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

Journal: Nature Communications

Article Title: A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma

doi: 10.1038/s41467-020-15318-5

Figure Lengend Snippet: a , b Stable isotope labeling with amino acids in cell culture (SILAC) was performed to identify novel binding partners. MET wt-tGFP and MET N375S-tGFP cells were labeled with heavy (H) and light (L) amino acids. The cutoff values for SILAC ratios, after normalizing with MET expression, were set at (>2, <0.5) respectively. a Scatter plot of transformed MET wt /MET N375S ratios of membrane-bound proteins. Both axes represent MET wt (H)/MET N375S (L) and MET wt (L)/MET N375S (H) ratios, respectively. b List of various membranous proteins identified in SILAC analysis found to be associated with MET N375S . c Interaction of ectopic MET and endogenous HER2 in H2170 MET wt-tGFP and MET N375S-tGFP cells was detected with immunoprecipitation and immunoblotting. Left, input controls. Total MET and HER2 band intensities, normalized to input controls and relative to MET wt , are shown below ( n = 5). d , e Detection of MET/HER2 co-localization (red) in EV, MET wt-tGFP , and MET N375S-tGFP cells with proximity ligation assay (PLA). Representative images are shown ( d ), with the PLA signals quantified ( e ) and expressed as the number of signals/cell ± SD ( n > 3). Scale bar, 20 µm. f Representative confocal microscopy images of MET (Alexa-488; green) and HER2 (Alexa-594; red) in isogenic H2170 clones ( n = 2). DAPI (blue) was used as nuclear counter stain. Co-localized proteins appeared in yellow. The smaller panels are detailed views of the outlined (white) squares in the respective images. Scale bar, 20 µm. The total MET fluorescence that co-localized with HER2 signal in each variant was tabulated on the right, data presented as mean ± SD (five fields in one representative experiment). g MET/HER2 interaction in H2170 MET wt-tGFP and MET N375S-tGFP tumors shown in Fig. was detected with immunoprecipitation and immunoblotting. IgG was used as a loading control. h – k The role of Sema domain in MET N375S-tGFP cells was examined with recombinant Sema proteins, wild-type, rSema wt ; N375S mutant, rSema N375S . Cell viability of MET wt-tGFP ( h ) and MET N375S-tGFP ( i ) cells after treatment with 1, 5, 10 µg/ml of rSema wt or rSema N375S for 72 h, presented as mean ± SD ( n = 3). Two-tailed Student’s t test; * P < 0.05, ** P < 0.01, *** P < 0.001. j Immunoblots showing the total and phosphorylated expressions of MET, HER2, Src, Akt, and ERK1/2 in lysates of the indicated cell lines after treatment with 10 µg/ml rSema. β-Actin was used as a loading control. k MET/HER2 interaction in H2170 MET N375S-tGFP cells was detected with immunoprecipitation and immunoblotting after treatment with 10 µg/ml rSema. Left, input controls. l MET/HER2 interaction in H2170 MET N375S-tGFP cells was detected after serum starvation (0.1% FBS), or co-incubated with HGF (0.1% FBS + 10 ng/ml HGF). Left, input controls. m HEK293 cells were transfected with 1 µg of either pCMV6-EV-tGFP vector, MET-wt, N375S, ∆Sema, N375Q, M1268T, or Y1248H plasmid, together with pCMV6-ERBB2-DDK plasmid, for 24 h. MET/HER2 interaction in HEK293 cells was detected with immunoprecipitation and immunoblotting. Total MET and HER2 band intensities, relative to MET wt , are shown below ( n = 3). Left, input controls and phosphorylated proteins. β-Actin was used as a loading control.

Article Snippet: pCMV6-AC-turboGFP (tGFP) vector, pCMV6-MET-tGFP, and pCMV6-ERBB2-DDK plasmids were obtained from Origene (#PS100010, #RG217003, and #RC12583).

