Journal: Molecular Neurobiology

Article Title: SirT2 Inhibition is Associated with Improvements in Depression-like Behavior and Memory Impairment in Olfactory Bulbectomized Mice

doi: 10.1007/s12035-026-05868-y

Figure Lengend Snippet: Effects of AK-7 on the SirT2/Ac-FoxO1/PPARγ signaling pathway in the PFC of OBX mice. A - F Expression levels of SirT2 ( A ), Ac-FoxO1 ( B ), p-FoxO1 ( C ), PPARγ ( D ), p-AKT (Thr308) ( E ), and p-AKT (Ser473) ( F ) in the PFC of OBX mice. G: Representative immunofluorescence images showing spatial overlap of SirT2, Ac-FoxO1, and PPARγ with the microglial marker Iba1 in the PFC of OBX mice treated with AK-7. H: Proposed mechanistic model of AK-7 action. A – F One-way ANOVA; A F (2, 24) = 8.965, p = 0.0012; B F (2, 24) = 7.719, p = 0.0026; C F (2, 24) = 5.336, p = 0.0121; D F (2, 24) = 8.487, p = 0.0016; E F (2, 18) = 6.321, p = 0.0083; F F (2, 18) = 13.48, p = 0.0003. All data are presented as mean ± SEM. *: p < 0.05, **: p < 0.01 vs. sham + vehicle group. #: p < 0.05, ##: p < 0.01 vs. OBX + vehicle group

Article Snippet: Membranes were blocked for 30 min in Tris-buffered saline containing 0.01% Tween-20 (TBST) supplemented with 5% skim milk and subsequently incubated overnight at 4 °C with primary antibodies against SirT1 (1:2000; Millipore, Billerica, MA, USA; 07–131), SirT2 (1:2000; Cell Signaling Technology [CST], Danvers, MA, USA; #12650), SirT3 (1:1000; CST; #5490), SirT4 (1:2000; Proteintech, Rosemont, IL, USA; 66543–1-Ig), SirT5 (1:1000; CST; #8782), SirT6 (1:1000; CST; #12486), SirT7 (1:1000; CST; #5360), ΔFosB (1:500; CST; #14695), HRD1 (1:500; Proteintech; 13473–1-AP), acetyl-FoxO1 (Lys294; 1:200; Invitrogen, Waltham, MA, USA; PA5-104560), phospho-FoxO1 (Ser256; 1:500; CST; #9461), total FoxO1 (1:500; CST; #2880), PPARγ (1:500; CST; #2435), phospho-AKT (Thr308; 1:200; CST; #13038), phospho-AKT (Ser473; 1:1000; CST; #4060), total AKT (1:1000; CST; #4691), CD86 (1:500; Proteintech; 13395–1-AP), inducible nitric oxide synthase (iNOS; 1:50000; Proteintech; 18985–1-AP), interleukin-1β (IL-1β; 1:500; Proteintech; 26048–1-AP), tumor necrosis factor-α (TNF-α; 1:100; Proteintech; 17590–1-AP), arginase-1 (1:500; Proteintech; 66129–1-Ig), transforming growth factor-β (TGF-β; 1:500; Proteintech; 21898–1-AP), myelin basic protein (MBP; 1:1000; CST; #78896), myelin-associated glycoprotein (MAG; 1:1000; CST; #9043), cyclicnucleotide phosphodiesterase (CNPase; 1:1000; CST; #5664), contactin-associated protein (Caspr; 1:1000; Abcam, Cambridge, UK; ab246501), and β-actin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA; SC-47778).

