from SMG in TP53 mutant female mice (n = 4) and analyzed by flow cytometry. EpCAMlowCD49fhigh-cells were sorted as ME cells (6.5%). (B) A schematic for integration of PiggyBac transposon vector plasmid. The Tet-On inducible gene expression system was used. FoxO1 expression was induced by doxycycline (Dox). (C) mCherry fluorescence merged with phase contrast in MEPB-FoxO1 cells treated with and without Dox (2 µg/mL) for 48 h. (D) Expression of FoxO1 mRNA in MEPB-FoxO1 cells treated with and without Dox for 24 h. *P < 0.05. n = 3. (E) Immunoblotting for FoxO1, αSMA, Krt14, Krt5, and β-actin in MEPB-FoxO1 cells treated with and without Dox for 72 h. (F) FoxO1 luciferase assay in the presence of FoxO1 inhibitor (Inh.; AS1842856) at the indicated concentrations. pGL4 luciferase reporter vector (upper) was constructed to include three FoxO1-binding elements (daf16:TTGTTTA and mdaf16:TTGCTTA). FoxO1 transcriptional activity was measured. pRL-TK was used as internal control. The Renilla luciferase normalized the firefly luciferase. #P < 0.05 vs. control (Ctrl). *P < 0.05 vs. Dox. n = 5. (G) Expression of αSMA mRNA in ME cells treated with and without FoxO1 inhibitor (Inh.; AS1842856, 1 μM) for 72 h. *P < 0.05. n = 3. (H) Expression of FoxO1 and αSMA mRNA in siRNA-mediated knockdown of FoxO1 (siFoxO1) or control (si Ctrl) in ME cells. *P < 0.05. n = 3. (I) Immunoblotting for NF-κB/p65 and phospho-NF-κB/p65 in MEPB-FoxO1 cells treated with and without Dox at the indicated time-points. The signal intensity of phospho-NF-κB/p65 was normalized to that of NF-κB/p65 (ratio). All data were representative of three independent experiments. See also Supplementary Figs. S2 and S3.

Article Snippet: Cells (2 × 105) were transfected with FoxO1 Mouse siRNA Oligo Duplex (#SR427332/SR418715, OriGene) and control siRNA duplex (#SR30002, OriGene) at a final concentration of 100 nmol/L using Lipofectamine RNAiMax reagent (Invitrogen, #13778-100) without antibiotics according to the manufacturer’s instructions.

Techniques: Over Expression, Isolation, Mutagenesis, Flow Cytometry, Plasmid Preparation, Gene Expression, Expressing, Fluorescence, Western Blot, Luciferase, Construct, Binding Assay, Activity Assay, Control, Knockdown

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 3. Transcriptome profiling of FoxO1-expressing ME cells. The RNA samples were prepared from MEPB-FoxO1 cells treated with and without Dox for 72 h. (A) Heatmap of differentially expressed genes (control vs. FoxO1). (B) The number of up- and down-regulated genes based on fold change of comparison pair (FoxO1/ control ≥ 2, P < 0.05). (C–F) Enrichment of Gene Ontology terms for biological processes associated with up- (C) and down- (D) regulated genes. (E,F) Data from the gene set enriched analysis (GSEA). The top gene lists of normalized enrichment score (NES) are shown in (E). *P < 0.001. Enrichment plot of cyclin A B1 B2 associated events during G2 M transition are shown in (F). See also Supplementary Tables S2–S5.

Article Snippet: Cells (2 × 105) were transfected with FoxO1 Mouse siRNA Oligo Duplex (#SR427332/SR418715, OriGene) and control siRNA duplex (#SR30002, OriGene) at a final concentration of 100 nmol/L using Lipofectamine RNAiMax reagent (Invitrogen, #13778-100) without antibiotics according to the manufacturer’s instructions.

