her2-ddk tp322909  (OriGene)

Journal: bioRxiv

Article Title: CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells

doi: 10.1101/2024.02.06.579138

Figure Lengend Snippet: A-B) Nanoparticle Tracking Analysis (NTA) of particles secreted by transfected HEK293T cells. Representative size distribution profiles of Mock, CD81-GFP, anti-HER2 samples. The black curve indicates the mean of three measurements, with SE in red. Mode and Mean diameters, and particle concentration are plotted. Error bars include at least three biological replicates. Significance * is P<0.05, *** P<0.001, vs Mock condition. C) Representative Cryo-EM images of Mock, CD81-GFP and antiHER2 EV samples confirming the vesicular structure and size heterogeneity of recovered vesicles. The indicated scale bar is 100 nm. D) Plot of the observed diameter of vesicles in Cryo-EM images (n=35 for Mock, n=99 for CD81-GFP, n=74 for antiHER2) and lamellarity, expressed as percentage of unilamellar and multilamellar vesicles over the observed bulk EV populations.

Article Snippet: The resulting product was pre-incubated with 30 nM HER2-DDK (TP322909, OriGene) for 30 min before addition to the other components: 10 nM tGFP Ab (TA150041, OriGene), anti-FLAG donor and protein G coated acceptor beads (10 ng/μl final concentration; AS103D and AL102C, PerkinElmer).

Techniques: Transfection, Concentration Assay, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells

doi: 10.1101/2024.02.06.579138

Figure Lengend Snippet: A) Representative immunoblotting of cell and EV lysates (1 μg proteins/well). EVs are positive to transmembrane (CD9) and cytosolic proteins (SYNTENIN, TSG101), while negative to CALNEXIN, and with low detectable levels of GAPDH compared to cell lysates. B) . Dot plots of imaging flow cytometry to detect GFP-positive EVs. The green fluorescent signal (Ch 2, 488 nm laser) was detected as sub-gating of EVs labeled with Cell Mask Deep Red (CMDR, in orange, Ch 11, 635 nm) to side-scatter (Ch 6). Non-fluorescent, calibrator SpeedBeads, Amnis (1 µm) were continuously run during acquisitions. The graph shows the quantification of double-positive particles. Mean and error bars derive from three independent experiments. C) Sandwich designed for the AlphaLISA competitive assay. CD81-GFP and antiHER2 EVs were tested for competition with HER2-DDK. Image created with BioRender.com. The graph shows the measured alpha counts normalized to the GFP-positive EV population as calculated by NTA and imaging flow cytometry. Mean and SD derive from three independent experiments (significance is **** P<0.0001). D) Representative western blotting of recombinant EVs immunoprecipitation with HER2-DDK or anti-GFP antibody in serum-free DMEM. GFP-positive fusion proteins are enclosed in the yellow box above the antibody heavy chains (black arrow), SYNTENIN, and HER2-DDK. Controls of beads flow through with HER2-DDK (*) or anti-GFP antibody (**) are shown on the right, indicating saturation of the beads’ surface to avoid non-specific binding. The graph shows the densitometric quantification of antiHER2 EVs captured by both HER2-DDK and anti-GFP Ab, with a competition effect of Trastuzumab. Mean and SD refer to two independent experiments.

Article Snippet: The resulting product was pre-incubated with 30 nM HER2-DDK (TP322909, OriGene) for 30 min before addition to the other components: 10 nM tGFP Ab (TA150041, OriGene), anti-FLAG donor and protein G coated acceptor beads (10 ng/μl final concentration; AS103D and AL102C, PerkinElmer).

Techniques: Western Blot, Imaging, Flow Cytometry, Labeling, Recombinant, Immunoprecipitation, Binding Assay

Journal: bioRxiv

Article Title: CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells

doi: 10.1101/2024.02.06.579138

Figure Lengend Snippet: A) Immunofluorescence of MDA-MB-231 and SK-BR-3 breast cancer cell lines as HER2 negative or positive cells, respectively. HER2 receptor is in red (Alexa Fluor 633), nuclei are shown in cyan (Hoechst). The indicated scale bar is 50 μm. B) Left: HER2 protein detection by Dot blot in lysates from wild-type or transfected (OE) MDA-MB-231 cells. Right: Immunoblot for checking the selection of SK-BR-3 cells with HER abrogation ( ERBB2 -KO, or KO). C) Representative confocal time lapse of recombinant EVs incubated with live cells (time points are indicated). GFP-EVs are shown in green, lysosomes are shown in magenta (Lysotracker red), and nuclei in cyan (Hoechst). The white squares highlight the co-localization between EVs and lysosomes (white arrowhead). The indicated scale bar is 20 μm. D) Representative confocal image of fixed SK-BR-3 cells recognizing HER2 (Alexa Fluor 633) and nuclei (Hoechst) after 4 hr incubation with CD81-GFP EVs (green spots). White arrows or the arrowhead indicate different localization of EVs upon cell interaction. Indicated scale bar is 20 μm. E-F) Quantification of recombinant EVs with recipient breast cancer cell lines. MDA-MB-231 (WT and HER2 OE) and SK-BR-3 (WT and KO) were incubated with EVs for 4 hours, then washed with PBS before fixation and HER2 immunofluorescence. Fourteen Z-stacks were acquired within around 11 μm of total Z-size and the Maximum Intensity Projections have been analyzed with an automated pipeline (using CellProfilerTM 4.0.7). Graphs report Mean and SD of the spot distribution from three independent experiments (* if P<0.05, ** if P<0.01, *** if P<0.001).

Article Snippet: The resulting product was pre-incubated with 30 nM HER2-DDK (TP322909, OriGene) for 30 min before addition to the other components: 10 nM tGFP Ab (TA150041, OriGene), anti-FLAG donor and protein G coated acceptor beads (10 ng/μl final concentration; AS103D and AL102C, PerkinElmer).

Techniques: Immunofluorescence, Dot Blot, Transfection, Western Blot, Selection, Recombinant, Incubation, IF-P