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. 2018 Dec 5;6(3):1801458. doi: 10.1002/advs.201801458

Figure 2.

Figure 2

siRNA transfection and protein knockdown of neural cells. a,b) Percentage of transfected cells (blue graph) and amount of internalized fluorescently labeled siRNA–Alexa488 per cell (green graph) in a) astroglial and b) neuronal cell cultures. c,d) Percentage of cell survival in c) astroglial and d) neuronal cell cultures 24 h after transfection. e,h) Percentage of e) astroglial and h) neuronal cells transfected with P4 particles after their preincubation with inhibitors of three endocytic pathways: chlorpromazine (chlorprom or C), an inhibitor of clathrin‐mediated intracellular transport; filipin (F), an inhibitor of cholesterol‐dependent caveolar cell transport; and amiloride (A), an inhibitor of Na+/H+ exchange in ligand‐independent, nonselective transport by macropinocytosis. f,i) Confocal images of f) astroglial i) and neuronal cells stained with Lysotracker (red, endosomes), siRNA–Alexa488 (green, siRNA), and 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue, nucleus) 24 h post‐transfection. g,j) Graph representing the Pearson's coefficient for siRNA/Lysotracker colocalization in g) astroglial and j) neuronal cell culture (−1 ≤ Rr ≤ 1, where 1 = maximum colocalization and −1 = maximal exclusion). k) Confocal images of astroglial cells stained with GFAP (green) and neuronal cells stained with synaptophysin (Syp, green), Tuj‐1 (red), and DAPI (blue) 24 h after transfection. l–o) Western blots and densitometry (intensity values normalized to actin) of l,m) GFAP and n,o) synaptophysin marker in neural cultures transfected with different P4 doses (doses 1, 2, 3 and 4 have concentrations of P4 supramolecular particles of 34 × 10−6, 52 × 10−6, 78 × 10−6, and 104 × 10−6 m, respectively, and of 65 × 10−9, 100 × 10−9, 150 × 10−9, and 200 × 10−9 m for siRNA, respectively) 24 h post transfection. *P < 0.05, ** P < 0.01, and **P < 0.001, least significant difference test (LSD) test (n = 7). All the experiments were performed using a dose 4 (P4: 104 × 10−6 m, siRNA: 200 × 10−9 m) except for western Blot analysis where four doses were used.