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: As a negative control, a lentivirus expressing scrambled shRNA, which does not target any mammalian gene, was used to transduce BEAS-2B cells (scrambled shRNA) (Addgene #1864).

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: As a negative control, a lentivirus expressing scrambled shRNA, which does not target any mammalian gene, was used to transduce BEAS-2B cells (scrambled shRNA) (Addgene #1864).

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: As a negative control, a lentivirus expressing scrambled shRNA, which does not target any mammalian gene, was used to transduce BEAS-2B cells (scrambled shRNA) (Addgene #1864).

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: As a negative control, a lentivirus expressing scrambled shRNA, which does not target any mammalian gene, was used to transduce BEAS-2B cells (scrambled shRNA) (Addgene #1864).

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: As a negative control, a lentivirus expressing scrambled shRNA, which does not target any mammalian gene, was used to transduce BEAS-2B cells (scrambled shRNA) (Addgene #1864).

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: As a negative control, a lentivirus expressing scrambled shRNA, which does not target any mammalian gene, was used to transduce BEAS-2B cells (scrambled shRNA) (Addgene #1864).

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

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a HMEC were transfected with scrambled-siRNA (sc-siRNA) or CHFR-siRNA. At 72 h post-transfection, cells stimulated with rAng1 (400 ng/ml) for different time intervals were used for IB analysis to determine phosphorylation of Akt1 at S473 and T308 ( n = 3 independent experiments). b Control HMEC and CHFR knockdown HMEC stimulated with rAng1 as above were stained with phospho-473-Akt1 specific antibody. c Control HMEC and CHFR knockdown HMEC were transfected with the Akt1 biosensor plasmid and then stimulated with rAng1 as above to assess live cell Akt1 activity by measuring the FRET ratio ( p <0.0001) ( left panel shows basal Akt activity; right panel shows Akt activity in response to rAng1 challenge). d-f Control HMEC and CHFR-siRNA treated HMEC stimulated with rAng1 were used for IB analysis to determine tyrosine phosphorylation Tie2 ( d ), phosphorylation of GSK3β at S9 and phosphorylation of β-catenin ( e ), ( n = 3 independent experiments). a, d-e Shown are mean values ± SEM ( n = 3 independent experiments; two-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: Scrambled-siRNA (Sc-siRNA) and human ( h )-specific siRNA1 against CHFR (A pool of 3 target-specific siRNAs, sense: 5′-CUCUGUGGCAAGUGAUGAAtt-3′; sense: 5′-GAAGAAGAUGUGCAAAGUAtt; sense: 5′-GGUAUAGUAUGGUAUUUGAtt-3′) were obtained from Santa Cruz Biotechnology. pEGFP-C2 plasmid (Addgene catalog #61853) encoding wild-type human CHFR (WT h -CHFR), h CHFR mutant lacking forkhead-associated domain (ΔFHA-CHFR), h CHFR mutant lacking RING finger domain (ΔRF-CHFR), mutant lacking cysteine-rich domain (ΔCR-CHFR), and mutant lacking poly-ADP ribose binding zinc-finger domain (ΔPBZ-CHFR) were custom prepared by Genscript (Piscataway, NJ). pmcherry-C1 plasmid (Addgene catalog #86631) encoding wild-type human Akt1 (WT h -Akt1), phopsho-defective h Akt1 (T308A and S473A) mutant and ubiquitylation-defective h Akt1 (K30R, K39R, K39R+154R and K30R+K39R+K154R+K268R) mutants were custom prepared by Genscript (Piscataway, NJ).

Techniques: Transfection, Phospho-proteomics, Control, Knockdown, Staining, Plasmid Preparation, Activity Assay

