Journal: Nature Communications

Article Title: Calcium transients regulate the apical emergence of basally located progenitors during Xenopus skin development

doi: 10.1038/s41467-025-61610-7

Figure Lengend Snippet: A Representative MIP image of the apical surface area of a MCC expressing calmodulin-GFP. Calmodulin displays colocalization with the basal bodies and the apical actin network. B Schematic depicting the localization of calmodulin in a MCC in relation to the basal bodies and the apical actin network. C Representative images of the skin epithelium from stage 24 control and calmodulin inhibitor-treated embryos. MCC fail to fully integrate into the superficial skin epithelium when calmodulin activity is blocked, as evident by their small apical surface area. D Quantification of the apical surface area of MCCs from control ( n = 100 MCCs) and calmodulin inhibitor-treated embryos ( n = 100 MCCs), 5 different embryos. Two-sided unpaired student’s t test; **** p < 0.0001; mean ± SEM. E Representative images of the skin neuroepithelium from embryos expressing wild-type calmodulin or Ca 2+ binding deficient calmodulin mutant (CALM1234). Expression of CALM1234 results in defective MCC apical emergence (green arrows) while expression of WT calmodulin does not affect MCC apical emergence (white arrows). F Quantification of MCC apical emergence upon expression of WT and Ca 2+ binding deficient mutant calmodulin. Two-sided χ 2 test; ** * * p < 0.0001. G Quantification of the apical surface area of MCCs expressing WT and Ca 2+ binding deficient mutant calmodulin. N = 80 control MCCs, 46 MCCs expressing WT calmodulin and 20 MCCs expressing mutant calmodulin from 4 embryos injected with WT calmodulin DNA and 4 embryos injected with mutant CALM1234 DNA. Two-sided unpaired student’s t test; **** p < 0.0001; mean ± SEM. Scale bars: A : 2 μm, D , F : 20 μm. Source data are provided as a Source data file.

Article Snippet: For microinjection of DNA constructs, atub:UtrGFP, atub:GECO-RED, PACR (Addgene #55774), atub:PACR, CALM-GFP (Addgene #47602), CALMWT (Addgene #111499), CALM1234 (Addgene #111518).

Techniques: Expressing, Control, Activity Assay, Binding Assay, Mutagenesis, Injection

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet:

Article Snippet: pCaM12(D20A/D56A)/pIRES2-eGFP , Addgene , Cat#111512.

Techniques: Control, Protease Inhibitor, Mutagenesis, DNA Purification, Recombinant, Software

Journal: Cellular and Molecular Life Sciences

Article Title: Structure and function of the N-terminal extension of the formin INF2

doi: 10.1007/s00018-022-04581-y

Figure Lengend Snippet:

Article Snippet: pCaM34(D93A/D129A)/pIRES2-eGFP , Addgene , Cat#111517.

Techniques: Control, Protease Inhibitor, Mutagenesis, DNA Purification, Recombinant, Software

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: RvD1 prevents LPS-induced SHP2 oxidation and its inactivation in the protection of AJ integrity. A. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in presence and absence of RvD1 (200 ng/ml) for 30 min and PTP activity was measured using PTP-specific phosphopeptide as a substrate. B & C. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in presence and absence of RvD1 (200 ng/ml) for the indicated time periods, cell extracts were prepared and equal amounts of protein from control and each treatment were immunoprecipitated with VE-cadherin or SHP2 antibodies and the immunocomplexes were analyzed by IB for the indicated proteins. D. All the conditions were same as in panel B except that after immunoprecipitation with SHP2 antibodies the immunocomplexes were assayed for SHP2 activity as described in panel A. E. Quiescent HUVECs that were treated with and without LPS (500 ng/ml) in the presence and absence of Allopurinol (50 μM) for 30 min were analyzed for SHP2 activity as described in panel D. F. All the conditions were same as in panel B except the cell extracts were immunoprecipitated with SHP2 antibodies and the immunocomplexes were immunoblotted for Cys sulphonate to measure SHP2 Cys oxidation and the blot was reprobed for total SHP2 levels. G. All the conditions were the same as in panel E except that the cell extracts were analyzed for SHP2 Cys oxidation as described in panel F and the blot was reprobed for total SHP2 levels. H. HUVECs were transiently transfected with empty vector (EV) or Myc-tagged recombinant SHP2 expression vector (WT and C459S mutant), grown to confluence, quiesced, treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min and cell extracts were prepared. An equal amount of protein from control and each treatment was immunoprecipitated with Cysteine sulphonate antibody and the immunocomplexes were analyzed by IB for Myc to show SHP2 Cysteine oxidation. An equal amount of protein from control and each treatment was also analyzed by WB for Myc to show the overexpression of SHP2. I. HUVECs that were transfected with WT or mutant SHP2 expression vector and quiesced were treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min and analyzed for SHP2 activity as described in panel D.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Activity Assay, Phospho-proteomics, Control, Immunoprecipitation, Transfection, Plasmid Preparation, Recombinant, Expressing, Mutagenesis, Over Expression

