KEGG pathway analysis.

(DOCX)</p

Proteomic analysis of EVs from uninfected and baculovirus infected <i>Tnms42</i> cells.

(A) The volcano plot shows differentially abundant proteins in EVs from infected compared to uninfected cells. The horizontal dashed grey line represents the set significance threshold (> 15) and significantly differentially abundant proteins are highlighted in black. (B) The bar chart shows the KEGG pathway analysis and the number of matched objects for the top 15 categories with the most matched objects, including metabolic pathways, endocytosis and signalling pathways.</p

Schematic overview of the employed EV isolation protocol.

Tnms42 cells were grown uninfected and infected with baculovirus at MOI 5 in parallel. EVs were isolated from conditioned media 48 hpi. Several low-speed centrifugation steps eliminated cells, cell debris and large vesicles. A filtration step was applied to additionally clarify the conditioned media. Via ultracentrifugation EVs were first caught in a sucrose cushion, washed, and pelleted via another ultracentrifugation step.</p

Physical characterization of <i>Tnms42</i> cell EVs.

TEM pictures of negative stained EVs from uninfected (A) and infected (B) cells showed cup-shaped vesicles (arrows). Bars 100 nm. Western blot analysis (C) revealed incorporation of EV marker proteins Hsp90 and Hsp70 into Tnms42 cell EVs and an enrichment of Hsp90 in EVs upon baculovirus infection.</p

Comparison proteins in EVs.

(XLSX)</p

Protein identification and quantification.

(XLSX)</p

GSEA of the top 100 most significantly differentially abundant proteins.

(A) The graph shows enriched terms upon baculovirus infection for the GO category cellular component with a significant depletion of the term ECM. (B) The bar chart presents enriched categories upon baculovirus infection utilizing KEGG pathway as functional database and a significant depletion of the term ECM-receptor interaction (FDR < 0.05).</p

Binding of insect cell expressed 3D6 antibodies to 3D6 epitope and human FcγRI.

<p><i>Sf</i>9 cells presenting the 3D6 epitope were incubated with purified antibodies and target binding was analysed by FACS. Antibodies expressed in Hi5 and <i>Tnao</i>38 cells using standard baculoviral vectors (Hi5, <i>Tnao</i>38) as well as SweetBac (Hi5 Glyco, <i>Tnao</i>38 Glyco) show a highly specific binding. The broadening of the peaks can be explained by different amounts of 3D6 epitope displayed on <i>Sf</i>9 cells (A). Binding of antibodies to human Fc gamma receptor I was measured by incubating 3D6 antibodies with U937 cells. FACS analysis showed that all antibodies bound FcγRI present on the cellular surface, but the binding of SweetBac expressed variants (Hi5 Glyco, <i>Tnao</i>38 Glyco) was significantly increased.</p

S1 Raw images -

(PDF)</p

Lectin Blot using <i>Ricinus communis</i> agglutinin I.

<p>Cellular supernatants were harvested 96 hpi and purified 3D6 antibodies were separated on SDS-PAGE and binding of biotinylated <i>Riccinus communis</i> agglutinin I to antibody heavy chains was tested. The lectin only bound SweetBac expressed antibodies (Hi5Glyco, Tn38Glyco), because of terminal galactose present on N-glycans. CHO and Mimic insect cell expressed antibodies were used as controls.</p