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: Journal of Cellular and Molecular Medicine

Article Title: Wild‐type p53 enhances endothelial barrier function by mediating RAC 1 signalling and RhoA inhibition

doi: 10.1111/jcmm.13460

Figure Lengend Snippet: ( A ) Western blot analysis of p53 levels in mouse microvascular endothelial cells ( MMVEC ) isolated from lungs of wild‐type ( WT ) and super p53 mice. Cells were treated in vitro with LPS or vehicle and pre‐treated with 17 AAG or vehicle (10% DMSO ) for 16 h. Blot shown is representative of 3 experiments. Signal intensity was analysed by densitometry. Protein levels were normalized to β‐actin. * P < 0.05 versus vehicle, **P < 0.01 versus vehicle. Means ± S.E.M. ( B ) Western blot analysis of p53 levels in MMVEC derived from lungs of WT and super p53 mice 24 hrs after treatment with LPS or vehicle and pre‐treated for 16 h with 17 AAG or vehicle (10% DMSO ). Blot shown is representative of 3 experiments per group. Signal intensity was analysed by densitometry. Protein levels were normalized to β‐actin. * P < 0.05 versus vehicle, ** P < 0.01 versus vehicle, ***** P < 0.0001 versus vehicle. Means ± S.E.M. ( C ) Western blot analysis of MDM 2 levels in MMVEC isolated from lungs of WT and super p53 mice. The endothelial cells were treated in vitro with LPS or vehicle and pre‐treated for 16 hrs with 17 AAG or vehicle (10% DMSO ). Blot shown is representative of 3 experiments. Signal intensity was analysed by densitometry. Protein levels were normalized to β‐actin. *P < 0.05 versus vehicle, ** P < 0.01 versus vehicle. Means ± S.E.M. ( D ) LPS was added to the media of MMVEC derived from the lungs of WT or super p53 mice. A gradual increase in endothelial permeability (reduced TEER ) was observed in both LPS ‐treated groups. However, WT cells were much more susceptible to LPS than those derived from super p53 mice. n = 4 per group. Means ± S.E. ( E ) Equal numbers of MMVEC derived from WT or super p53 mice were seeded on ECIS golden plated arrays and were allowed to form confluent monolayers. Cells derived from super p53 mice demonstrated an increased potential to reach confluence compared to WT cells. n = 4 per group. Means ± S.E.

Article Snippet: Ad‐p53‐GFP (1260) and ad‐GFP (1060) were obtained from Vector Biolabs (Malvern, PA USA).

Techniques: Western Blot, Isolation, In Vitro, Derivative Assay, Permeability

Journal: Journal of Cellular and Molecular Medicine

Article Title: Wild‐type p53 enhances endothelial barrier function by mediating RAC 1 signalling and RhoA inhibition

doi: 10.1111/jcmm.13460

Figure Lengend Snippet: ( A ) Western blot analysis of active Rac1, P53, Rac1 and β‐actin after 8 hrs of treatment with either vehicle ( DMSO ; VEH ) or 1 μΜ 17ΑΑG, or 1 μΜ Α UY 922 of human lung microvascular endothelial cells ( HLMVEC ). Blot is representative of 3 independent experiments. Signal intensity of active Rac1, P53, Rac1 was analysed by densitometry. Protein levels were normalized to Rac1 or β‐actin, as indicated. * P < 0.05 versus vehicle, *****P < 0.0001 versus vehicle. Means ± S.E. ( B ) HLMVEC were transfected with control (irrelevant) si RNA (si CTR ) or PAK si RNA (si PAK ) at t = 0. PAK si RNA ‐treated cells exhibited reduced TEER values; n = 4 per group. Means ± S.E. In additional experiments, similarly treated HLMVEC were exposed to vehicle (0.1% DMS 0) or AUY 922. Bars indicate normalized TEER values 50 hrs after transfection. n = 4 per group. ***P < 0.001 versus vehicle‐treated cells; Means ± S.E. (lower left panel). Western blot analysis of PAK expression in HLMVEC 24 h an 48 h after si CTR or si PAK transfection. Blot shown is representative of 3 independent experiments. Signal intensity of PAK was analysed by densitometry. Protein levels were normalized to β‐actin. ***P < 0.001 versus control si RNA . Means ± S.E. (lower right panel). ( C ) HLMVEC were transfected with si CTR or si PAK and were consequently exposed to vehicle or AUY 922 for 16 hrs. Western blot analysis demonstrates p53 expression levels of transfected cells after 8 hrs of treatment with either vehicle or 17ΑΑG. Blot shown is representative of 3 independent experiments. Signal intensity of p53 was analysed by densitometry. Protein levels were normalized to β‐actin. *P < 0.05 versus vehicle. Means ± S.E. (left panel).