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a HLMVEC were transfected with Sc-siRNA or CHFR-siRNA. At 72 h post-transfection, cells challenged with LPS (5 μg/ml) for different time periods were used for IB analysis to determine expression of CHFR and Akt1. Shown are mean values ± SEM ( n = 3 independent experiments; two-way ANOVA followed by Tukey’s post-hoc test). b-c HLMVEC were transfected with Sc-siRNA or CHFR-siRNA. At 72 h post transfection, cells challenged with LPS (5 μg/ml) for different time periods were stained with antibodies specific to VE-cadherin, Akt1, and FoXO1. Confocal imaging shows that CHFR depletion in EC prevents LPS-induced degradation of Akt1 and VE-cadherin and nuclear accumulation of FoxO1. d CHFR depletion prevents LPS-induced expression of FoxO1 and Ang-2. HLMVEC were transfected with Sc-siRNA or CHFR-siRNA as above and exposed to LPS for different time periods were used for IB to determine expression of FoxO1 and Ang-2. e LPS-induced time-course expression of FoxO1, CHFR, and Ang-2 in HLMVEC. HLMVEC exposed to LPS (5 μg/ml) for 0, 1, 2, 4, and 6 h were used for IB. d-e Shown are mean values ± SEM ( n = 3 independent experiments; two-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: Scrambled-siRNA (Sc-siRNA) and human ( h )-specific siRNA1 against CHFR (A pool of 3 target-specific siRNAs, sense: 5′-CUCUGUGGCAAGUGAUGAAtt-3′; sense: 5′-GAAGAAGAUGUGCAAAGUAtt; sense: 5′-GGUAUAGUAUGGUAUUUGAtt-3′) were obtained from Santa Cruz Biotechnology. pEGFP-C2 plasmid (Addgene catalog #61853) encoding wild-type human CHFR (WT h -CHFR), h CHFR mutant lacking forkhead-associated domain (ΔFHA-CHFR), h CHFR mutant lacking RING finger domain (ΔRF-CHFR), mutant lacking cysteine-rich domain (ΔCR-CHFR), and mutant lacking poly-ADP ribose binding zinc-finger domain (ΔPBZ-CHFR) were custom prepared by Genscript (Piscataway, NJ). pmcherry-C1 plasmid (Addgene catalog #86631) encoding wild-type human Akt1 (WT h -Akt1), phopsho-defective h Akt1 (T308A and S473A) mutant and ubiquitylation-defective h Akt1 (K30R, K39R, K39R+154R and K30R+K39R+K154R+K268R) mutants were custom prepared by Genscript (Piscataway, NJ).

Techniques: Transfection, Expressing, Staining, Imaging

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a-b CHFR deficiency in EC prevents LPS-induced ubiquitylation of Akt1. HLMVEC transfected with sc-siRNA or CHFR-siRNA. At 72 h post-transfection, cells were challenged with LPS (5 μg/ml) for 6 h in the presence of the proteasomal inhibitor MG132 (10 μM). Cell lysates were immunoprecipitated with anti-Akt1 mAb and blotted with antibodies specific to K 48 -linked poly-Ub or K 63 -linked poly-Ub ( n = 2 independent experiments) ( a ). Chfr fl/fl (WT) and Chfr ΔEC mice were challenged with LPS (10 mg/kg; i.p.) for 6 h. After the LPS challenge, lungs harvested were used to determine ubiquitylation of Akt1 as above in a (b) . c-f CHFR induces ubiquitylation of activated Akt1. c Control and CHFR-depleted HLMVEC were pretreated with inhibitors of PDK1 and mTORC2 for 30 min and then exposed to LPS (5 μg/ml). Thereafter, cell lysates were used for IB to assess phosphorylation of Akt1. d-e HLMVEC pretreated with or without PDK1 and mTORC2 inhibitors for 1 h and stimulated with LPS (6 h) in the presence of MG123 (10 μM) showed that CHFR binds and ubiquitylates phosphorylated Akt1. f HEK-293T cells were transfected with HA-tagged ubiquitin (HA-Ub) (0.5 μg/ml) alone or co-transfected with N-terminal GFP-tagged WT-CHFR (1.5 μg/ml), N-terminal pmCherry-tagged WT-Akt1 (1.5 μg/ml), and phosphorylation-defective Akt1 mutant (Akt1T308A/S473A) (1.5 μg/ml) plasmids. Thirty-six hours after transfection, cells were incubated with MG123 (10 μM) for 3h, and cell lysates were used to determine phosphorylation-dependent ubiquitylation of Akt1.

Article Snippet: Scrambled-siRNA (Sc-siRNA) and human ( h )-specific siRNA1 against CHFR (A pool of 3 target-specific siRNAs, sense: 5′-CUCUGUGGCAAGUGAUGAAtt-3′; sense: 5′-GAAGAAGAUGUGCAAAGUAtt; sense: 5′-GGUAUAGUAUGGUAUUUGAtt-3′) were obtained from Santa Cruz Biotechnology. pEGFP-C2 plasmid (Addgene catalog #61853) encoding wild-type human CHFR (WT h -CHFR), h CHFR mutant lacking forkhead-associated domain (ΔFHA-CHFR), h CHFR mutant lacking RING finger domain (ΔRF-CHFR), mutant lacking cysteine-rich domain (ΔCR-CHFR), and mutant lacking poly-ADP ribose binding zinc-finger domain (ΔPBZ-CHFR) were custom prepared by Genscript (Piscataway, NJ). pmcherry-C1 plasmid (Addgene catalog #86631) encoding wild-type human Akt1 (WT h -Akt1), phopsho-defective h Akt1 (T308A and S473A) mutant and ubiquitylation-defective h Akt1 (K30R, K39R, K39R+154R and K30R+K39R+K154R+K268R) mutants were custom prepared by Genscript (Piscataway, NJ).

Techniques: Transfection, Immunoprecipitation, Control, Phospho-proteomics, Ubiquitin Proteomics, Mutagenesis, Incubation