Techniques: Expressing, Immunofluorescence, Marker

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK2-mediated thermogenic gene expression may occur through the JNK/FOXO1/UCP1 axis (A) Western blotting and quantification of FOXO1 in sWAT from male WT mice housed at room or cold temperature for 3 h ( n = 4 mice per group). (B) Ucp1 expression in sWAT from male WT mice housed at 4 °C for 0, 3, 6, or 12 h ( n = 4 mice). (C) Ucp1 expression in sWAT harvested from control mice and then treated with 40 μM isoproterenol (Iso) and/or 4 μM FOXO1 inhibitor (AS1842856) for 6 h ( n = 4 wells per group). (D) Foxo1 expression in sWAT from male control and AdR2KO mice housed at RT or cold for 3 h ( n = 4 mice). (E) Western blotting and quantification of nuclear FOXO1 (nFOXO1), cytoplasmic FOXO1 (cFOXO1), and total FOXO1 (tFOXO1) in sWAT from male control and AdR2KO mice housed at RT or cold for 3 h ( n = 3 mice). (F) Western blotting and quantification of phosphorylated JNK (pJNK) and total JNK (tJNK) in sWAT from male WT mice housed at RT or cold for 3 h ( n = 3 mice). (G) Foxo1 expression in sWAT harvested from control mice and then treated with 40 μM isoproterenol (Iso) and/or 100 μM JNK inhibitor (SP600125) for 6 h ( n = 6 wells per group). (H) Western blotting and quantification of pJNK and tJNK in sWAT from control and AdR2KO mice housed at RT or cold for 3 h ( n = 3 mice). (I) Foxo1 and (J) Ucp1 expression in differentiated primary adipocytes from sWAT of control and AdR2KO mice. After differentiation for 8 days, mature adipocytes were treated with or without 10 μM isoproterenol (Iso), 1 μM AS1842856, or 50 μM SP600125 for 6 h n = 5–6 wells in (I); n = 3–8 wells in (J). (K) The mRNA levels of Mapk8 (encodes JNK1), Foxo1 , Ppargc1a , and Ucp1 in mature mouse adipocyte cells, 3T3-L1. Adipocytes were transfected with siRNAs for 72 h and then treated with 10 μM isoproterenol (Iso) for 6 h ( n = 6 wells per group). (L) The mRNA levels of Foxo1 , Ppargc1a , and Ucp1 in differentiated mouse primary adipocytes from sWAT of control and AdR2KO mice. Adipocytes were transduced with low ( Foxo1L ) and high ( Foxo1H ) MOI of AAV-8 containing mouse Foxo1 plasmid for 7 days and then treated with 20 μM isoproterenol (Iso) for 16 h ( n = 8 wells per group). The immunoblot images are representative of at least three independent experiments. p values were determined by the two-tailed Student’s t test (A and F) or one-way ANOVA with Tukey’s multiple comparisons test (B-E and G-L). All data are presented as the mean ± SEM of at least three independent biological replicates. See also and . AS: AS1842856; SP: SP600125.

Article Snippet: The PVDF membranes were blocked with 5% nonfat dry milk (Research Products International; M17200 ) in TBST for 1 hour and then incubated overnight at 4°C with primary antibodies against ROCK1 (BD Biosciences; 611136), ROCK2 (BD Biosciences; 610623), phospho-Thr 850 MBS (Millipore; 36-003), total MBS (BioLegend; 925101), UCP1 (Abcam; ab23841), phospho-FOXO1 (Ser 256 , Cell Signaling; 9461), FOXO1 (Cell Signaling; 2880), phospho-SAPK/JNK (Thr 183 /Tyr 185 , Cell Signaling; 9251), phospho-AKT (Ser473, Cell Signaling Technology; 4060), AKT (Cell Signaling; 9272), total JNK (SAPK/JNK, Cell Signaling; 9252), Lamin A/C (Cell Signaling; 4777), and GAPDH (Cell Signaling; 2118).

Techniques: Gene Expression, Western Blot, Expressing, Control, Transfection, Transduction, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK2 deletion decreased the oxygen consumption rate in AdR2KO mice after cold exposure The whole-body oxygen consumption rate (VO 2 ), carbon dioxide production rate (VCO 2 ), and respiratory exchange rate (RER) in 10- to 12-week-old male control and AdR2KO mice housed at (A) room temperature (RT) or (B) cold (4 °C) for 72 h ( n = 6 mice per group). (C) The mRNA expression of brown fat-selective genes ( Ppargc1a , Ucp1 , and Cidea ) and general adipocyte genes ( Adipoq and Slc2a4 ) in the sWAT of male control and adipocyte-specific constitutively active ROCK (AdcaRK) mice ( n = 6 mice per group). (D) Immunohistochemistry for UCP1 in sections of sWAT from control and AdcaRK mice housed at RT or 4 °C for 3 days ( n = 3 mice per group). High-magnification images are shown in the boxed squares. The scale bars are 5 mm and 100 μm in the low- and high-magnification images, respectively. The images are representative of three independent experiments. (E) The mRNA level of Ucp1 in sWAT harvested from control and AdcaRK mice and then treated with 40 μM isoproterenol (Iso) and 4 μM FOXO1 inhibitor (AS1842856) for 6 h ( n = 6 wells per group). (F) The mRNA levels of Foxo1 and Irf4 in sWAT from male control and AdcaRK mice housed at RT or cold for 3 days ( n = 6 mice per group). p values were determined by two-tailed Mann-Whitney U tests (A and B) or one-way ANOVA with Tukey’s multiple comparisons test (C, E, and F). All data are presented as the mean ± SEM of at least three independent biological replicates. See also . AS: AS1842856.