Article Snippet: Ad‐p53‐GFP (1260) and ad‐GFP (1060) were obtained from Vector Biolabs (Malvern, PA USA).

Techniques: Western Blot, Transfection, Control, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Wild‐type p53 enhances endothelial barrier function by mediating RAC 1 signalling and RhoA inhibition

doi: 10.1111/jcmm.13460

Figure Lengend Snippet: ( A ) Western blot analysis of p53 and β‐actin expression in HLMVEC after 48‐h treatment with ad‐ GFP , si RNA for p53 (siP53), or ad‐p53‐ GFP . Blot shown is representative of 3 independent experiments. Signal intensity of p53 was analysed by densitometry. Protein levels were normalized to β‐actin. *P < 0.05 versus ad‐ GFP , ***P < 0.001 versus ad‐ GFP . Means ± S.E. (left panel). Western blot analysis of pLIMK and LIMK levels in HLMVEC after 48‐h treatment with ad‐ GFP , siP53 or ad‐p53‐ GFP . Blot shown is representative of 3 independent experiments. Signal intensity of pLIMK was analysed by densitometry. Protein levels were normalized to LIMK . ***P < 0.001 versus ad‐ GFP . Means ± S.E. (right panel). ( B ) Western blot analysis of pLIMK , LIMK and β‐actin levels in HLMVEC after 48‐h treatment with vehicle (veh) (0.1% DMSO ) or Nutlin. Blot shown is representative of 3 independent experiments. Signal intensity of pLIMK was analysed by densitometry. Protein levels were normalized to LIMK . *P < 0.05 versus VEH . Means ± S.E. ( C ) Western blot analysis of pLIMK , LIMK and β‐actin levels in HLMVEC 1 hr after treatment with LPS or vehicle and pre‐treated for 16 h with 17 AAG or vehicle. Blot shown is representative of 3 independent experiments. Signal intensity of pLIMK was analysed by densitometry. Protein levels were normalized to LIMK . *P < 0.05 versus LPS . Means ± S.E.

Article Snippet: Ad‐p53‐GFP (1260) and ad‐GFP (1060) were obtained from Vector Biolabs (Malvern, PA USA).

Techniques: Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Wild‐type p53 enhances endothelial barrier function by mediating RAC 1 signalling and RhoA inhibition

doi: 10.1111/jcmm.13460

Figure Lengend Snippet: ( A ) Western blot analysis of pC ofilin, cofilin and β‐actin levels in HLMVEC treated with LPS or vehicle and pre‐treated with 17 AAG or vehicle (0.01% DMSO ). Blot shown is representative of 3 independent experiments. Signal intensity of pC ofilin was analysed by densitometry. Protein levels were normalized to cofilin. *P < 0.05 versus LPS , **P < 0.01 versus LPS , # P < 0.05 versus vehicle. Means ± S.E. ( B ) Western blot analysis of pcofilin, cofilin, p53 and β‐actin levels in HLMVEC treated with vehicle or Nutlin. Blot shown is representative of 3 independent experiments. Signal intensity of pC ofilin and p53 was analysed by densitometry. Protein levels were normalized to cofilin or β‐actin. *P < 0.05 versus vehicle, *** P < 0.001 versus vehicle. Means ± S.E. ( C ) Western blot analysis of pcofilin, cofilin and β‐actin levels in HLMVEC transfected with either irrelevant si RNA (si CTR ) or si RNA for p53 (siP53) and consequently treated with vehicle (0.01% DMSO ) or 17 AAG . The blot shown is representative of 3 independent experiments. Signal intensity of pC ofilin was analysed by densitometry. Protein levels were normalized to cofilin. *** P < 0.001 versus vehicle, # P < 0.05 versus si CTR . Means ± S.E. ( D ) Western blot analysis of pcofilin and cofilin in HLMVEC transfected with irrelevant si CTR , siP53 or ad‐p53‐ GFP . Blot shown is representative of 3 independent experiments. Signal intensity of pC ofilin was analysed by densitometry. Protein levels were normalized to cofilin. *** P < 0.001 versus si CTR . Means ± S.E.

Article Snippet: Ad‐p53‐GFP (1260) and ad‐GFP (1060) were obtained from Vector Biolabs (Malvern, PA USA).

Techniques: Western Blot, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Wild‐type p53 enhances endothelial barrier function by mediating RAC 1 signalling and RhoA inhibition

doi: 10.1111/jcmm.13460

Figure Lengend Snippet: ( A ) Phospho‐cofilin expression levels in MMVEC isolated from lungs of wild‐type ( WT ) and super p53 mice. Cells were treated in vitro with LPS or vehicle and pre‐treated with 17 AAG or vehicle (0.01% DMSO ) for 16 h. Blot shown is representative of 3 experiments. Signal intensity was analysed by densitometry. Protein levels were normalized to cofilin. ****P < 0.001 versus vehicle. Means ± S.E.M. ( B ) Western blot analysis of p cofilin, cofilin, p53, P190 RHOGAP and β‐actin levels in lungs retrieved from WT and super p53 mice. Signal intensity was analysed by densitometry. Protein levels were normalized to cofilin or β‐actin. **P < 0.01 vs controls (wild type). Means ± S.E.

