3.2 SAC，DADSand DTS treatment induces autophagic f...
An increased number of autophagosomes in cells does not correspond to increased cellular autophagic activity.It is also possible that the increased number of autophagosomes and LC3-II protein levels in SAC-，DADSand DTS-treated cells is the result of the blocking of autophagosome fusion with lysosomes.To test whether SAC，DADSor DTS treatment specifically induces autophagy，we measured the activity of mTOR，a serine/threonine protein kinase whose activity is inhibited during the induction of autophagy.Phospho-p70S6 Kinase(phospho-p70S6K)was used as a marker for m TOR activity and it was found that the level of phospho-p70S6K was greatly decreased after treatment with 1μg/m L SAC，1μg/mL DADS or 0.1μg/mL DTS(Figure 3A，B).We also measured autophagic flux，which is used to denote the process of autophagosome formation，by monitoring the degradation of the substrate SQSTM1/p62.It was found that 1μg/mL SAC，1μg/mL DADS or 0.1μg/mL DTS caused rapid degradation of SQSTM1/p62 in RAW264.7 cells(Figure 3C，D).
Figure 1 DATSand DAS does not induce autophagy in RAW264.7 cells
(A)The structures of DADS，SAC，DTS，DATS and DAS.(B)RAW264.7 cells were treated with or without 5μg/mL DATS，25μg/mL DATS，1μg/mL DASor 0.1μg/mL DAS for 4h.Cells were stained with anti-LC3 antibody and analyzed by confocal microscopy.Scale bars，10μm.(C)Cells from(B)were quantified to determine the number of autophagosomes.At least 30 cells were counted for each treatment，and at least three replicates were performed.Data are presented as the mean±SD.P＞0.05 compared with control cells based on one-way ANOVA.
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Figure 2 DADS，SACand DTSincrease the number of autophagosomes in RAW264.7
cells(A)RAW264.7 cells treated with or without 1μg/mL DADS，1μg/mL SAC or 0.1μg/mL DTS for 4h were stained with anti-LC3 antibody and analyzed by confocal microscopy.Scale bars，10μm.(B)Cells from(A)were quantified for the number of autophagosomes.At least 30 cells were counted for each treatment，and at least three replicates were performed.Data are presented as the mean±SD；**P＜0.01 compared with control cells by one-way ANOVA.(C)Extracts from Raw 264.7 cells treated with or without 1μg/mL DADS，1μg/mL SAC or 0.1μg/mL DTS for 4h were immunoblotted with antibodies against LC3.(D)RAW264.7 cells treated with or without 1μg/mL DADS，1μg/mL
SAC or 0.1μg/mL DTS for 8h were stained with anti-LC3 antibody and analyzed by confocal microscopy.Scale bars，10μm.(E)Cells from(D)were quantified for the number of autophagosomes.At least 30 cells were counted for each treatment，and at least three replicates were performed.Data are presented as the mean±SD；**P＜0.01 compared with control cells by one-way ANOVA.(F)Primary macrophages treated with or without 1μg/mL DADS，1μg/mL SAC or 0.1μg/mL DTS for 4h were stained with anti-LC3 antibody and analyzed by confocal microscopy.Scale bars，10μm.(G)Cells from(F)were quantified for the number of autophagosomes.At least 30 cells were counted for each treatment，and at least three replicates were performed.Data are presented as the mean±SD；**P＜0.01 compared with control cells by one-way ANOVA.
To further confirm that SAC，DADSor DTSspecifically induce autophagy，the lysosome degradation capacity was tested by labeling cells with derivative-quenched bovine serum albumin(DQ-BSA)which is a self-quenched lysosome degradation indicator.Lysosomal degradation of DQ-BSA results in the release of non-quenched protein fragments that are brightly fluorescent.As shown in Figure 3E，F，SAC，DADS or DTS treatment did not impair lysosome degradation capacity.
Autophagic flux was also measured by monitoring LC3 turnover.This assay is based on the observation that LC3-Ⅱis degraded in lysosomes.We determined the amount of LC3-Ⅱdelivered to lysosomes by comparing LC3-Ⅱlevels in the presence and absence of the lysosomal protease inhibitor leupeptin.Higher levels of LC3-Ⅱaccumulation in the SAC-，DADSor DTS-treated cells in the presence of leupeptin may indicate the enhancement of autophagic flux.It was found that SAC，DADS or DTS treatment alone resulted in an increase of the LC3-Ⅱform of the endogenous LC3 protein，indicating the activation of autophagy.Furthermore，the levels of LC3-Ⅱwere higher in the presence of leupeptin(Figure 3G，H)，suggesting that autophagic flux was increased after treatment with SAC，DADS or DTS.
Taken together，these results further support our conclusion that SAC，DADSand DTSinduce autophagy by inhibiting m TOR phosphorylation activity.