Techniques: Labeling, Cell Culture, Binding Assay, Expressing, Transformation Assay, Immunoprecipitation, Western Blot, Proximity Ligation Assay, Confocal Microscopy, Clone Assay, Staining, Fluorescence, Variant Assay, Recombinant, Mutagenesis, Two Tailed Test, Incubation, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma

doi: 10.1038/s41467-020-15318-5

Figure Lengend Snippet: a – c Cellular invasion on isogenic wild-type (MET wt-tGFP ) and N375S mutant (MET N375S-tGFP ) clones, treated with kinase inhibitors or siRNA silencing of MET or HER2, were being evaluated. a H2170 MET N375S-tGFP cells were treated with crizotinib (10 µM), lapatinib (0.3 µM) or a crizotinib/lapatinib combination for 36 h. Representative images of cell invasion are shown. b H2170 MET N375S-tGFP cells were co-incubated with 10 nM of the indicated siRNA overnight, and seeded in Matrigel invasion chambers for 36 h. Representative images are shown. Scale bar: 200 μm. c Percentage of invaded cells relative to vehicle control (0.1% DMSO) or Scr siRNA control were expressed as mean ± SD ( n = 3). One-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001. Left, immunoblots of siRNA-treated H2170 MET N375S-tGFP cells. For immunoblots, cells were harvested 48 h after siRNA treatment, and β-actin was used as a loading control. d – f Calu-1 MET wt-tGFP and MET N375S-tGFP cells were co-incubated with 10 nM of the indicated siRNA overnight, and seeded in Matrigel invasion chambers for 24 h. Representative images of cell invasion for MET wt-tGFP ( d ) and MET N375S-tGFP cells ( e ) are shown. Scale bar: 200 μm. f Percentage of invaded cells relative to Scr siRNA control in Calu-1 MET wt-tGFP and MET N375S-tGFP cells were expressed as mean ± SD ( n = 3). Left, immunoblots of siRNA-treated Calu-1 MET N375S-tGFP cells. For immunoblots, cells were harvested 48 h after siRNA treatment, and β-actin was used as a loading control. One-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001. g , h Anchorage-independent colony formation on isogenic H2170 MET N375S-tGFP cells, treated with kinase inhibitors or siRNA silencing of MET or HER2, were being evaluated. g H2170 MET N375S-tGFP cells were co-incubated with 10 nM of the indicated siRNA overnight, and seeded in soft agar for 4 weeks. Representative images are shown. h The number of colonies were quantified after treatment with crizotinib, lapatinib, trastuzumab, or in combination. Data were presented as percentage of colonies relative to vehicle control (0.1% DMSO) ± SD ( n = 3). i – l Efficacies of HER2 inhibitors were evaluated in xenograft models. Tumor growth of MET wt-tGFP ( i ) and MET N375S-tGFP ( j ) xenografts after treatment with trastuzumab, pertuzumab, lapatinib, and ASLAN001 were expressed at mean ± SEM ( n = 5). Two-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001. k Immunohistochemistry staining showing the changes in p-MET after treatment with trastuzumab in MET wt-tGFP and MET N375S-tGFP tumors. Representative images are shown. Scale bar, 50 µm. l Expression of p-MET was quantified and expressed at mean of positive-staining/100 cells ± SD ( n = 5). Two-tailed Student’s t test; * P < 0.05, ** P < 0.01, *** P < 0.001. m HEK293 cells were transfected with 1 µg of either pCMV6-ERBB2-wt, ∆D1, ∆D2, ∆D3, ∆D4, or ∆TK plasmid, together with pCMV6-MET-N375S plasmid, for 24 h. MET/HER2 interaction in HEK293 cells was detected with immunoprecipitation and immunoblotting. Left, input controls and phosphorylated proteins. β-Actin was used as a loading control. n , o Isogenic Calu-1 MET wt-tGFP ( n ) and MET N375S-tGFP ( o ) cells were engrafted into SCID mice, and treated daily with vehicle ( n = 6) or 15 mg/kg afatinib ( n = 5) starting 14 days after inoculation. Arrows indicate treatment start date. Kaplan–Meier analyses of the mice are shown.

Article Snippet: pCMV6-AC-turboGFP (tGFP) vector, pCMV6-MET-tGFP, and pCMV6-ERBB2-DDK plasmids were obtained from Origene (#PS100010, #RG217003, and #RC12583).

Techniques: Mutagenesis, Clone Assay, Incubation, Western Blot, Immunohistochemistry, Staining, Expressing, Two Tailed Test, Transfection, Plasmid Preparation, Immunoprecipitation