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. 2021 Oct 18;2021:7716201. doi: 10.1155/2021/7716201

Figure 4.

Figure 4

Puerarin inhibited the RagA/mTOR/p70S6K pathway and the lysosomal localization of mTORC1 in the NSCs. (a) Detection of RagA, mTOR, and p70S6K. The protein expression of RagA, mTOR, and p70S6K were analyzed by Western blot in LPS-challenged neural stem cells. After 24 h treatment with 200 ng/ml LPS alone or in combination with puerarin (P) (10, 25, and 50 μM), NSCs were analyzed by Western blot for the protein expression of RagA, phospho-mTOR (p-mTOR), mTOR, phospho-p70S6K (p-p70S6K), and p70S6K. (b) Quantitation of the blots in (a). The blots in (a) were quantified by the densitometric method. The protein value was shown as means ± SD (n = 3). ###p < 0.001 (LPS vs. control); ∗∗∗p < 0.001 (LPS+P vs. LPS). (c) Immunofluorescence staining of mTORC1 in LPS-stimulated neural stem cells. After 24 h treatment with 200 ng/ml LPS or in combination with P (10, 25, and 50 μM), NSCs were incubated with mTOR and lysosomal biomarker LAMP2 antibodies, and DAPI was used to stain nuclear. The images were captured with a Zeiss fluorescence microscope (Jena, Germany). Scale bar, 20 μm. (d) Quantification of mTOR and LAMP2. Colocalization of mTOR and LAMP2 was quantified by Pearson's R value. The results were shown as means ± SD (n = 3). ∗p < 0.05.