Pitstop 2

Jurkat and Nalm-6 cells were seeded at 2 × 10⁵ cells/mL and treated with endocytosis inhibitors. To inhibit caveolin-dependent endocytosis Nystatin (80 µM, 30 mins, Sigma-Aldrich, Australia), and Methyl-β-Cyclodextrin (MβCD) (4 mM, 40 mins, Sigma-Aldrich, Australia) were used. To inhibit clathrin-mediated endocytosis (CME): Pitstop^®2 (20 µM, 10 mins, Abcam, UK) and. Dyngo^®4a (30 µM, 30 mins Abcam, UK) were used. To inhibit macropinocytosis: 1 hr treatment with Wortmannin (10 µM Sigma-Aldrich, Australia) or Amiloride (25 µM, Sigma-Aldrich, Australia) was used. The cells were then exposed to the star polymers for 20 mins and thereafter washed three times with PBS before analysis by flow cytometry as described. For confocal microscopy analysis, the cells were seeded at 2 × 10⁵ cells/mL on a FluoroDish™ (World Precision Instruments, Inc.) and imaged with Leica TCS SP8 DLS (Leica Microsystems) using the 63× oil immersion objective. Hoechst 33342 (ThermoFisher Scientific) was used for nuclei staining. All experiments were performed in triplicate.

The following primary antibodies were used; CD11a Alexa Fluor^™ 594 clone HI111 (Biolegend, San Diego, CA), rabbit polyclonal anti-Rab5, anti-Rab11A, anti-Rab7, or anti-Lamp1 antibody (Abcam, Cambridge, UK), anti-beta-actin antibody (AnaSpec, Fremont, CA) (1:1000), and anti-LtxA monoclonal antibody 107A3A3 [75 (link)] in hybridoma supernatants (1:10 dilution). The following secondary antibodies were used: goat anti-rabbit IgG Alexa Fluor^®488 (1:1000); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Fc) or (HRP)-goat anti-rabbit (Pierce, Rockford, IL) (1:10,000). Transferrin labeled with Alexa Fluor®555 was from Invitrogen (Waltham, MA, USA). Dynamin inhibitor Dynole 34-2 and its inactive control, Dynole 31-2, were purchased from SigmaAldrich (St. Louis, MO), Dynasore and Pitstop 2 (Abcam, Cambridge, UK). The inhibitors were used in the following concentrations: 10 μM Dynole 34-2; 10 μM Dynole 31-2; 10 μM Dynasore; 5 μM Pitstop 2.

To determine the EPC-EX uptake mechanisms by ASCs, we focused on the endocytic uptake pathways, including macropinocytosis, clathrin and caveolin-dependent pathways. For this, the cells were pre-treated for 30 min with various inhibitors: 80 μM dynasore (dynamin inhibitor, Sigma-Aldrich), 5 μM LY290042 (macropinocytosis inhibitor, Enzo), 10 μM pitstop 2 (clathrin inhibitor, Abcam) or 200 μM genistein (caveolin inhibitor, EMD Millipore). The concentration of the drugs was determined by following our previous studies [24 (link)]. After treatment, the cells were washed twice with PBS and then the PKH-labelled EPC-EXs were co-incubated with the ASCs for 24 h. Fluorescent images were obtained by a fluorescence microscope (EVOS, NY) and fluorescent intensity was quantified by flow cytometry analysis (Accuri C6 flow cytometer).

MG-132 (Calbiochem no. 474790), MDL 28170 (Tocris Bioscience no. 1146/10), MK-801 (Tocris Bioscience no. 924), okadaic acid (Tocris Bioscience no. 1136), tautomycetin (Tocris Bioscience no. 2305), FK-506 (Tocris Bioscience no. 3631), Dynasore (Tocris Bioscience no. 2897), PitStop2 (Abcam no. ab120687), APV (Tocris Bioscience no. 0106), NMDA (Sigma-Aldrich no. 3263), and DHPG (Tocris no. 0805). All chemicals were diluted in a modified Tyrode’s solution (pH 7.4) containing 25 mM Hepes, 119 mM NaCl, 2.4 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, and 30 mM glucose. For NMDA experiments, neurons were first incubated for 5 min in modified Tyrode’s solution, and then NMDA 2× (diluted in modified Tyrode’s at 100 μM) was added to the wells. For controls experiments, Tyrode’s solution was applied twice. After 4 min, neurons were dipped in Tyrode’s solution to wash them and returned to their original medium. NMDA treatment combined with live imaging was performed the same way, and all incubation and washing steps were performed on stage. For DHPG experiment, the same procedure was used as for NMDA-LTD, but neurons were incubated for 5 min in 100 μM DHPG. For Triton X-100 extraction experiment, live DIV14 hippocampal neurons were incubated for 5 min at RT in 0.25% Triton X-100 preheated at 37°C. Neurons were fixed directly after extraction.