Journal: PloS one

Article Title: FoxO3a (Forkhead Box O3a) deficiency protects Idiopathic Pulmonary Fibrosis (IPF) fibroblasts from type I polymerized collagen matrix-induced apoptosis via caveolin-1 (cav-1) and Fas.

doi: 10.1371/journal.pone.0061017

Figure Lengend Snippet: Figure 1. Low FoxO3a confers IPF fibroblasts with resistance to polymerized collagen-mediated apoptosis. A & B. Serum starved IPF and control fibroblasts (n = 5, each) were permitted to attach to polymerized collagen for 48 h in the absence of serum and cell morphology (A) and cell viability (B) were examined. Shown are 50X magnification images of cell morphology. C & D. IPF and control fibroblasts infected with adenovirus expressing FoxO3a (F3) or empty vector GFP were cultured on collagen for 48 h in serum free medium and cell morphology (C) and cell viability (D) were examined. Shown is 50X magnification of cell morphology. Note that forced expression of FoxO3a significantly increased apoptosis in IPF fibroblasts on collagen matrix. *p = 0.003 versus GFP control. The assay was obtained from triplicate. doi:10.1371/journal.pone.0061017.g001

Article Snippet: Adenovirus expressing GFP tagged wild type FoxO3a, dominant negative FoxO3a with the deletion of the transactivation domain from the c-terminus and empty vector were also purchased from Vector BioLabs.

Techniques: Control, Infection, Expressing, Plasmid Preparation, Cell Culture

Journal: PloS one

Article Title: FoxO3a (Forkhead Box O3a) deficiency protects Idiopathic Pulmonary Fibrosis (IPF) fibroblasts from type I polymerized collagen matrix-induced apoptosis via caveolin-1 (cav-1) and Fas.

doi: 10.1371/journal.pone.0061017

Figure Lengend Snippet: Figure 2. FoxO3a regulates cav-1 mRNA and protein expression. A. Upper, shown is a representative Western blot analysis for cav-1 protein expression in control (n = 4) and IPF fibroblasts (n = 4) cultured on type I polymerized collagen matrix in serum free medium for 24 h. Lower, serum starved IPF (n = 8) and control fibroblasts (n = 6) were cultured under the same condition. Shown is cav-1/actin expression ratio of control and IPF fibroblasts on polymerized collagen. *p = 0.02 versus control fibroblasts. B. ChIP assay was carried out with primers as described in the Materials and Methods. Putative FoxO3a consensus binding sequences were underlined within C1 region. 1/25 volume (2 ml) of recovered DNA with primers flanking C1 region was used for PCR and 139 bp of ChIP-PCR product was amplified. ChIP-PCR without recovered DNA was carried out using C1 forward and reverse primers as a negative control (ND). ChIP-PCR without FoxO3a antibody using C1 forward and reverse primers was also carried out as an antibody control (NAB). ChIP-PCR from immunoprecipitate with normal rabbit IgG using C1 forward and reverse primers was performed as an IgG control (IgG). Image was obtained from a different gel. M: 1kb DNA ladder. C. Quantitative RT-PCR was carried out using primers as described in Materials and Methods. Shown is quantitative RT-PCR normalized to 18S rRNA in FoxO3a2/2 and FoxO3a+/+ cells. *p = 0.002. The assay was obtained from triplicate. D. Upper panel, FoxO3a 2/2 cells were infected with adenovirus expressing wild type FoxO3a (F3) or empty vector GFP, and cultured on polymerized collagen for 24 hrs. Cav-1 protein expression was measured by Western analysis. GAPDH was used as a loading control. Lower panel. FoxO3a 2/2 cells transfected with constructs expressing wild type FoxO3a (WT), or S253A mutant FoxO3a (S253A) or a FoxO3a construct with all Akt phosphorylation residues mutated with alanine (TM) were cultured on polymerized collagen for 24 hrs. Cav-1 protein levels were measured by Western blot analysis. Vec: empty vector. Images were obtained from same Western blot. doi:10.1371/journal.pone.0061017.g002

Article Snippet: Adenovirus expressing GFP tagged wild type FoxO3a, dominant negative FoxO3a with the deletion of the transactivation domain from the c-terminus and empty vector were also purchased from Vector BioLabs.

