Journal: Metabolism: clinical and experimental

Article Title: BMI-Related Progression of Atypical PKC-dependent Aberrations in Insulin Signaling Through IRS-1, Akt, FoxO1 and PGC-1α in Livers of Obese and Type 2 Diabetic Humans

doi: 10.1016/j.metabol.2015.08.011

Figure Lengend Snippet: Alterations in enzyme activity of aPKC and Akt (panel a), phosphorylation of aPKC, Akt and FoxO1 (panel b), association of aPKC and Akt with immunoprecipitable WD40/ProF (panel c) and triglyceride levels (panel d) in livers of lean, obese and T2D humans. Values are mean ± SEM of (N) patients. Asterisks indicate: *, P<0.05; **, P<0.01; and ***, P<0.001 (ANOVA). Representative blots are shown in panels b and c (upper bands, phosphoproteins; lower bands, total proteins).

Article Snippet: Western analyses and immunoprecipitations were conducted as described ( 3 , 4 , 16 , 17 , 19 , 20 , 27 ) using: anti-phospho-serine-473-Akt, anti-glyceraldehyde-phosphate dehydrogenase (GAPDH), anti-WD40/ProF, anti-PEPCK, anti-G6Pase, anti-FAS, and anti-aPKC (Santa Cruz Biotechnologies, Santa Cruz, CA); antiphospho-threonine-560/555-PKC-ζ/λ/ι (Invitrogen, Carlsbad, CA); anti-p-serine-256-FoxO1, anti-FoxO1 (Abnova, Walnut, CA); anti-PKC-λ/ι (Transduction Antibodies, Bedford, MA); anti-phospho-serine-9-GSK3β, anti-GSK3β, anti-phospho-serine-2248-mTOR, anti-mTOR, anti-ACC and mouse anti-Akt Mab (Cell Signaling Technologies, Danvers, MA); anti-IRS-1, anti-IRS-2 (Millipore, Temecula, CA); anti-SREBP (Thermo Fisher Scientific, Freemont, CA); anti-PKC-ζ (Dr. Todd C. Sacktor, State University of New York, NY); and anti-PGC-1α (Genetex, Irvine, CA), a C-terminally-directed antiserum that measures 77kDa and 91kDa PGC-1α isoforms; however, we report only on the much more abundant 77kDa isoform, which, is specifically present in human liver, and strongly influenced by FoxO1 ( 11 ).

Techniques: Activity Assay

Journal: Metabolism: clinical and experimental

Article Title: BMI-Related Progression of Atypical PKC-dependent Aberrations in Insulin Signaling Through IRS-1, Akt, FoxO1 and PGC-1α in Livers of Obese and Type 2 Diabetic Humans

doi: 10.1016/j.metabol.2015.08.011

Figure Lengend Snippet: BMI-related alterations in: (top row), phosphorylation of aPKC and expression and abundance of aPKCs and IRS-2; (2nd row), phosphorylation of Akt, FoxO1, mTOR and GSK3β, and IRS-1 levels; (3rd row), protein levels of gluconeogenic and lipogenic enzymes and PGC-1α; and (4th row) mRNA levels of gluconeogenic and lipogenic enzymes and PGC-1α in livers of lean group-1 [BMI=20-25, N=12], overweight group-2 [BMI=25-30, N=9], obese group-3 [BMI=30-35, N=8], obese group-4 [BMI=36-40, N=9]; obese group-5 [BMI=40-45, N=6], and T2D group-6 [BMI=23-44, N=5]. Relative values are mean ± SEM. Asterisk indicates: *, P<0.05 (ANOVA) versus lean control value. Representative blots are shown in Fig 4.

