Proteome profiler cytokine array

Conditioned media were gathered from cultured MCF10A expressing EV, ErbB2^amp, or PI3Kα^H1047R. Protein concentration was normalized between samples using a colorimetric BCA assay (Pierce) and medium was concentrated using Amicon Ultra 0.5 ml 3 kDa filters (MillporeSigma). For functional conditioned media experiments, 80 μl of basal assay medium supplemented with 200 ng/ml of rhIL-6 (R&D Systems), conditioned EV medium, conditioned PI3Kα^H1047R medium, or conditioned EV medium + 200 ng/ml rhIL-6 was added from the duct port to stable endothelial vessels two days after seeding and cultured overnight before fixation or permeability analysis. For secretome analysis, conditioned media were treated with 1:100 Halt Protease and Phosphatase Inhibitor (Thermo), snap frozen in liquid nitrogen, and stored at −80 °C prior to western blot analysis. A Proteome Profiler Cytokine Array (R&D systems) was used to profile secreted cytokine content according to manufacturer instructions. To compare between membranes, densiometric intensities were normalized and compared to EV secretion using Eq. (1) $\frac{\mathrm{Cytokine}{\left(i\right)}_{\mathrm{Mutant}}-{\mathrm{Background}}_{\mathrm{Mutant}}}{\mathrm{Cytokine}{\left(i\right)}_{\mathrm{EV}}-{\mathrm{Background}}_{\mathrm{EV}}}\times \frac{\mathrm{Protein}\mathrm{Content}{\mathrm{Reference}}_{\mathrm{EV}}}{\mathrm{Protein}\mathrm{Content}{\mathrm{Reference}}_{\mathrm{Mutant}}}.$

Supernatant, serum, or urine protein was analyzed by ELISA kits specific for IL-6 (BD Biosciences), ICAM-1 (R&D Systems), NGAL, and TREM-1 (Invitrogen, Thermo Fischer Scientific) according to manufacturers’ instructions. HRGEC were dissolved in 100 μL of in-house prepared RIPA lysis buffer. The kidney tissue was crushed in liquid nitrogen and ∼100 mg of powdered tissue was dissolved in 500 μl of in-house prepared RIPA lysis buffer. All samples were sonicated on ice and protein concentration was determined by Bradford protein assay reagent (Bio-Rad). 40 μg of protein from HRGEC was used for assessment of IL-6 and 50 μg of kidney protein was used for assessment of ICAM-1 using respective ELISA kits. Supernatant from HRGEC was analyzed for 105 cytokines using a proteome profiler cytokine array (R&D Systems) per manufacturer’s instructions and resultant pixel density was calculated using ImageJ software.

Proteome profiler cytokine array (R&D systems, Minneapolis, MN) was used to screen relative levels of inflammatory cytokines following lymphatic injury and gel implantation according to the manufacturer’s protocol. There were four groups, as follows (i) sham injury: in the sham group, a skin incision was made without injuring lymphatic vessels; (ii) lymphatic injury: in the injury group, a section of afferent lymphatic vessel to the popliteal lymph node was surgically removed, but the gel was not implanted; (iii) sham injury plus gel, a skin incision was made with a scalpel, gel was implanted at the site of the injury without injuring lymphatic vessels; ( iv) injury plus gel, a section of afferent lymphatic vessel to the popliteal lymph node was surgically removed and the fibrin gel was implanted. After 10 days, tissues were harvested from the saphenous region and homogenized using a handheld homogenizer according to the manufacturer’s protocol. Tissue lysates were used for cytokine arrays, as directed by the manufacturer. Cytokine expression was semi-quantitively determined via densitometric analysis by Protein Array Tool in MATLAB (37 ). The data were presented as fold-change in comparison to the sham group (see Figure S3 for array images).