Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness

doi: 10.1152/ajplung.00405.2009

Figure Lengend Snippet: Analysis of CD44 and caveolin-1 regulation of high-molecular-weight hyaluronan (HMW-HA) binding to human pulmonary endothelial cells (EC). A: EC were grown to confluency and serum-starved for 1 h, and Triton X-100-soluble, Triton X-100-insoluble, and OptiPrep fractions were prepared. The 20% OptiPrep fraction represents the caveolin-enriched microdomain (CEM) fraction. Fractions were run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-caveolin-1 (a), anti-fibrillarin (b), anti-cyclooxygenase (COX) IV (c), anti-lysosomal-associated membrane glycoprotein 2 precursor (LAMP2b, d), anti-Golgi reassembly stacking protein 65 (GRASP65, e), or anti-VEGF receptor (anti-VEGFR, f). B: EC were grown to confluency, serum-starved for 1 h, and either left untreated (control) or treated with 100 nM HMW-HA (5 min) or the CEM-abolishing cholesterol-depletion agent methyl-β-cyclodextrin (MβCD, 5 mM) for 1 h prior to 100 nM HMW-HA treatment (5 min). Cellular material was solublized in 4°C 1% Triton X-100, and soluble and insoluble fractions were obtained. Triton X-100-insoluble fraction was overlaid with 60%, 40%, 30%, and 20% OptiPrep and centrifuged at 35,000 rpm in an SW60 rotor for 12 h at 4°C. Triton X-100-soluble material and OptiPrep fractions were run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-caveolin-1 (a), anti-CD44s (IM-7, standard domain, b), or anti-VEGF receptor 2 (anti-VEGFR2, c) antibody. The 20% OptiPrep fraction is the CEM fraction. Experiments were performed in triplicate, with highly reproducible findings, and representative data are shown. C: immunoblot analysis of small interfering RNA (siRNA)-treated or untreated human EC. Cellular lysates from untransfected (control, no siRNA), scramble siRNA (siRNA that does not target any known human mRNA), caveolin-1 siRNA, or CD44 siRNA transfection were analyzed using immunoblotting with anti-caveolin-1 (a), anti-CD44 (IM-7, b), or anti-actin (c) antibody. Experiments were performed in triplicate, each with similar results, and representative data are shown. D: quantitation of fluorescein-conjugated HMW-HA binding to scramble siRNA-, annexin A11 siRNA-, CD44 siRNA-, or caveolin-1 siRNA-treated EC. Fluorescein-conjugated HMW-HA (100 nM) was added for 15 min to EC in serum-free medium, cells were washed 3 times in serum-free medium, and fluorescence intensity was quantified. Cells were counted utilizing a hemocytometer.

Article Snippet: The 20% OptiPrep fraction represents the caveolin-enriched microdomain (CEM) fraction.

Techniques: High Molecular Weight, Binding Assay, SDS Page, Membrane, Control, Western Blot, Small Interfering RNA, Transfection, Quantitation Assay, Fluorescence

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness

doi: 10.1152/ajplung.00405.2009

Figure Lengend Snippet: Role of HMW-HA-induced recruitment of annexin A2 and protein S100-A10 to human EC CEM. A: HMW-HA induces caveolin-1 redistribution to EC-EC junctions. Human EC were grown to confluency, serum-starved for 1 h, and either left untreated (control) or treated with 100 nM HMW-HA (15 min), fixed in 4% paraformaldehyde, and stained with anti-caveolin-1 antibody, anti-vascular endothelial (VE)-cadherin antibody, or 4′,6-diamidino-2-phenylindole (DAPI). Overlay is a merged image of caveolin-1, VE-cadherin, and DAPI fluorescence, with yellow color indicating colocalization of caveolin-1 and VE-cadherin. B and C: EC were grown to confluency, serum-starved for 1 h, and either left untreated (control) or treated with 100 nM HMW-HA (5 min) and CEM fractions (20% OptiPrep layer). B: CEM fractions were run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-annexin A2 (a), anti-protein S100-A10 (b), anti-filamin A (c), anti-filamin B (d), or anti-caveolin-1 (e) antibody. Experiments were performed in triplicate, with highly reproducible findings, and representative data are shown. C: CEM fractions were solublized in immunoprecipitation (Ippt) buffer and immunoprecipitated with anti-annexin A2 antibody. Resulting immunobeads were run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-phosphotyrosine (a) or anti-annexin A2 (b) antibody. Experiments were performed in triplicate, with highly reproducible findings, and representative data are shown. D: EC were treated with no siRNA (control), scramble siRNA, annexin A2 siRNA, or protein S100-A10 siRNA for 48 h. EC lysates were obtained and run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-annexin A2 (a), anti-protein S100-A10 (b), or anti-actin (c) antibody. Experiments were performed in triplicate, with highly reproducible findings, and representative data are shown. E: percent inhibition of maximal HMW-HA-induced TER response in human EC with scramble, annexin A11, annexin A2, protein S100-A10, or annexin A2 + protein S100-A10 siRNA treatment. Silencing both annexin II and protein S100-A10 is required for maximal inhibition of HMW-HA-induced TER in EC.