Techniques: Expressing, Control, Comparison

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 4. FoxO1 suppressed ME cell proliferation via cell cycle arrest. (A) Viability of MEPB-FoxO1 cells treated with and without Dox (2 µg/mL) at the indicated time-points. (B,C) Cell proliferation rates were measured by BrdU incorporation assay. BrdU positive/DAPI (%, left) with and without Dox (2 µg/mL) for 24 h (B) or with and without transfection of siRNA for FoxO1 for 48 h (C). Immunofluorescent images were showed on the right (BrdU; green, DAPI; blue). (D–F) Expression of p27(KIP1) in MEPB-FoxO1 cells. Cells were treated with and without Dox (2 µg/mL) (D), pretreated with and without FoxO1 inhibitor (Inh.; AS1842856, 1 μM) (E) and transfected with siRNA for FoxO1 (F) in the presence of Dox (2 µg/mL) for 48h. The expression data of p21(CIP/WAF1) were shown in Fig. S4. (G) Chromatin immunoprecipitation-quantitative real-time PCR (ChIP-qPCR) analysis of the DNA binding activity of FoxO1 in ME cells. DNA sample was prepared from MEPB-FoxO1 cells treated with Dox (2 µg/mL) for 72 h. The associated DNA at the promoter regions of p21CIP/WAF1 (− 1722 to − 1712) and p27KIP1 (− 1036 to − 1026), after incubation with FoxO1 antibody-conjugated protein G beads, were immunoprecipitated and analyzed by qPCR. *P < 0.05. n = 3. All data were representative of three independent experiments. (H) A schematic for FoxO1-induced cell growth inhibition. See also Supplementary Fig. S4.

Article Snippet: Cells (2 × 105) were transfected with FoxO1 Mouse siRNA Oligo Duplex (#SR427332/SR418715, OriGene) and control siRNA duplex (#SR30002, OriGene) at a final concentration of 100 nmol/L using Lipofectamine RNAiMax reagent (Invitrogen, #13778-100) without antibiotics according to the manufacturer’s instructions.

Techniques: BrdU Incorporation Assay, Transfection, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Binding Assay, Activity Assay, Incubation, Immunoprecipitation, Inhibition

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 5. FoxO1 induced Eda/Eda2r expression in ME cells through NF-κB activation. (A,B) Gene expression (A) and immunoblotting (B) of Eda and Eda2r in MEPB-FoxO1 cells treated with and without Dox (2 µg/mL) for 24 h. (C) Expression of Eda and Eda2r in Dox-treated (2 µg/mL, 72 h) MEPB-FoxO1 cells with and without FoxO1 inhibitor (Inh.; AS1842856, 10 μM) pretreatment for 24 h. (D) Expression of Eda and Eda2r in Dox-treated (2 µg/mL, 72 h) MEPB-FoxO1 cells with and without NF-κB inhibitor (MG132, 20 μM) pretreatment for 6 h. *P < 0.05. n = 3. All data were representative of three independent experiments. See also Supplementary Fig. S5.

Article Snippet: Cells (2 × 105) were transfected with FoxO1 Mouse siRNA Oligo Duplex (#SR427332/SR418715, OriGene) and control siRNA duplex (#SR30002, OriGene) at a final concentration of 100 nmol/L using Lipofectamine RNAiMax reagent (Invitrogen, #13778-100) without antibiotics according to the manufacturer’s instructions.

Techniques: Expressing, Activation Assay, Gene Expression, Western Blot

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 6. Inhibition of FoxO1 inhibited development of the primitive epithelium of SMG ex vivo. Epithelia of SMG rudiments on E14.5 (n = 4), were mounted in Matrigel drops and cultured in the presence of FGF1 and FGF7 with and without FoxO1 inhibitor (Inh.; AS1842856, 10 μM) for 3 days. (A) Phase contrast images. Bar = 500 μm. (B) The image of immnofluorescent of αSMA, Eda, Eda2r, and phospho-NF-κB after 3 days of culture. Nuclei were stained with DAPI. Bar = 20 μm. (C) Expression of Eda and Eda2r after 3 days of culture. *P < 0.05. All data were representative of three independent experiments. See also Supplementary Figs. S6 and S7.