Article Snippet: Western analyses and immunoprecipitations were conducted as described ( 3 , 4 , 16 , 17 , 19 , 20 , 27 ) using: anti-phospho-serine-473-Akt, anti-glyceraldehyde-phosphate dehydrogenase (GAPDH), anti-WD40/ProF, anti-PEPCK, anti-G6Pase, anti-FAS, and anti-aPKC (Santa Cruz Biotechnologies, Santa Cruz, CA); antiphospho-threonine-560/555-PKC-ζ/λ/ι (Invitrogen, Carlsbad, CA); anti-p-serine-256-FoxO1, anti-FoxO1 (Abnova, Walnut, CA); anti-PKC-λ/ι (Transduction Antibodies, Bedford, MA); anti-phospho-serine-9-GSK3β, anti-GSK3β, anti-phospho-serine-2248-mTOR, anti-mTOR, anti-ACC and mouse anti-Akt Mab (Cell Signaling Technologies, Danvers, MA); anti-IRS-1, anti-IRS-2 (Millipore, Temecula, CA); anti-SREBP (Thermo Fisher Scientific, Freemont, CA); anti-PKC-ζ (Dr. Todd C. Sacktor, State University of New York, NY); and anti-PGC-1α (Genetex, Irvine, CA), a C-terminally-directed antiserum that measures 77kDa and 91kDa PGC-1α isoforms; however, we report only on the much more abundant 77kDa isoform, which, is specifically present in human liver, and strongly influenced by FoxO1 ( 11 ).

Techniques: Expressing, Control

Journal: Metabolism: clinical and experimental

Article Title: BMI-Related Progression of Atypical PKC-dependent Aberrations in Insulin Signaling Through IRS-1, Akt, FoxO1 and PGC-1α in Livers of Obese and Type 2 Diabetic Humans

doi: 10.1016/j.metabol.2015.08.011

Figure Lengend Snippet: BMI-related alterations in: phosphorylation status of aPKC, Akt, FoxO1, mTOR, and GSK3β; and levels of PKC-ι, aPKC, Akt, IRS-1, IRS-2, FoxO1, mTOR, GSK3β, PEPCK, G6Pase, active nuclear fragment of SREBP-1c, FAS, ACC, GAPDH and PGC-1α in livers of lean control, obese and T2D humans. Representative blots are shown here. See Fig 3 for group details and mean group values.

Article Snippet: Western analyses and immunoprecipitations were conducted as described ( 3 , 4 , 16 , 17 , 19 , 20 , 27 ) using: anti-phospho-serine-473-Akt, anti-glyceraldehyde-phosphate dehydrogenase (GAPDH), anti-WD40/ProF, anti-PEPCK, anti-G6Pase, anti-FAS, and anti-aPKC (Santa Cruz Biotechnologies, Santa Cruz, CA); antiphospho-threonine-560/555-PKC-ζ/λ/ι (Invitrogen, Carlsbad, CA); anti-p-serine-256-FoxO1, anti-FoxO1 (Abnova, Walnut, CA); anti-PKC-λ/ι (Transduction Antibodies, Bedford, MA); anti-phospho-serine-9-GSK3β, anti-GSK3β, anti-phospho-serine-2248-mTOR, anti-mTOR, anti-ACC and mouse anti-Akt Mab (Cell Signaling Technologies, Danvers, MA); anti-IRS-1, anti-IRS-2 (Millipore, Temecula, CA); anti-SREBP (Thermo Fisher Scientific, Freemont, CA); anti-PKC-ζ (Dr. Todd C. Sacktor, State University of New York, NY); and anti-PGC-1α (Genetex, Irvine, CA), a C-terminally-directed antiserum that measures 77kDa and 91kDa PGC-1α isoforms; however, we report only on the much more abundant 77kDa isoform, which, is specifically present in human liver, and strongly influenced by FoxO1 ( 11 ).