Techniques: Expressing, Western Blot, Control, Cell Culture, Binding Assay, Amplification, Negative Control, Quantitative RT-PCR, Infection, Plasmid Preparation, Transfection, Construct, Mutagenesis, Phospho-proteomics

Journal: PloS one

Article Title: FoxO3a (Forkhead Box O3a) deficiency protects Idiopathic Pulmonary Fibrosis (IPF) fibroblasts from type I polymerized collagen matrix-induced apoptosis via caveolin-1 (cav-1) and Fas.

doi: 10.1371/journal.pone.0061017

Figure Lengend Snippet: Figure 3. FoxO3a deficiency suppresses cav-1 expression in IPF fibroblast on collagen matrix. A. IPF and control fibroblasts infected with adenovirus expressing wild type FoxO3a (F3), dominant negative FoxO3a (DF) or empty vector (GFP) were cultured on polymerized collagen for 24 h in serum free medium. After total RNA was isolated, quantitative RT-PCR was carried out with cav-1 primers as described in the Materials and Methods. Shown is quantitative RT-PCR for cav-1 mRNA levels normalized to 18S rRNA in IPF and control fibroblasts. *p = 0.03 versus GFP in control fibroblasts, **p = 0.02 versus GFP in IPF fibroblasts. ***p = 0.016 versus GFP in IPF fibroblast. The assay was obtained from triplicate from 2 control and 1 IPF fibroblasts. B. IPF and control fibroblasts expressing wild type FoxO3a (F3), mutant FoxO3a (DF) or empty vector (GFP) were transfected with a luciferase construct containing consensus Forkhead binding sites (FHRE-Luc). Cells were attached to polymerized collagen in serum free medium for 24 h and luciferase activity was measured as described in the Materials and Methods. Non: cells without transfection of FHRE-Luc construct. The assay was obtained from triplicate. C. Left upper panel, shown is representative Western blot analysis (WB) of cav-1 protein expression in IPF fibroblasts expressing wild type (F3), dominant negative FoxO3a (DF) or GFP control and cultured on polymerized collagen for 24 h. Left middle panel, nuclear fraction was isolated from IPF fibroblasts cultured on collagen as described in the Materials and Methods and unphosphorylated FoxO3a protein level was measured. NC : nuclear fraction. Lamin A was used as a loading control. Left lower panel : cytoplasmic fraction was isolated from IPF fibroblasts cultured on collagen and phosphorylated FoxO3a (p-FoxO3a) was measured. CP : cytoplasmic fraction. GAPDH is shown as a loading control. Right panel, densitometric analysis of cav-1/GAPDH expression ratio. D. Left panel, control fibroblasts expressing wild type (F3), dominant negative FoxO3a (DF) or GFP control and cultured on polymerized collagen for 24 h and cav-1 protein expression was measured. GAPDH is shown as loading control. Right panel, densitometric analysis of cav-1/GAPDH expression ratio. All blots represent 3 independent assays. doi:10.1371/journal.pone.0061017.g003

Article Snippet: Adenovirus expressing GFP tagged wild type FoxO3a, dominant negative FoxO3a with the deletion of the transactivation domain from the c-terminus and empty vector were also purchased from Vector BioLabs.