Article Snippet: The PVDF membranes were blocked with 5% nonfat dry milk (Research Products International; M17200 ) in TBST for 1 hour and then incubated overnight at 4°C with primary antibodies against ROCK1 (BD Biosciences; 611136), ROCK2 (BD Biosciences; 610623), phospho-Thr 850 MBS (Millipore; 36-003), total MBS (BioLegend; 925101), UCP1 (Abcam; ab23841), phospho-FOXO1 (Ser 256 , Cell Signaling; 9461), FOXO1 (Cell Signaling; 2880), phospho-SAPK/JNK (Thr 183 /Tyr 185 , Cell Signaling; 9251), phospho-AKT (Ser473, Cell Signaling Technology; 4060), AKT (Cell Signaling; 9272), total JNK (SAPK/JNK, Cell Signaling; 9252), Lamin A/C (Cell Signaling; 4777), and GAPDH (Cell Signaling; 2118).

Techniques: Control, Expressing, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK2-mediated thermogenic gene expression may occur through the JNK/FOXO1/UCP1 axis (A) Western blotting and quantification of FOXO1 in sWAT from male WT mice housed at room or cold temperature for 3 h ( n = 4 mice per group). (B) Ucp1 expression in sWAT from male WT mice housed at 4 °C for 0, 3, 6, or 12 h ( n = 4 mice). (C) Ucp1 expression in sWAT harvested from control mice and then treated with 40 μM isoproterenol (Iso) and/or 4 μM FOXO1 inhibitor (AS1842856) for 6 h ( n = 4 wells per group). (D) Foxo1 expression in sWAT from male control and AdR2KO mice housed at RT or cold for 3 h ( n = 4 mice). (E) Western blotting and quantification of nuclear FOXO1 (nFOXO1), cytoplasmic FOXO1 (cFOXO1), and total FOXO1 (tFOXO1) in sWAT from male control and AdR2KO mice housed at RT or cold for 3 h ( n = 3 mice). (F) Western blotting and quantification of phosphorylated JNK (pJNK) and total JNK (tJNK) in sWAT from male WT mice housed at RT or cold for 3 h ( n = 3 mice). (G) Foxo1 expression in sWAT harvested from control mice and then treated with 40 μM isoproterenol (Iso) and/or 100 μM JNK inhibitor (SP600125) for 6 h ( n = 6 wells per group). (H) Western blotting and quantification of pJNK and tJNK in sWAT from control and AdR2KO mice housed at RT or cold for 3 h ( n = 3 mice). (I) Foxo1 and (J) Ucp1 expression in differentiated primary adipocytes from sWAT of control and AdR2KO mice. After differentiation for 8 days, mature adipocytes were treated with or without 10 μM isoproterenol (Iso), 1 μM AS1842856, or 50 μM SP600125 for 6 h n = 5–6 wells in (I); n = 3–8 wells in (J). (K) The mRNA levels of Mapk8 (encodes JNK1), Foxo1 , Ppargc1a , and Ucp1 in mature mouse adipocyte cells, 3T3-L1. Adipocytes were transfected with siRNAs for 72 h and then treated with 10 μM isoproterenol (Iso) for 6 h ( n = 6 wells per group). (L) The mRNA levels of Foxo1 , Ppargc1a , and Ucp1 in differentiated mouse primary adipocytes from sWAT of control and AdR2KO mice. Adipocytes were transduced with low ( Foxo1L ) and high ( Foxo1H ) MOI of AAV-8 containing mouse Foxo1 plasmid for 7 days and then treated with 20 μM isoproterenol (Iso) for 16 h ( n = 8 wells per group). The immunoblot images are representative of at least three independent experiments. p values were determined by the two-tailed Student’s t test (A and F) or one-way ANOVA with Tukey’s multiple comparisons test (B-E and G-L). All data are presented as the mean ± SEM of at least three independent biological replicates. See also and . AS: AS1842856; SP: SP600125.