Article Snippet: Ad‐p53‐GFP (1260) and ad‐GFP (1060) were obtained from Vector Biolabs (Malvern, PA USA).

Techniques: Expressing, Isolation, In Vitro, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Wild‐type p53 enhances endothelial barrier function by mediating RAC 1 signalling and RhoA inhibition

doi: 10.1111/jcmm.13460

Figure Lengend Snippet: ( A ) Western blot analysis of PDXP , p53 and β‐actin protein expression in HLMVEC transfected with irrelevant si RNA (si CTR ) or si RNA which targets p53 gene expression (siP53). Cells were then treated with vehicle (0.01% DMSO ) or 17 AAG . Blot shown is representative of 3 experiments. Signal intensity was analysed by densitometry. Protein levels of P53 and PDXP were normalized to β‐actin. *P < 0.01 vs vehicle, **P < 0.01 vs vehicle. Means ± S.E. ( B ) Cells were transfected with si CTR ) or si PDXP and were then exposed to LPS . A gradual increase in endothelial permeability (reduced TEER ) was observed in the LPS ‐treated cells which were transfected with si CTR . Cells exposed to the si PDXP were not sensitive to LPS . n = 4 per group. Western blot analysis of PDXP and β‐actin protein expression in HLMVEC transfected with si CTR or si PDXP . Blot shown is representative of 3 experiments. Signal intensity of PDXP was analysed by densitometry. Protein levels were normalized to β‐actin. **P < 0.01 vs si CTR . Means ± S.E. ( C ) Western blot analysis of PDXP and β‐actin protein expression in HLMVEC treated with LPS or vehicle (left), or pre‐treated with Nutlin prior to LPS or vehicle treatment (right). Blot shown is representative of 3 experiments. Signal intensity was analysed by densitometry. Protein levels of PDXP were normalized to β‐actin. *P < 0.05 vs vehicle. Means ± S.E.

Article Snippet: Ad‐p53‐GFP (1260) and ad‐GFP (1060) were obtained from Vector Biolabs (Malvern, PA USA).

Techniques: Western Blot, Expressing, Transfection, Gene Expression, Permeability

Journal: Journal of Cellular and Molecular Medicine

Article Title: Wild‐type p53 enhances endothelial barrier function by mediating RAC 1 signalling and RhoA inhibition

doi: 10.1111/jcmm.13460

Figure Lengend Snippet: ( A ) Cells were transfected with irrelevant si RNA (si CTR ) or si RNA targeting the p190 RHOGAP gene expression and were then exposed to LPS . A gradual increase in endothelial permeability (reduced TEER ) was observed in both LPS ‐treated groups. However, cells exposed to si P190 RHOGAP were more susceptible to LPS than those transfected with si CTR , n = 4 per group. Western blot analysis of P190Rho GAP and β‐actin protein expression in HLMVEC transfected with si CTR or si P190 RHOGAP . Blot shown is representative of 3 experiments. Signal intensity of P190 RHOGAP was analysed by densitometry. Protein levels were normalized to β‐actin. ### P < 0.001 vs si CTR . Means ± S.E. (right panel). ( B ) Western blot analysis of P190 RHOGAP and β‐actin in HLMVEC treated with LPS or vehicle and pre‐treated with AUY 922 ( AUY ) or vehicle (0.1% DMSO ). Blot shown is representative of 3 independent experiments. Signal intensity of P190 RHOGAP was analysed by densitometry. Protein levels were normalized to β‐actin. *P < 0.05 versus LPS , ***P < 0.001 versus LPS . Means ± S.E. ( C ) Phospho‐ MLC 2 expression levels in MMVEC isolated from the lungs of wild‐type and super p53 mice. Cells were treated in vitro with LPS or vehicle and pre‐treated with 17 AAG or vehicle (0.01% DMSO ) for 16 hrs. Blot shown is representative of 3 experiments. Signal intensity was analysed by densitometry. Protein levels were normalized to cofilin. * P < 0.05 versus vehicle. Means ± S.E. ( D ) Schematic presentation of the proposed mechanism by which p53 regulates pulmonary barrier function by mediating Rac1 protective signalling and inhibiting barrier disruptive RhoA activation.

Article Snippet: Ad‐p53‐GFP (1260) and ad‐GFP (1060) were obtained from Vector Biolabs (Malvern, PA USA).

Techniques: Transfection, Gene Expression, Permeability, Western Blot, Expressing, Isolation, In Vitro, Activation Assay