Techniques: Expressing, Control, Infection, Dominant Negative Mutation, Plasmid Preparation, Cell Culture, Isolation, Quantitative RT-PCR, Mutagenesis, Transfection, Luciferase, Construct, Binding Assay, Activity Assay, Western Blot

Journal: PloS one

Article Title: FoxO3a (Forkhead Box O3a) deficiency protects Idiopathic Pulmonary Fibrosis (IPF) fibroblasts from type I polymerized collagen matrix-induced apoptosis via caveolin-1 (cav-1) and Fas.

doi: 10.1371/journal.pone.0061017

Figure Lengend Snippet: Figure 4. Aberrant function of the PTEN/Akt axis inactivates FoxO3a and suppresses cav-1 expression. A. Left panel. IPF and control fibroblasts infected with adenovirus expressing hyperactive Akt (HA), dominant negative Akt (DA) or empty vector GFP were cultured on polymerized collagen and cav-1 protein expression was examined by Western blot analysis. GAPDH is shown as a loading control. Right panel, cav-1/GAPDH expression ratio was quantified by densitometry. *p = 0.017, **p = 0.008 versus GFP control. B. Control fibroblasts infected with adenovirus co- expressing either wild type FoxO3a & hyperactive Akt or empty GFP & hyperactive Akt were cultured on polymerized collagen in serum free medium for 24 h, and Western analysis was carried out to measure cav-1, p-Akt, FoxO3a and GAPDH levels. Note that cav-1 level is increased when FoxO3a and hyperactive Akt were co-expressed. C. Left, IPF fibroblasts infected with adenovirus expressing wild type PTEN (WP) or empty vector GFP were cultured on polymerized collagen for 24 h and cav-1 protein expression was examined by Western blot analysis. GAPDH is shown as loading control. Right, cav-1/GAPDH levels were quantified by densitometry. D. Left, control fibroblasts infected with adenovirus expressing mutant PTEN (MP) or empty vector GFP were cultured on polymerized collagen for 24 h and cav-1 protein levels were examined by Western blot analysis. GAPDH is shown as loading control. Right, cav-1/GAPDH levels were quantified by densitometry. All blots represent at least 3 independent assays. doi:10.1371/journal.pone.0061017.g004

Article Snippet: Adenovirus expressing GFP tagged wild type FoxO3a, dominant negative FoxO3a with the deletion of the transactivation domain from the c-terminus and empty vector were also purchased from Vector BioLabs.

Techniques: Expressing, Control, Infection, Dominant Negative Mutation, Plasmid Preparation, Cell Culture, Western Blot, Mutagenesis

Journal: PloS one

Article Title: FoxO3a (Forkhead Box O3a) deficiency protects Idiopathic Pulmonary Fibrosis (IPF) fibroblasts from type I polymerized collagen matrix-induced apoptosis via caveolin-1 (cav-1) and Fas.

doi: 10.1371/journal.pone.0061017

Figure Lengend Snippet: Figure 5. Low FoxO3a and cav-1 function confers IPF fibroblasts with an apoptotic-resistant phenotype via Fas suppression. A & B. IPF and control fibroblasts over-expressing cav-1 or empty vector GFP were cultured on collagen for 48 h in serum free medium and cell morphology (A) and cell viability (B) were examined. Shown is 50X magnification of cell morphology. C. Serum starved IPF and control fibroblasts were cultured on polymerized collagen for 24 h in the absence of serum, and Fas/GAPDH protein expression level and caspase-3/7 activity were measured as described in the Materials and Methods (n = 4 lines each). *p = 0.02, **p = 0.015 versus control fibroblasts. D. Upper panel. IPF and control fibroblasts were cultured on collagen as a function of time, and Fas and GAPDH protein expression was measured. Lower panel. Shown is the Fas/GAPDH protein expression ratio in IPF and control fibroblasts cultured on polymerized collagen as a function of time (n = 2 lines each). E. 26105 control and IPF fibroblasts cultured on polymerized collagen in the absence of serum were ligated with 0–2 mg of human Fas activating antibody for 18 h. Viable cells were measured as described in the Materials and Methods. Shown are the % viable cells exposed to the various concentrations of Fas activating antibody compared with that of untreated cells. *p = 0.016 versus non treated cells. Results were obtained from triplicates. doi:10.1371/journal.pone.0061017.g005

Article Snippet: Adenovirus expressing GFP tagged wild type FoxO3a, dominant negative FoxO3a with the deletion of the transactivation domain from the c-terminus and empty vector were also purchased from Vector BioLabs.