Article Snippet: The PVDF membranes were blocked with 5% nonfat dry milk (Research Products International; M17200 ) in TBST for 1 hour and then incubated overnight at 4°C with primary antibodies against ROCK1 (BD Biosciences; 611136), ROCK2 (BD Biosciences; 610623), phospho-Thr 850 MBS (Millipore; 36-003), total MBS (BioLegend; 925101), UCP1 (Abcam; ab23841), phospho-FOXO1 (Ser 256 , Cell Signaling; 9461), FOXO1 (Cell Signaling; 2880), phospho-SAPK/JNK (Thr 183 /Tyr 185 , Cell Signaling; 9251), phospho-AKT (Ser473, Cell Signaling Technology; 4060), AKT (Cell Signaling; 9272), total JNK (SAPK/JNK, Cell Signaling; 9252), Lamin A/C (Cell Signaling; 4777), and GAPDH (Cell Signaling; 2118).

Techniques: Gene Expression, Western Blot, Expressing, Control, Transfection, Transduction, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK2 deletion decreased the oxygen consumption rate in AdR2KO mice after cold exposure The whole-body oxygen consumption rate (VO 2 ), carbon dioxide production rate (VCO 2 ), and respiratory exchange rate (RER) in 10- to 12-week-old male control and AdR2KO mice housed at (A) room temperature (RT) or (B) cold (4 °C) for 72 h ( n = 6 mice per group). (C) The mRNA expression of brown fat-selective genes ( Ppargc1a , Ucp1 , and Cidea ) and general adipocyte genes ( Adipoq and Slc2a4 ) in the sWAT of male control and adipocyte-specific constitutively active ROCK (AdcaRK) mice ( n = 6 mice per group). (D) Immunohistochemistry for UCP1 in sections of sWAT from control and AdcaRK mice housed at RT or 4 °C for 3 days ( n = 3 mice per group). High-magnification images are shown in the boxed squares. The scale bars are 5 mm and 100 μm in the low- and high-magnification images, respectively. The images are representative of three independent experiments. (E) The mRNA level of Ucp1 in sWAT harvested from control and AdcaRK mice and then treated with 40 μM isoproterenol (Iso) and 4 μM FOXO1 inhibitor (AS1842856) for 6 h ( n = 6 wells per group). (F) The mRNA levels of Foxo1 and Irf4 in sWAT from male control and AdcaRK mice housed at RT or cold for 3 days ( n = 6 mice per group). p values were determined by two-tailed Mann-Whitney U tests (A and B) or one-way ANOVA with Tukey’s multiple comparisons test (C, E, and F). All data are presented as the mean ± SEM of at least three independent biological replicates. See also . AS: AS1842856.

Article Snippet: The PVDF membranes were blocked with 5% nonfat dry milk (Research Products International; M17200 ) in TBST for 1 hour and then incubated overnight at 4°C with primary antibodies against ROCK1 (BD Biosciences; 611136), ROCK2 (BD Biosciences; 610623), phospho-Thr 850 MBS (Millipore; 36-003), total MBS (BioLegend; 925101), UCP1 (Abcam; ab23841), phospho-FOXO1 (Ser 256 , Cell Signaling; 9461), FOXO1 (Cell Signaling; 2880), phospho-SAPK/JNK (Thr 183 /Tyr 185 , Cell Signaling; 9251), phospho-AKT (Ser473, Cell Signaling Technology; 4060), AKT (Cell Signaling; 9272), total JNK (SAPK/JNK, Cell Signaling; 9252), Lamin A/C (Cell Signaling; 4777), and GAPDH (Cell Signaling; 2118).