Article Snippet: The 20% OptiPrep fraction represents the caveolin-enriched microdomain (CEM) fraction.

Techniques: Control, Staining, Fluorescence, SDS Page, Immunoprecipitation, Inhibition

Journal: Cancer discovery

Article Title: Pharmacological suppression of B7-H4 glycosylation restores antitumor immunity in immune-cold breast cancers

doi: 10.1158/2159-8290.CD-20-0402

Figure Lengend Snippet: (A) SKBR3 cells were treated with 10 μM doxorubicin and/or 10 μM NGI-1 for 24 h. Membrane CALR, HSP70 and HSP90 were measured by flow cytometry. (B) MDA-MB-468-vector and MDA-MB-468-B7-H4 knockout cells were established and treated with 5 μM doxorubicin for 24 h. Immunofluorescence staining of the immunogenic cell death markers CALR on the cell surface was performed. Mean fluorescence index of CALR was quantified by ImageJ. Representative images are shown. (C-D) SKBR3, MDA-MB-468, MDA-MB-468-vector, MDA-MB-468-B7-H4 knockout cells were treated with 1 or 10 μM doxorubicin and/or 10 μM NGI-1 for 24 h. p-eIF2a and actin were examined by immunoblotting. Scale bar, 100 μm. (E) Representative paired immunohistochemistry staining of B7-H4 and phospho eIF2α (Ser51) in tissue array BC081120. Statistical analysis of immunohistochemical staining indicates B7-H4 expression is negatively correlated with p-eIF2α expression in breast cancer (r = −0.249, p =8.71x10−3). (F) MDA-MB-468-Flag-hB7-H4 were treated in the presence or absence of doxorubicin (10 μM) and/or NGI-1 (10 μM). Then Flag-hB7-H4 was immunoprecipitated followed by immunoblot. The indicated proteins were examined. (G) Schematic diagram of the procedure of OptiPrep density gradient assay with 24 collected fractions from low to high density is shown. MDA-MB-468-vector and MDA-MB-468-hB7-H4 knockout cells were treated with 10 μM doxorubicin for 24 hr followed by OptiPrep density gradient assay. HSP90, CALR, eIF2α and p-eIF2α in fraction 1 to 13 were examined by immunoblotting. (H) eIF2a was immunoprecipitated in fraction 13 in both MDA-MB-468-vector and MDA-MB-468-B7-H4 knockout cells followed by immunoblotting. PERK, eIF2α and p-eIF2α were examined.

Article Snippet: OptiPrep density gradient protein fractionation assay To prepare the iodixanol gradient, a 50% (w/v) and 5% (w/v) solution of iodixanol were made by diluting the stock solution (60% (w/v) aqueous iodixanol from StemCell Technologies with 0.25 M sucrose, 6 mM EDTA, 60 mM Tris-HCl pH7.4.

Techniques: Flow Cytometry, Plasmid Preparation, Knock-Out, Immunofluorescence, Staining, Fluorescence, Western Blot, Immunohistochemistry, Immunohistochemical staining, Expressing, Immunoprecipitation