Supplementary Figure 3 from Prometastatic NEDD9 Regulates Individual Cell Migration via Caveolin-1–Dependent Trafficking of Integrins
Year: 2023
Type: supplementary-materials
Abstract: Supplementary Figure 3. (A). Quantification of volume of early endosomes based on 3D reconstructed confocal images of BT459-siCon, -siN2, -siN3) cells stained with EEA1 antibodies; mean volume (%) normalized to siCon+S.E.M; n=3, 10 cells/treatment; one-way ANOVA: *p<0.0004 for siCon/siN2 or /siN3 (EEA1). (B). Representative confocal images of MDA-MB-231LN-siCon, -siN2) cells stained with anti-EEA1 and anti-Integrin b1 antibodies; inserts-the enlarged areas of individual cells indicated by rectangles in the main panel; scale bar, 10µm; (C). WB analysis of lipid raft fractionation of MDA-MB-231 cells constitutively expressing shNEDD9 (N1, N4) with anti-b1 integrin, -transferin receptor (TrfR), -EGFR, -Ras and -caveolin-1 (CAV1) antibodies; (D). Representative confocal images of MDA-MB-231 cells expressing GFP-NEDD9 and stained with µ2 AP2; no colocolization found; scale bar, 10µm; (E). Western blot analysis of immunoprecipitated (IP) NEDD9 from shNEDD9 (N1)-MDA-MB-231LN cells with or without Nedd9 re-expression (cDNA-Nedd9), non-specific IgG -control; staining for subunits of AP2-complex part of clathrin trafficking machimery: adaptin a and g, pµ2 AP2; (F). WB of BT459-siCon and -siN2,-N3 cells with anti-pTyr14-CAV1, -NEDD9 and -GAPDH antibodies. (G). Flow cytometry analysis of integrin b4 surface staining in control (siCon) or siNEDD9 (N2, N3) MDA-MB-231cells treated or untreated with siCaveolin-1 (siCAV1); graphs are mean values of RFU % to siCon (assigned as 100%) +/- S.E.M; one-way ANOVA; *p<0.002 for siCon/siN2 or N3; *p for siCon/siCAV1+siN2 or N3 is not significant (ns).
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4362529693