Techniques: Control, Expressing, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK2-mediated thermogenic gene expression may occur through the JNK/FOXO1/UCP1 axis (A) Western blotting and quantification of FOXO1 in sWAT from male WT mice housed at room or cold temperature for 3 h ( n = 4 mice per group). (B) Ucp1 expression in sWAT from male WT mice housed at 4 °C for 0, 3, 6, or 12 h ( n = 4 mice). (C) Ucp1 expression in sWAT harvested from control mice and then treated with 40 μM isoproterenol (Iso) and/or 4 μM FOXO1 inhibitor (AS1842856) for 6 h ( n = 4 wells per group). (D) Foxo1 expression in sWAT from male control and AdR2KO mice housed at RT or cold for 3 h ( n = 4 mice). (E) Western blotting and quantification of nuclear FOXO1 (nFOXO1), cytoplasmic FOXO1 (cFOXO1), and total FOXO1 (tFOXO1) in sWAT from male control and AdR2KO mice housed at RT or cold for 3 h ( n = 3 mice). (F) Western blotting and quantification of phosphorylated JNK (pJNK) and total JNK (tJNK) in sWAT from male WT mice housed at RT or cold for 3 h ( n = 3 mice). (G) Foxo1 expression in sWAT harvested from control mice and then treated with 40 μM isoproterenol (Iso) and/or 100 μM JNK inhibitor (SP600125) for 6 h ( n = 6 wells per group). (H) Western blotting and quantification of pJNK and tJNK in sWAT from control and AdR2KO mice housed at RT or cold for 3 h ( n = 3 mice). (I) Foxo1 and (J) Ucp1 expression in differentiated primary adipocytes from sWAT of control and AdR2KO mice. After differentiation for 8 days, mature adipocytes were treated with or without 10 μM isoproterenol (Iso), 1 μM AS1842856, or 50 μM SP600125 for 6 h n = 5–6 wells in (I); n = 3–8 wells in (J). (K) The mRNA levels of Mapk8 (encodes JNK1), Foxo1 , Ppargc1a , and Ucp1 in mature mouse adipocyte cells, 3T3-L1. Adipocytes were transfected with siRNAs for 72 h and then treated with 10 μM isoproterenol (Iso) for 6 h ( n = 6 wells per group). (L) The mRNA levels of Foxo1 , Ppargc1a , and Ucp1 in differentiated mouse primary adipocytes from sWAT of control and AdR2KO mice. Adipocytes were transduced with low ( Foxo1L ) and high ( Foxo1H ) MOI of AAV-8 containing mouse Foxo1 plasmid for 7 days and then treated with 20 μM isoproterenol (Iso) for 16 h ( n = 8 wells per group). The immunoblot images are representative of at least three independent experiments. p values were determined by the two-tailed Student’s t test (A and F) or one-way ANOVA with Tukey’s multiple comparisons test (B-E and G-L). All data are presented as the mean ± SEM of at least three independent biological replicates. See also and . AS: AS1842856; SP: SP600125.

Article Snippet: anti-Phospho-Ser 256 FOXO1 , Cell Signaling Technology , Cat# 9461; RRID: AB_329831.

Techniques: Gene Expression, Western Blot, Expressing, Control, Transfection, Transduction, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK2 deletion decreased the oxygen consumption rate in AdR2KO mice after cold exposure The whole-body oxygen consumption rate (VO 2 ), carbon dioxide production rate (VCO 2 ), and respiratory exchange rate (RER) in 10- to 12-week-old male control and AdR2KO mice housed at (A) room temperature (RT) or (B) cold (4 °C) for 72 h ( n = 6 mice per group). (C) The mRNA expression of brown fat-selective genes ( Ppargc1a , Ucp1 , and Cidea ) and general adipocyte genes ( Adipoq and Slc2a4 ) in the sWAT of male control and adipocyte-specific constitutively active ROCK (AdcaRK) mice ( n = 6 mice per group). (D) Immunohistochemistry for UCP1 in sections of sWAT from control and AdcaRK mice housed at RT or 4 °C for 3 days ( n = 3 mice per group). High-magnification images are shown in the boxed squares. The scale bars are 5 mm and 100 μm in the low- and high-magnification images, respectively. The images are representative of three independent experiments. (E) The mRNA level of Ucp1 in sWAT harvested from control and AdcaRK mice and then treated with 40 μM isoproterenol (Iso) and 4 μM FOXO1 inhibitor (AS1842856) for 6 h ( n = 6 wells per group). (F) The mRNA levels of Foxo1 and Irf4 in sWAT from male control and AdcaRK mice housed at RT or cold for 3 days ( n = 6 mice per group). p values were determined by two-tailed Mann-Whitney U tests (A and B) or one-way ANOVA with Tukey’s multiple comparisons test (C, E, and F). All data are presented as the mean ± SEM of at least three independent biological replicates. See also . AS: AS1842856.

