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
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