Techniques: Control, Expressing, Plasmid Preparation, Cell Culture, Activity Assay

Journal: PloS one

Article Title: FoxO3a (Forkhead Box O3a) deficiency protects Idiopathic Pulmonary Fibrosis (IPF) fibroblasts from type I polymerized collagen matrix-induced apoptosis via caveolin-1 (cav-1) and Fas.

doi: 10.1371/journal.pone.0061017

Figure Lengend Snippet: Figure 6. Forced expression of FoxO3a increases Fas expression via cav-1 in IPF fibroblasts and promotes apoptosis on polymerized collagen matrix. A. IPF fibroblasts infected with adenovirus over-expressing FoxO3a (F3), cav-1 (C-1) or empty vector GFP were cultured on polymerized collagen for 48 h in serum free medium. Left panel, Fas, FoxO3a, cav-1 and GAPDH expression were measured by Western blot analysis. Three independent assays are represented. Right panel. Fas/GAPDH protein expression ratio was quantified. *p = 0.02 versus GFP.

Article Snippet: Adenovirus expressing GFP tagged wild type FoxO3a, dominant negative FoxO3a with the deletion of the transactivation domain from the c-terminus and empty vector were also purchased from Vector BioLabs.

Techniques: Expressing, Infection, Plasmid Preparation, Cell Culture, Western Blot

Journal: PloS one

Article Title: FoxO3a (Forkhead Box O3a) deficiency protects Idiopathic Pulmonary Fibrosis (IPF) fibroblasts from type I polymerized collagen matrix-induced apoptosis via caveolin-1 (cav-1) and Fas.

doi: 10.1371/journal.pone.0061017

Figure Lengend Snippet: Figure 7. Fibroblasts within the fibroblastic foci of IPF patient lung specimens express inactive FoxO3a and a-smooth muscle actin but not cav-1 and cleaved caspase-3. Upper upper panel, frozen serial sections of IPF patient lung specimens (F/F: fibroblastic foci) were immunostained with anti-a smooth muscle actin antibody (upper left), phosphorylated FoxO3a (inactive FoxO3a, upper middle), or anti-cav-1 (upper right), and IHC was carried out as described in the Materials and Methods (n = 3). Upper lower panel, shown are IHC images obtained from control tissue without primary antibodies as negative controls (N/C). Lower upper panel. IHC analysis of IPF (left) and control (middle) lung tissue was performed using anti-cleaved caspase-3 antibodies. N/C : IHC images obtained from control tissue without cleaved caspase-3 antibody as a negative control. Lower lower panel. Fas expression was measured from frozen sections of IPF (left) and control (middle) lung specimens. N/C : IHC images obtained from control tissue without Fas antibody as a negative control. Note that cav-1, cleaved caspase-3 and Fas expression were absent or very low in cells within the fibroblastic foci. doi:10.1371/journal.pone.0061017.g007

Article Snippet: Adenovirus expressing GFP tagged wild type FoxO3a, dominant negative FoxO3a with the deletion of the transactivation domain from the c-terminus and empty vector were also purchased from Vector BioLabs.

Techniques: Control, Negative Control, Expressing

Journal: PloS one

Article Title: FoxO3a (Forkhead Box O3a) deficiency protects Idiopathic Pulmonary Fibrosis (IPF) fibroblasts from type I polymerized collagen matrix-induced apoptosis via caveolin-1 (cav-1) and Fas.