Article Snippet: anti-Phospho-Ser 256 FOXO1 , Cell Signaling Technology , Cat# 9461; RRID: AB_329831.

Techniques: Control, Expressing, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY

Journal: PLOS One

Article Title: FOXO1 transcription factor modulates airway epithelial responses to viral infection

doi: 10.1371/journal.pone.0345169

Figure Lengend Snippet: A) FOXO1 mRNA expression was analyzed by RT-qPCR in BEAS-2B cells transduced with FOXO1 shRNA or scrambled shRNA (n = 8). B) Representative Western blot and C) densitometry quantification (n = 6) of FOXO1 protein levels in cells treated with FOXO1 shRNA lentivirus, scrambled shRNA lentivirus and untransduced cells (control). Statistical analysis with t-test *p < 0.05, **p < 0.01. FOXO3 (D) and FOXO4 (E) expression levels in FOXO1 shRNA and scrambled shRNA lentivirus transfected cells were assessed by RT-qPCR (n = 5). Mean fluorescence intensity of FOXO1 localized to the nucleus (F) and whole cell expression (G) was quantified with Volocity software. For each group 40-60 cells per slide were analyzed. Statistical Analysis was performed using Anova **P < 0.01, **** P < 0.0001 H) Immunofluorescence staining for FOXO1 in BEAS-2B cells transduced with FOXO1 shRNA or scrambled shRNA. FOXO1 (red) was detected using an anti-FOXO1 monoclonal antibody, nuclei were stained with DAPI (blue), and the cytoskeleton was visualized with phalloidin (green). Images were captured at 60 × magnification using a DeltaVision Confocal Microscope.

Article Snippet: Gene expression was analyzed using TaqMan assays (Thermo Fisher Scientific, Waltham, MA) for FOXO1 (Hs00231106_m1), FOXO3 (Hs00818121_m1), FOXO4 (Cat#Hs00172973_m1), TLR3 (Hs01551078_m1), RIG-1/DDX58 (Hs01061436_m1), MAVS (Hs00920075_m1), MYD88 (Hs01573837_g1) and GapdH (Hs02758991_g1) as internal control.

Techniques: Expressing, Quantitative RT-PCR, Transduction, shRNA, Western Blot, Control, Transfection, Fluorescence, Software, Immunofluorescence, Staining, Microscopy

Journal: PLOS One

Article Title: FOXO1 transcription factor modulates airway epithelial responses to viral infection

doi: 10.1371/journal.pone.0345169

Figure Lengend Snippet: WST-1 proliferation assay (A) and total cell counts (B) for 5 days following plating of BEAS-2B cells treated with FOXO1 shRNA lentivirus, scrambled shRNA lentivirus and untransduced cells (control) (n = 3). Representative flow cytometry plot (D) and percent of dead (C) and apoptotic (E) BEAS-2B cells analyzed with Annexin V and PI dye (n = 3). Statistical Analysis was performed with ANOVA. F) BEAS-2B monolayer average resistance tracings at 4000 Hz in response to wounding challenge measured with ECIS1600; n = 4 cell replicates per group. Quantification of resistance after wounding (G) and at the end-point (H) shows a significant difference between scrambled shRNA and FOXO1 deficient BEAS-2B cells. Statistical Analysis with t-test* p < 0.05.

Article Snippet: Gene expression was analyzed using TaqMan assays (Thermo Fisher Scientific, Waltham, MA) for FOXO1 (Hs00231106_m1), FOXO3 (Hs00818121_m1), FOXO4 (Cat#Hs00172973_m1), TLR3 (Hs01551078_m1), RIG-1/DDX58 (Hs01061436_m1), MAVS (Hs00920075_m1), MYD88 (Hs01573837_g1) and GapdH (Hs02758991_g1) as internal control.