Techniques: Control

Journal: Metabolism: clinical and experimental

Article Title: BMI-Related Progression of Atypical PKC-dependent Aberrations in Insulin Signaling Through IRS-1, Akt, FoxO1 and PGC-1α in Livers of Obese and Type 2 Diabetic Humans

doi: 10.1016/j.metabol.2015.08.011

Figure Lengend Snippet: Correlations between aPKC activity (p-thr-555/560-PKC-ι/ζ), PKC-ι mRNA, IRS-1 levels, Akt activity (p-ser-473-Akt), p-ser-256-FoxO1, PGC-1α mRNA, PEPCK mRNA, G6Pase mRNA, SREBP-1c mRNA and FAS mRNA versus BMI in livers of lean control (blue diamonds), obese (red squares) and type 2 diabetic (green triangles) humans.

Article Snippet: Western analyses and immunoprecipitations were conducted as described ( 3 , 4 , 16 , 17 , 19 , 20 , 27 ) using: anti-phospho-serine-473-Akt, anti-glyceraldehyde-phosphate dehydrogenase (GAPDH), anti-WD40/ProF, anti-PEPCK, anti-G6Pase, anti-FAS, and anti-aPKC (Santa Cruz Biotechnologies, Santa Cruz, CA); antiphospho-threonine-560/555-PKC-ζ/λ/ι (Invitrogen, Carlsbad, CA); anti-p-serine-256-FoxO1, anti-FoxO1 (Abnova, Walnut, CA); anti-PKC-λ/ι (Transduction Antibodies, Bedford, MA); anti-phospho-serine-9-GSK3β, anti-GSK3β, anti-phospho-serine-2248-mTOR, anti-mTOR, anti-ACC and mouse anti-Akt Mab (Cell Signaling Technologies, Danvers, MA); anti-IRS-1, anti-IRS-2 (Millipore, Temecula, CA); anti-SREBP (Thermo Fisher Scientific, Freemont, CA); anti-PKC-ζ (Dr. Todd C. Sacktor, State University of New York, NY); and anti-PGC-1α (Genetex, Irvine, CA), a C-terminally-directed antiserum that measures 77kDa and 91kDa PGC-1α isoforms; however, we report only on the much more abundant 77kDa isoform, which, is specifically present in human liver, and strongly influenced by FoxO1 ( 11 ).

Techniques: Activity Assay, Control

Journal: Metabolism: clinical and experimental

Article Title: BMI-Related Progression of Atypical PKC-dependent Aberrations in Insulin Signaling Through IRS-1, Akt, FoxO1 and PGC-1α in Livers of Obese and Type 2 Diabetic Humans

doi: 10.1016/j.metabol.2015.08.011

Figure Lengend Snippet: Effects of aPKC inhibitor, ACPD, on phosphorylation/activation of aPKC (a) and Akt (b), PGC-1α protein levels (c), IRS-1 protein levels, (d), phosphorylation of FoxO1, (e) PGC-1α mRNA levels (f) and PKC-ι mRNA levels (g) in hepatocytes of lean control and T2D humans. Hepatocytes were incubated 24 hours ± 1µM insulin (Ins) ± 1µM ACPD, as described (17, 27) [note that, as reported (17) 1µM insulin was needed to maintain full activation of aPKC and Akt over the course of a 24-hour incubation]. Relative values are mean±SEM of 4 patients. Asterisks indicate P<0.05 (ANOVA). Representative blots of phosphoproteins are shown in upper bands, and loading controls in lower bands.

