Ab120192

Two inhibitors of NP internalization were used: Dynasore (ab120192, Abcam) and Pitstop2 (SML-1169, Sigma-Aldrich). Both inhibitors were used at a concentration of 40 μM/dish and they remained active 3 h prior to NP treatment. Dynasore inhibits dynamin, an important factor in receptor-mediated endocytosis, while Pitstop2 blocks clathrin-mediated endocytosis in the receptor-mediated endocytosis pathway. Both inhibitors were added to MSCs for 30 min before treatment with positively charged PCS NPs. CID 1067700 (cat. 2184, Axon Medchem, Groningen, Netherlands) was used to block Rab7 by competitive inhibition.

SH-SY5Y cells were transfected with the late-endosome marker GFP-Rab7a as described above. Sixteen hours after transfection, cells were treated with 0, 40, 80, or 120 µM of the endocytosis inhibitor dynasore (Abcam, ab120192) and 5 μM CLR16. Before imaging the cells, the culture medium was removed and the cells were washed with PBS to remove any CLR16 in the medium or on the surface of the cell. Cells were imaged every hour between 3 and 6 h. For quantitation, three areas were chosen randomly from different fields of view for each time point and each well. For collection of haze-reduced images, specific settings (Image/Image filter/Haze-reduction/setting-5) of the microscope were used to remove any background fluorescence including fluorescence in the cytoplasm, so that only bright, punctate fluorescence was visible. The percentage of CLR16 colocalized with Rab7a was calculated by dividing the number of yellow puncta (CLR16 in late endosomes/endolysosomes) by the total number of yellow and green puncta (Rab7a) × 100.

Cells were washed five times with serum-free media (DMEM) to remove any traces of serum. Furthermore, cells were incubated with Dynasore (80 μM; Abcam, Ab120192) or Pitstop 2 (15 μM; Abcam, ab120687) in serum-free media for 30 or 20 min, respectively. DMSO was used as a control for Dynasore and Pitstop 2 negative control (Abcam, ab120688). After incubation, drug-containing media was replaced with spike protein (3 μg per 200 μl of media) and further incubated for 5 min to allow internalization. Finally, cells were processed for immunofluorescence labeling.