Techniques: Proliferation Assay, shRNA, Control, Flow Cytometry

Journal: PLOS One

Article Title: FOXO1 transcription factor modulates airway epithelial responses to viral infection

doi: 10.1371/journal.pone.0345169

Figure Lengend Snippet: A) TLR3 mRNA expression was assessed by RT-qPCR in BEAS-2B cells transduced with FOXO1 shRNA or scrambled shRNA lentivirus. Expression levels were normalized to housekeeping gene GAPDH and expressed relative to untransduced cells (n = 12). Statistical analysis was performed with t-test ****p < 0.0001. B) TLR4 mRNA expression was analyzed by RT-qPCR in BEAS-2B cells transduced with FOXO1 shRNA and scrambled shRNA lentivirus. Expression was normalized to GAPDH and expressed relative to untransduced cells (n = 4). Statistical analysis with t-test. Representative Western blot (C) and densitometry analysis (D) of TLR3 expression for FOXO1 deficient BEAS-2B cells compared to controls, B-actin was used as a loading control (n = 6). Statistical Analysis with t-test, **p < 0.01. FOXO1 deficient cells and scrambled shRNA cells were stimulated with 50 µg/mL Poly(I:C) for 24 hours and release of IL6 (E),CCL2 (F), GM-CSF (G), IFN-λ (H), CXCL10 (I), IL8 (J), TNF-α (K), and TSLP (L) was tested with an MSD assay (n = 3). TLR3 mRNA expression was assessed by RT-qPCR in BEAS-2B (M) and NHBE (N) cells stimulated with Poly(I:C) for 8 hours in the presence or absence of the FOXO1 inhibitor AS1842856 (1 µM) (n = 5). Statistical analysis was performed using ANOVA *p < 0.05. **p < 0.01, **** P < 0.0001.

Article Snippet: Gene expression was analyzed using TaqMan assays (Thermo Fisher Scientific, Waltham, MA) for FOXO1 (Hs00231106_m1), FOXO3 (Hs00818121_m1), FOXO4 (Cat#Hs00172973_m1), TLR3 (Hs01551078_m1), RIG-1/DDX58 (Hs01061436_m1), MAVS (Hs00920075_m1), MYD88 (Hs01573837_g1) and GapdH (Hs02758991_g1) as internal control.

Techniques: Expressing, Quantitative RT-PCR, Transduction, shRNA, Western Blot, Control

Journal: PLOS One

Article Title: FOXO1 transcription factor modulates airway epithelial responses to viral infection

doi: 10.1371/journal.pone.0345169

Figure Lengend Snippet: A) RT-qPCR showed increased TLR3 mRNA expression for BEAS-2B transfected with a CA-FOXO1 plasmid compared to vector control (cells transfected with an empty plasmid); GAPDH was used as a housekeeping gene (n = 6). Representative Western blot (B) and densitometry analysis (C) of TLR3 expression for BEAS-2B transfected with CA-FOXO1 plasmid compared to vector control, β-actin was used as a loading control (n = 6). Statistical Analysis with t-test, **p < 0.01. D + E) Immunofluorescence staining for BEAS-2B transduced with CA-FOXO1 shows increased FOXO1 protein in the nucleus. FOXO1 (red) was detected using an anti-FOXO1 antibody with a red-fluorescent secondary antibody, F-actin (green) with phalloidin, and nuclei (blue) with DAPI. Images were taken with an Olympus IX81 epifluorescence microscope using a 20X objective lens. Volocity Analysis was used to quantify nuclear localization of FOXO1 by measuring the mean fluorescence intensity of FOXO1 staining colocalized with DAPI. For each group 40−60 cells per slide were analyzed. Statistical Analysis was conducted with ANOVA **** p < 0.001. BEAS-2B cells transduced with FOXO1 or scrambled shRNA lentivirus were analyzed by RT-qPCR for DDX58 (RIG-I, F), MAVS (G), and MYD88 (H) mRNA expression at baseline and after Poly(I:C) stimulation (8 h and 24 h). Expression was normalized to GAPDH and expressed relative to unstimulated scrambled controls (n = 3; ANOVA). (I) NHBE cells were infected with SARS-CoV-2 in the presence or absence of a FOXO1 inhibitor. Total RNA was collected 24 h post-infection, and viral RNA levels were quantified by qRT-PCR, normalized to ACTB, and expressed relative to mock-infected cells (n = 3; paired t-test).

