Fig. 4

Autophagy mediates the quiescence of NPSCs and regulates the expression of P27. (1) Quiescent NPSCs induced by serum starvation upregulate autophagy and the expression of P27. Representative images of GFP and mRFP puncta under normal and serum starvation conditions are shown (a), together with the quantification of autophagosomes and autolysosomes (b). n = 5. c, d Western blot analysis was performed to examine the expression of P27, LC3 and P62 at the protein level. GAPDH served as an internal control. Data are expressed as the mean ± standard deviation, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. NC normal culture condition, SS serum starvation condition. (2) Autophagy promotes NPSC quiescence. Representative images of GFP and mRFP puncta under serum starvation conditions with or without rapamycin are shown (e), together with the quantification of autophagosomes and autolysosomes (f). n = 5. g, h Western blot analysis was performed to examine the expression of P27, LC3 and P62 under serum starvation conditions with or without rapamycin. i, j Ki67 staining showed that NPSC proliferation was further inhibited after 48 h of rapamycin treatment under serum starvation conditions. Data are expressed as the mean ± standard deviation, n = 3. *p < 0.05, **p < 0.01. SS serum starvation condition, Rapa rapamycin. (3) autophagy inhibition disturbs the quiescence of NPSCs. Representative images of GFP and mRFP puncta under serum starvation conditions with or without chloroquine are shown (k), together with the quantification of autophagosomes and autolysosomes (l). n = 5. m, n Western blot analysis was performed to examine the expression of P27, LC3 and P62 under serum starvation conditions with or without chloroquine. o, p Ki67 staining showed that NPSC proliferation increased after 48 h of chloroquine treatment under serum starvation conditions. Data are expressed as the mean ± standard deviation, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. SS serum starvation condition, CQ chloroquine, DAPI 4′,6-diamidino-2-phenylindole