A21070

1 d before immunostaining, ZMEL-GFP cells and ZMEL-mCherry cells that express WT-Paxillin were plated on either glass bottom dishes (FD35-100; World Precision Instruments) or collagen-coated dishes (0.5 kPa, SV3520-COL-0.5; 50 kPa, SV3520-COL-50; Matrigen). Cells were fixed with 4% PFA and permeabilized in 0.1% Triton X-100/TBS for 5 min and then blocked with 1% BSA + 1% FBS for 1 h. Cells were incubated with primary antibodies overnight at 4°C and secondary antibodies for 1 h at room temperature. Primary antibodies used were chicken anti-GFP (1:500, ab13970; Abcam) and rabbit anti-pY118 Paxillin (1:500, NBP2-24459; Novus Biologicals). Secondary antibodies used were Alexa Fluor 488 goat anti-chicken IgY(H + L) secondary antibody (1:500, 103-545-155; Jackson Immunoresearch), Alexa Fluor 633 goat anti-rabbit IgG(H + L) secondary antibody (1:500, A-21070; Thermo Fisher Scientific), and DAPI (D9542; Sigma-Aldrich). Imaging was performed using either a Zeiss LSM 880 (Carl Zeiss) with a 63×/1.40 oil immersion objective with three photomultiplier tubes and two GaSP detectors or a Leica Yokogawa CSU-W1 spinning disc confocal microscope with a Leica PL APO 63×/1.40 oil immersion objective and an iXon Life 888 EMCCD camera at room temperature.

An anti-CD63 mouse monoclonal antibody, Alexa Fluor 633 conjugated (#H5C6, Biolegend, San Diego, CA, USA) and the lipophilic dyes PHK26 or PHK67 (Merck, Darmstadt, Germany) were used to stain the membranes of M-NVs or exosomes. A goat anti-rabbit Alexa Fluor 633 conjugated secondary antibody (0.1 μg) (#A-21070, Thermofisher) was used to assay its intravesicular incorporation into nascent M-NVs (~2 × 10⁷ particles). CD63 antibody (0.1 μg) was centrifuged for 10 min at 17,000× g before use to eliminate aggregates and then incubated for 1 h at room temperature with M-NVs or exosomes (both ~2 × 10⁷ particles) with 1% BSA (v/v). Then, stained particles were purified from unstained dyes using exosome spin columns (MW 3000, Thermofisher) as recommended. M-NVs or exosomes (both ~2 × 10⁷ particles) were stained for 5 min at room temperature in agitation with PHK26 or PHK67 dyes (0.5 μL each in a final volume of 100 μL using diluent buffer C) and columns purified as described above.

MCF10CA1a cells were cultured in a #1.5H-N high performance glass bottom 12 well plate (Cellvis) and processed for immunofluorescence microscopy. The cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with BlockAid (Invitrogen, B10710). Cells were probed with antibodies for PLIN3, SQLE, and NSDHL (Sigma, HPA006427; SantaCruz Biotechnologies, sc-271651; Atlas Antibodies, HPA000571, respectively). Proteins were detected using secondary AlexaFluor antibodies (Life Technologies, A-21070 and A-21052), and cells were counterstained for neutral lipids using 1 μg/mL 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503; Life Technologies, Grand Island, NY, United States), and for nuclei using 300 nM 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI; Invitrogen, D1306). Samples were imaged using a Nikon A1R-MP inverted confocal microscope (Nikon Instruments Inc., Melville, NY, United States). Images were acquired using the Plan Apo λ 100x Oil objective, 76.63 µm pinhole size, and DAPI, FITC, and Cy5 lasers. All image processing was conducted using Nikon NIS-Elements AR acquisition and analysis software. A Landweber 2D deconvolution algorithm was implemented, with point scan confocal modality, clear noise, and 12, 12, 12 iterations.

For mouse ESCs, a similar protocol was followed for all staining procedures. Fixed samples were permeabilized with 0.25% Triton X-100 for 15 min. Excess Triton was washed in PBS and blocked with TNB for one hour. The primary antibodies were incubated at 4 °C o/n. After several washes with 0.1% Triton X-100, secondary antibodies and DAPI were incubated for 1 h at room temperature. Cells were then washed at least 5 times with 0.1% Triton X-100 and mounted in Vectashield mounting medium. To analyze GFP-MYC and TFP-MYC endogenous fluorescence, cells were fixed, permeabilized, and incubated for 1 h only with DAPI at RT. Primary antibodies were: phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (rabbit, 1:200, Cell Signaling, #4370 #9106), Anti-SOX2 antibody (rabbit, 1:200, Abcam, #ab97959) and Oct-3/4 Antibody (C-10) (mouse, 1:200, Santa Cruz, #sc-5279). Secondary antibodies: 633 goat anti-rabbit (1:500, Life Tech, #A-21070) and 568 goat anti-mouse (1:500, Life Tech, #A-11004).

For embryos, the same procedure was followed for all the stages and staining procedures. Embryos were permeabilized in 0.5% Triton X-100 for 30 min. Excess triton was washed in PBS for 15 min. Then, embryos were blocked with a TNB-blocking reagent (Perkin Elmer, FP1012-X) for 1 h. Primary antibodies were incubated overnight at 4 °C. Afterward, embryos were washed several times with 0.1% Triton X-100 and incubated with secondary antibodies and DAPI 1 h at room temperatures. After secondary antibodies incubation, embryos were washed with 0.1% Triton X-100 at least 5 times. Primary and secondary antibodies were diluted in the blocking solution. Primary antibodies: GFP Goat Polyclonal Antibody (Goat, 1:200, OriGene, #R1091AP), Human/Mouse Brachyury Antibody (mouse, 1:250, R&D systems, #AF2085-SP), and Anti-SOX2 antibody (rabbit, 1:200, Abcam, #ab97959). Secondary antibodies: 648 donkey anti-goat (1:500, Life Tech, #A-21447), 488 goat anti-mouse (1:500, Life Tech, #A32723) and 633 goat anti-rabbit (1:500, Life Tech, #A-21070).