Article Snippet: Western analyses and immunoprecipitations were conducted as described ( 3 , 4 , 16 , 17 , 19 , 20 , 27 ) using: anti-phospho-serine-473-Akt, anti-glyceraldehyde-phosphate dehydrogenase (GAPDH), anti-WD40/ProF, anti-PEPCK, anti-G6Pase, anti-FAS, and anti-aPKC (Santa Cruz Biotechnologies, Santa Cruz, CA); antiphospho-threonine-560/555-PKC-ζ/λ/ι (Invitrogen, Carlsbad, CA); anti-p-serine-256-FoxO1, anti-FoxO1 (Abnova, Walnut, CA); anti-PKC-λ/ι (Transduction Antibodies, Bedford, MA); anti-phospho-serine-9-GSK3β, anti-GSK3β, anti-phospho-serine-2248-mTOR, anti-mTOR, anti-ACC and mouse anti-Akt Mab (Cell Signaling Technologies, Danvers, MA); anti-IRS-1, anti-IRS-2 (Millipore, Temecula, CA); anti-SREBP (Thermo Fisher Scientific, Freemont, CA); anti-PKC-ζ (Dr. Todd C. Sacktor, State University of New York, NY); and anti-PGC-1α (Genetex, Irvine, CA), a C-terminally-directed antiserum that measures 77kDa and 91kDa PGC-1α isoforms; however, we report only on the much more abundant 77kDa isoform, which, is specifically present in human liver, and strongly influenced by FoxO1 ( 11 ).

Techniques: Activation Assay, Control, Incubation

Journal: Metabolism: clinical and experimental

Article Title: BMI-Related Progression of Atypical PKC-dependent Aberrations in Insulin Signaling Through IRS-1, Akt, FoxO1 and PGC-1α in Livers of Obese and Type 2 Diabetic Humans

doi: 10.1016/j.metabol.2015.08.011

Figure Lengend Snippet: Aberrations in insulin signaling and consequences thereof in human obesity and T2DM. Note that inhibition of Akt-dependent FoxO1 phosphorylation on the WD40/ProF platform (defect #1) presumably precedes IRS-1 loss and impairment in Akt activation (defect #2).

Article Snippet: Western analyses and immunoprecipitations were conducted as described ( 3 , 4 , 16 , 17 , 19 , 20 , 27 ) using: anti-phospho-serine-473-Akt, anti-glyceraldehyde-phosphate dehydrogenase (GAPDH), anti-WD40/ProF, anti-PEPCK, anti-G6Pase, anti-FAS, and anti-aPKC (Santa Cruz Biotechnologies, Santa Cruz, CA); antiphospho-threonine-560/555-PKC-ζ/λ/ι (Invitrogen, Carlsbad, CA); anti-p-serine-256-FoxO1, anti-FoxO1 (Abnova, Walnut, CA); anti-PKC-λ/ι (Transduction Antibodies, Bedford, MA); anti-phospho-serine-9-GSK3β, anti-GSK3β, anti-phospho-serine-2248-mTOR, anti-mTOR, anti-ACC and mouse anti-Akt Mab (Cell Signaling Technologies, Danvers, MA); anti-IRS-1, anti-IRS-2 (Millipore, Temecula, CA); anti-SREBP (Thermo Fisher Scientific, Freemont, CA); anti-PKC-ζ (Dr. Todd C. Sacktor, State University of New York, NY); and anti-PGC-1α (Genetex, Irvine, CA), a C-terminally-directed antiserum that measures 77kDa and 91kDa PGC-1α isoforms; however, we report only on the much more abundant 77kDa isoform, which, is specifically present in human liver, and strongly influenced by FoxO1 ( 11 ).

Techniques: Inhibition, Activation Assay

Journal: eLife

Article Title: TAZ inhibits glucocorticoid receptor and coordinates hepatic glucose homeostasis in normal physiological states

doi: 10.7554/eLife.57462

Figure Lengend Snippet:

Article Snippet: Antibody , (Rabbit monoclonal) anti-p-FoxO1 (Ser256) , Cell Signaling Technology , Cat. #: 84192 RRID: AB_2800035 , PMID: 31583122 IB (1:1000).

Techniques: Blocking Assay, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Reporter Assay, Mutagenesis, cDNA Synthesis, Extraction, Glucose Oxidase Assay, Recombinant, Construct, Control, Knockdown, Generated, Sequencing, Cell Culture, In Vivo, Protease Inhibitor, SYBR Green Assay, Membrane, Transfection

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