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: Pharmacological inhibition of SHP2 blunts the capacity of RvD1 in the attenuation of LPS-induced Frk activation, α-catenin and VE-cadherin Tyr phosphorylation and AJ disruption. A. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) alone or in combination with or without PHPS1 (10 μM), a potent inhibitor of SHP2, for 30 min, cell extracts were prepared and an equal amount of protein from control and each treatment was immunoprecipitated with pTyr or VE-cadherin antibodies and the immunocomplexes were analyzed by IB for the indicated proteins. The same cell extracts were also analyzed for the indicated protein total levels. B. All the conditions were the same as in panel A except that the quiescent HUVEC monolayer after the treatments was stained double immunofluorescently for α-catenin and VE-cadherin as described in Figure legend 2E. C & D. Quiescent HUVEC monolayer was treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) alone or in combination with or without PHPS1 (10 μM) for 2 h or the indicated time periods and subjected to dextran flux (C) or TER (D) assays, respectively. The bar graphs represent Mean±SD values of three experiments. *, p<0.05 vs control; # , p<0.05 vs LPS.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Disruption, Control, Immunoprecipitation, Staining

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: Both ALX/FPR2 and GPR32 mediate the protective effects of RvD1 on LPS-induced endothelial AJ disruption and its barrier dysfunction. A. Cell extracts of control and various time periods of LPS (500 ng/ml)-treated HUVECs were analyzed by WB for ALX/FPR2 and GPR32 levels using their specific antibodies. B-D. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) in combination with and without Boc2 (3 μM), ALX/FPR2 inhibitor, for 30 min and XO activity (B), ROS production (C) and SHP2 activity (D) were measured. E. All the conditions were the same as in panel B except that cell extracts were prepared, and equal amounts of protein from control and each treatment were immunoprecipitated with pTyr or VE-Cadherin antibodies and the immunocomplexes were analyzed by IB for the indicated proteins using their specific antibodies. The same cell extracts were also analyzed by WB for the total levels of the indicated proteins. F & G. Quiescent HUVEC monolayer was treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) alone or in combination with and without Boc2 (3 μM) for 2 h or the indicated time periods and subjected to dextran flux (F) and TER (G) assays, respectively. H-J. Quiescent HUVECs were incubated with either control IgG or GPR32 IgG (10 μg/ml) alone or in combination with and without Boc2 (3 μM) for 30 min followed by treatment with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min and XO activity (H), ROS production (I) and SHP2 activity (J) were measured. K. All the conditions were the same as in panel H except that cell extracts were prepared and equal amounts of protein from control and each treatment were immunoprecipitated with pTyr or VE-cadherin antibodies and the immunocomplexes were analyzed by IB for the indicated proteins using their specific antibodies. The same cell extracts were also analyzed by WB for the total levels of the indicated proteins. L. The quiescent HUVEC monolayer that was incubated with either control IgG, GPR32 IgG (10 μg/ml), Boc2 (3 μM) alone or in combination for 30 min followed by treatment with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min was stained double immunofluorescently for α-catenin and VE-cadherin as described in Figure legend 2E. M & N. Quiescent HUVECs monolayer was incubated with either control IgG or GPR32 IgG (10 μg/ml) alone or in combination with and without Boc2 (3 μM) for 30 min followed by treatment with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 2 h or the indicated time periods and subjected to dextran flux (M) and TER (N) assays, respectively. The bar graphs represent Mean±SD values of three experiments. *, p<0.05 vs control or control IgG; # , p<0.05 vs LPS or con IgG+LPS.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Disruption, Control, Activity Assay, Immunoprecipitation, Incubation, Staining

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: RvD1 attenuates LPS-induced aortic endothelial AJ disruption and hyper-permeability via blocking XO activity and SHP2 inactivation. A. C57BL/6 mice which were kept on chow diet were administered intraperitoneally with RvD1 (10 μg/kg body weight) every 2 days for 3 times before injecting LPS (5 mg/kg body weight) and 24 h later the aortas were isolated, tissue extracts were prepared and an equal amount of protein from each condition was analyzed for XO activity as described in Figure legend 4B. B. All the conditions were the same as in panel A except that tissue extracts containing an equal amount of protein from each condition were immunoprecipitated with Cys sulphonate antibodies and the immunocomplexes were analyzed by IB for SHP2. The same tissue extracts were analyzed by WB for total SHP2 levels. C. All the conditions were the same as in panel A except that tissue extracts were analyzed for SHP2 activity as described in Figure legend 5D. D & E. All the conditions were the same as in panel A except that tissue extracts were immunoprecipitated with pTyr (D) or VE-cadherin (E) antibodies and the immunocomplexes were analyzed by IB for the indicated proteins using their specific antibodies. The same tissue extracts were analyzed by WB for the indicated protein total levels. F. All the conditions were same as in panel A except that after isolation the aortas were opened longitudinally, fixed, permeabilized, blocked and co-immunostained for α-catenin and VE-cadherin as described in Figure legend 2E. G. All the conditions were the same as in panel A except that mice were anesthetized and 0.1 ml of 1% Evans Blue (EB) dye was injected into the tail vein. After 20 min, the blood vessels were perfused with PBS through the left ventriculum and the aortas were isolated and photographed. After taking the pictures, the aortas were minced, incubated in formaldehyde solution at 55 °C for 24 h, centrifuged and the optical density of the supernatant was measured at 610 nm in SpectraMax 190 spectrophotometer (Molecular Devices). The aortic endothelial barrier permeability was expressed as ng of EB dye extravasated per mg aorta. The bar graphs represent Mean±SD values of three experiments with 2 animals/group or 5 animals minimum. *, p<0.05 vs control; # , p<0.05 vs LPS.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Disruption, Permeability, Blocking Assay, Activity Assay, Isolation, Immunoprecipitation, Injection, Incubation, Spectrophotometry, Control