doi: 10.1371/journal.pone.0061017

Figure Lengend Snippet: Figure 8. Schematic illustrating the mechanism by which FoxO3a deficiency confers IPF fibroblasts with resistance to polymerized collagen-mediated apoptosis. When control fibro- blasts are cultured on polymerized collagen, Akt activity is suppressed by elevated PTEN activity, resulting in an increase in FoxO3a activity. Active FoxO3a then transcriptionally increases cav-1 level and cells undergo apoptosis via high Fas expression and caspase-3/7 activity. In contrast, PTEN activity is low in response to IPF fibroblast attachment to collagen matrix, which activates Akt thereby suppressing FoxO3a function. Cav-1 transcription is low due to inactive FoxO3a, thereby suppressing Fas expression & caspase-3/7 activity, which subsequently protects IPF fibroblasts from polymerized collagen-induced apoptosis. doi:10.1371/journal.pone.0061017.g008

Article Snippet: Adenovirus expressing GFP tagged wild type FoxO3a, dominant negative FoxO3a with the deletion of the transactivation domain from the c-terminus and empty vector were also purchased from Vector BioLabs.

Techniques: Control, Cell Culture, Activity Assay, Expressing

Journal: Diabetes

Article Title: Glucose Induces Mouse β-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor

doi: 10.2337/db15-0529

Figure Lengend Snippet: IRS2 is required for glucose-induced β-cell proliferation in vivo and ex vivo. A–D: Mice with generalized deletion of IRS2 or littermate controls were infused intravenously with saline or glucose for 4 days. B and C: β-Cell proliferation was increased by hyperglycemia in WT and HT controls (n = 10–16) but not in IRS2-KO mice (n = 1, 4, and 7). D: β-Cell mass was reduced in diabetic IRS2-KO mice (n = 4–9, except for KO BG 116–138 for which n = 1). E and F: Glucose induced proliferation in WT- but not KO-dispersed mouse islet cells cultured for 72 h (n = 6–9). G and H: Acute knockdown of IRS2 by adenovirus-delivered shRNA targeting IRS2 (MOI 25) in mouse islet cell cultures diminished glucose-induced β-cell proliferation (n = 4). Arrows point to BrdU-positive β-cell nuclei. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. BG, blood glucose; ND, not determined; ns, not signficant.

Article Snippet: Adenovirus (Ad-shIRS2, Ad-Cre, Ad-CMV-GFP-Cyclin-D2, Ad-shCyclin-D2, and Ad-shIR; Vector Biolabs) was added the day after islet trypsinization; multiplicity of infection (MOI) is per figure legends.

Techniques: In Vivo, Ex Vivo, Saline, Cell Culture, Knockdown, shRNA

Journal: Diabetes

Article Title: Glucose Induces Mouse β-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor

doi: 10.2337/db15-0529

Figure Lengend Snippet: Glucose-induced proliferation in mouse islets does not require the insulin receptor (IR). A–L: IR blockers S961 (A–F; 100 nmol/L added 120 min before glucose or insulin [100 nmol/L] stimulation) or HNMPA (G–L; 10 μmol/L added 60 min before glucose stimulation) prevented activation of insulin signaling 2 min after glucose treatment (whole islets, n = 4; A and B and G and H) but did not prevent the glucose-induced increase in PCNA abundance after 72 h of glucose treatment (whole islets, n = 3–4; C and D and I and J) or the glucose-induced increase in the percent of β-cells incorporating BrdU (dispersed islets, n = 4–8; E and F and K and L). Adenovirus expressing an shRNA targeting the mouse IR (MOI 50) in dispersed islets reduced IR expression 72 h after glucose exposure (n = 5; M and N) but did not prevent the glucose-induced increase in PCNA abundance (n = 5; O and P) or β-cell BrdU incorporation (n = 4; Q and R). Arrows point to BrdU-positive β-cell nuclei. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. 15 mmol/L glucose control condition (marked by dotted line); #P < 0.05 vs. 5 mmol/L control. gluc, glucose; ns, not significant; p, phosphorylated; tot, total; veh, vehicle.

Article Snippet: Adenovirus (Ad-shIRS2, Ad-Cre, Ad-CMV-GFP-Cyclin-D2, Ad-shCyclin-D2, and Ad-shIR; Vector Biolabs) was added the day after islet trypsinization; multiplicity of infection (MOI) is per figure legends.