Article Snippet: Gene expression was analyzed using TaqMan assays (Thermo Fisher Scientific, Waltham, MA) for FOXO1 (Hs00231106_m1), FOXO3 (Hs00818121_m1), FOXO4 (Cat#Hs00172973_m1), TLR3 (Hs01551078_m1), RIG-1/DDX58 (Hs01061436_m1), MAVS (Hs00920075_m1), MYD88 (Hs01573837_g1) and GapdH (Hs02758991_g1) as internal control.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Immunofluorescence, Staining, Transduction, Microscopy, Fluorescence, shRNA, Infection

Journal: PLOS One

Article Title: FOXO1 transcription factor modulates airway epithelial responses to viral infection

doi: 10.1371/journal.pone.0345169

Figure Lengend Snippet: A) Representative set of Immunofluorescence staining for BEAS-2B cells stimulated with 50 µg/mL Poly(I:C) at different time points. FOXO1 (red) was detected using an anti-FOXO1 monoclonal antibody, nuclei were stained with DAPI (blue), and the cytoskeleton was visualized with phalloidin (green). Images were captured at 60 × magnification using a DeltaVision Confocal Microscope. B) Mean fluorescence intensity of FOXO1 localized to the nucleus was quantified with Volocity software. For each group 40–60 cells were analyzed. Statistical Analysis was performed using Anova ***P < 0.001, **** P < 0.0001.

Article Snippet: Gene expression was analyzed using TaqMan assays (Thermo Fisher Scientific, Waltham, MA) for FOXO1 (Hs00231106_m1), FOXO3 (Hs00818121_m1), FOXO4 (Cat#Hs00172973_m1), TLR3 (Hs01551078_m1), RIG-1/DDX58 (Hs01061436_m1), MAVS (Hs00920075_m1), MYD88 (Hs01573837_g1) and GapdH (Hs02758991_g1) as internal control.

Techniques: Immunofluorescence, Staining, Microscopy, Fluorescence, Software

Journal: PLOS One

Article Title: FOXO1 transcription factor modulates airway epithelial responses to viral infection

doi: 10.1371/journal.pone.0345169

Figure Lengend Snippet: A) The top panel presents FOXO1 ChIP-Seq peaks retrieved from Gene Expression Omnibus ( GSM3681486 ) in HUVEC cells and ( GSM5214707 ) in the HepG2 cell line. The bottom panel shows FOXO1 binding sites from the ChIP-Atlas visualized with IGV (Integrative Genomics Viewer). The FOXO1 motif from the HOCOMOCO database within the proximal promoter sequence of the TLR3 gene is highlighted below. B) EMSA with nuclear extracts from BEAS-2B cells incubated with FOXO1-TLR3 Promoter Oligos. Nuclear extracts from BEAS-2B cells transfected with CA-FOXO1 or plasmid control. Lane 1: dye only, Lane 2: probe only, Lane 3: nuclear extracts from CA-FOXO1 BEAS-2B cells, Lane 4: nuclear extracts from CA-FOXO1 BEAS-2B cells + cold competitor, Lane 5: nuclear extracts from vector control BEAS-2B cells, Lane 6: nuclear extracts from vector control BEAS-2B cells + cold competitor. Lane 3 shows complex I formation, which disappears in lane 4, indicating non-specific binding. C) Nuclear extracts of BEAS-2B cells transfected with CA-FOXO1 incubated with or without FOXO1 mAb shows the formation of complex I + II, but no supershift occurred. D) Incubation of protein extracts from BEAS-2B FOXO1 deficient and scrambled control lines show formation of complexes I and II but no difference is observed between cell lines.

Article Snippet: Gene expression was analyzed using TaqMan assays (Thermo Fisher Scientific, Waltham, MA) for FOXO1 (Hs00231106_m1), FOXO3 (Hs00818121_m1), FOXO4 (Cat#Hs00172973_m1), TLR3 (Hs01551078_m1), RIG-1/DDX58 (Hs01061436_m1), MAVS (Hs00920075_m1), MYD88 (Hs01573837_g1) and GapdH (Hs02758991_g1) as internal control.

Techniques: ChIP-sequencing, Gene Expression, Binding Assay, Sequencing, Incubation, Transfection, Plasmid Preparation, Control