Article Snippet: Cells (2 × 105) were transfected with FoxO1 Mouse siRNA Oligo Duplex (#SR427332/SR418715, OriGene) and control siRNA duplex (#SR30002, OriGene) at a final concentration of 100 nmol/L using Lipofectamine RNAiMax reagent (Invitrogen, #13778-100) without antibiotics according to the manufacturer’s instructions.

Techniques: Inhibition, Ex Vivo, Cell Culture, Staining, Expressing

Journal: BioMed Research International

Article Title: Characterization of Zebrafish Pax1b and Pax9 in Fin Bud Development

doi: 10.1155/2014/309385

Figure Lengend Snippet: The interaction between PAX1 and FOXO1. (a) FOXO1 is a new PAX1-interacting protein. HEK293FT cells were transfected with Myc-tagged FOXO1 and Flag-tagged PAX1 cloned from human cDNA. Cells were harvested for immunoprecipitation with anti-Flag affinity resin and immunoblotted with the indicated antibodies. (b) The interaction of Pax1 and FOXO1 was conserved. Myc-tagged FOXO1 was immunoprecipitated by Flag-tagged Pax1b or mouse Pax1. (c) PAX9 did not interact with FOXO1 in HEK293FT cells. zPax1b: zebrafish Pax1b; mPax1: mouse Pax1. TCL: total cell lysate. IP: immunoprecipitation.

Article Snippet: Antibodies used are the following: rabbit polyclonal antibody to Pax1 (83312 from Abcam), Uncx4.1 (ARP47548 from Aviva Systems Biology), Noggin3 (16054 from Abcam), and FOXO1 (sc-11350 from Santa Cruz) and mouse monoclonal antibody to Flag (F1804 from Sigma) or Myc (M4439 from Sigma).

Techniques: Transfection, Clone Assay, Immunoprecipitation

Journal: BioMed Research International

Article Title: Characterization of Zebrafish Pax1b and Pax9 in Fin Bud Development

doi: 10.1155/2014/309385

Figure Lengend Snippet: The interaction between PAX1 and FOXO1 is enhanced under hypoxia stress. (a) Colocalization of PAX1 and FOXO1 in HeLa cells. Myc-FOXO1 is distributed in the cytoplasm and nucleus while Flag-PAX1 is located in the nucleus only. The colocalization of PAX1 and FOXO1 was increased after treatment with 20 μ M CoCl 2 for 14 h. Myc-FOXO1 was cotransfected with Flag-PAX1 into HeLa cells. 36 h after transfection, cells were subjected to immunostaining using anti-Myc antibody, anti-Flag antibody, and DAPI and observed by microscopy. ((b)-(c)) 2 μ g Flag-tagged PAX1 and/or 0.5 μ g Myc-tagged FOXO1 were transfected into HEK293FT cells, respectively. Semiendogenous Co-IP revealed that the interaction of PAX1 and FOXO1 was strengthened when stimulated with CoCl 2 and the enhancement occurred in a dose-dependent manner.

Article Snippet: Antibodies used are the following: rabbit polyclonal antibody to Pax1 (83312 from Abcam), Uncx4.1 (ARP47548 from Aviva Systems Biology), Noggin3 (16054 from Abcam), and FOXO1 (sc-11350 from Santa Cruz) and mouse monoclonal antibody to Flag (F1804 from Sigma) or Myc (M4439 from Sigma).

Techniques: Transfection, Immunostaining, Microscopy, Co-Immunoprecipitation Assay

Journal: Cell reports

Article Title: Hepatic palmitoyl-proteomes and acyl-protein thioesterase protein proximity networks link lipid modification and mitochondria

doi: 10.1016/j.celrep.2023.113389

Figure Lengend Snippet:

Article Snippet: FoxO1 (D7C1H) , Cell Signaling Technology , Cat# 14952; RRID:AB_2722487.

Techniques: Virus, Recombinant, Staining, Bicinchoninic Acid Protein Assay, Reverse Transcription, Glucose Assay, Colorimetric Assay, Mass Spectrometry, Plasmid Preparation, Software, Transfection, Blocking Assay, Protease Inhibitor