Techniques: Activation Assay, Expressing, shRNA, BrdU Incorporation Assay, Control

Journal: Diabetes

Article Title: Glucose Induces Mouse β-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor

doi: 10.2337/db15-0529

Figure Lengend Snippet: IRS2 and MTOR but not the insulin receptor are required for glucose induction of cyclin D2; cyclin D2 is required for glucose-induced β-cell proliferation. A–C: IRS2-KO islets contained less IRS2 (n = 1–4; A) and cyclin D2 (n = 4–6; B–C). D and E: IRS2-KO islets failed to induce cyclin D2 protein when cultured in high glucose (n = 5–14). F–K: Blocking insulin receptor action using S961 (whole islets, n = 4; F–G), HNMPA (whole islets, n = 3; H–I), or adenovirus with shRNA targeting the insulin receptor (MOI 50) (dispersed islets, n = 5; J–K) did not prevent glucose induction of cyclin D2 protein. L and M: Treating whole islets with insulin (100 nmol/L) did not induce cyclin D2 protein expression (n = 4). N and O: Blocking MTOR but not PI3K or ERK in whole islets prevented glucose induction of cyclin D2 protein (n = 5–8). P–T: Reducing cyclin D2 expression by adenovirus with shRNA targeting cyclin D2 in dispersed islets (MOI 5, n = 3) prevented glucose induction of cyclin D2 (P and Q), PCNA (P and R), and proliferation (S and T). Panels D–T are at 72 h. Arrows point to BrdU-positive β-cell nuclei. Dotted line in O marks the high glucose control. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. cyc, cyclin; gluc, glucose; ins, insulin; IR, insulin receptor; PD, MEK inhibitor PD98059; RAP, rapamycin; veh, vehicle; WM, wortmannin.

Article Snippet: Adenovirus (Ad-shIRS2, Ad-Cre, Ad-CMV-GFP-Cyclin-D2, Ad-shCyclin-D2, and Ad-shIR; Vector Biolabs) was added the day after islet trypsinization; multiplicity of infection (MOI) is per figure legends.

Techniques: Cell Culture, Blocking Assay, shRNA, Expressing, Control

Journal: Diabetes

Article Title: Glucose Induces Mouse β-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor

doi: 10.2337/db15-0529

Figure Lengend Snippet: Restoring cyclin D2 expression rescues the proliferation defect due to loss of IRS2 and rapamycin (RAPA) treatment. A: In dispersed IRS2-WT islets, cyclin D2 overexpression (MOI 5) in 5 mmol/L glucose increased β-cell proliferation to levels achieved in 15 mmol/L glucose, and cyclin D2 overexpression in 15 mmol/L glucose further increased proliferation (n = 3–4). In dispersed IRS2-KO islets, 15 mmol/L glucose did not significantly increase β-cell proliferation compared with 5 mmol/L, but overexpression of cyclin D2 markedly increased proliferation in 15 mmol/L glucose. B and C: Reduced dispersed β-cell proliferation caused by adenovirus knockdown of IRS2 (MOI 50, n = 3–4; B) or RAPA treatment (n = 4–5; C) was rescued by overexpression of cyclin D2 (MOI 5). BrdU incorporation results were confirmed by a second proliferation measure, immunoblot for PCNA: loss of PCNA by IRS2 knockdown (dispersed islets, MOI 50, n = 8; D–F) or RAPA treatment (whole islets, n = 3; G–I) was rescued by overexpression of cyclin D2 (MOI 5). Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. cyc, cyclin; gluc, glucose; ns, not significant; p, phosphorylated; tot, total.

Article Snippet: Adenovirus (Ad-shIRS2, Ad-Cre, Ad-CMV-GFP-Cyclin-D2, Ad-shCyclin-D2, and Ad-shIR; Vector Biolabs) was added the day after islet trypsinization; multiplicity of infection (MOI) is per figure legends.

Techniques: Expressing, Over Expression, Knockdown, BrdU Incorporation Assay, Western Blot

Journal: Cell metabolism

Article Title: Targeting Peripheral CB 1 Receptors Reduces Ethanol Intake via a Gut-Brain Axis

doi: 10.1016/j.cmet.2019.04.012

Figure Lengend Snippet: (A) Time course and rate of increase in oleate-driven respiratory activity in MGN3–1 cells. Cells were treated with 10 or 100 nM JD5037, 2.5 M of FCCP (positive control) or vehicle in the presence of the fluorescent extracellular O2 consumption reagent. Oxidative respiration was recorded every 90 sec and the slope of the initial linear increase was calculated. Points and bars are means ± SEM from n = 11–15 experiments, as indicated. *P < 0.05 compared to vehicle. (B) Verification of Cnr1 knockdown by rtPCR in cells used in panel C. Cellular uptake of the constructs was verified by fluorescent microscopy (20x magnification) and the degree of knockdown was determined by rt-PCR, *P < 0.05, n = 4. (C) Oleate-driven oxidative activity in MGN3–1 cells with shRNA-mediated knockdown of Cnr1 expression and their mock-transfected controls, n = 3–8 experiments, as indicated, *P < 0.05.

Article Snippet: Scramble shRNA with GFP Adenovirus (Ad-scramble-shRNA) , Vector Biolabs , 1122.

Techniques: Activity Assay, Positive Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Construct, Microscopy, shRNA, Expressing, Transfection

Journal: Nature Communications

Article Title: Sox17 is required for endothelial regeneration following inflammation-induced vascular injury

doi: 10.1038/s41467-019-10134-y

Figure Lengend Snippet: HIF-1α activates transcription of Sox17. a Western blot analysis in control HLMVECs and HLMVECs for which CRISPR/Cas9 was used to delete HIFs. ECs were treated with the HIF prolyl hydroxylase inhibitor DMOG to induce HIF expression. DMOG (1 mM) increased HIF-1α and HIF-2α protein expression in control ECs but not in ECs lacking HIF-1α or HIF-2α. Induction of HIF expression was coupled to Sox17 upregulation. n = 3. b Quantification of a showed that protein expression of HIF-1α and HIF-2α in ECs was significantly increased by DMOG treatment. Sox17 showed a 2.5-fold increase in Sox17 expression control DMSO treated ECs. The increase in Sox17 was significantly reduced in HIF-1α-deleted ECs but preserved in HIF-2α-deleted ECs, indicating the importance of HIF-1α in mediating Sox17 expression. n = 3. c Representation of the SOX17 promoter region with 3 HREs indicated by circled numbers, and their respective sequences are displayed. d HLMVECs were exposed to normoxia or 1% O2 (hypoxia) for 8 h. Ch-IP assay followed by qPCR was performed to amplify the HRE regions in the SOX17 promoter. Studies were performed in ECs exposed to either normoxia or hypoxia. n = 3. e 293T cells were transfected with a HIF-1α overexpression plasmid containing SOX17 luciferase reporter constructs. Luciferase values were normalized to Renilla luciferase control reporter values. A schematic representation of corresponding deletion constructs is presented in the right panel. n = 3 and duplicates per sample. Results show that hypoxia activation of SOX17 HRE3 was required for Sox17 expression. ** P < 0.01 and *** P < 0.001. Data are shown as mean ± SEM. Analysis was performed using two-way ANOVA with Bonferroni post-tests for ( b , d , e )

Article Snippet: At 8 h later, the EGFP-tagged Cas9 adenovirus (Ad-GFP-Cas9, Vector Biolabs #1901) was also added at a MOI of 10 to transduce HLMVECs.

Techniques: Western Blot, Control, CRISPR, Expressing, Transfection, Over Expression, Plasmid Preparation, Luciferase, Construct, Activation Assay