3.3 Knockdown of ATG5 expression inhibits autophag...
Figure 3 SAC，DADSand DTSincrease autophagic flux(https://www.daowen.com)
(A)Raw264.7 cells were treated with or without 1μg/mL DADS，1μg/mL SAC，0.1μg/mL DTS or 100nm rapamycin for 4h.The cell extracts were subject to western blot analysis using antibodies against p70S6K or phospho-p70S6K.(B)The ratio of phospho-p70S6K/p70S6K for each sample in(A)was determined by IOD(integrated optical density)analysis using Image Pro-Plus 6.0 software；at least
three replicates were performed.(C)RAW264.7 cells were treated with or without 1μg/mL DADS，1μg/mL SAC or 0.1μg/mL DTS for 4h，and the total cell extracts were subject to western blot analysis using antibodies against SQSTM1/p62 or GAPDH.(D)The ratio of SQSTM1/GAPDH for each sample in(C)was determined by IOD analysis using Image Pro-Plus 6.0 software；at least three replicates were performed.(E)Lysosomes labeled with DQ-BSA.After 4 h of treatment with 1μg/mL DADS，1μg/mL SAC or 0.1μg/mL DTS，RAW264.7 cells were washed twice with PBS and then incubated in DMEM containing DQ Red BSA at a final concentration of 10μg/mL for 3h at 37℃.The cells were then washed twice with PBS to remove excess probe and analyzed by confocal microscopy.Fluorescent signal generated by lysosomal proteolysis of DQ-BSA was not changed in DADS-，SACor DTS-treated cells compared with untreated cells.(F)Fluorescence intensity of the brightly fluorescent fragments released by lysosomal degradation of DQ-BSA was IOD.At least 40 cells were analyzed for each treatment.(G)RAW264.7 cells were treated with or without 1μg/mL DADS，1μg/mL SAC，0.1μg/mL DTS，in the presence or absence of 5μg/mL leupeptin for 4h.Total cell extracts were subject to western blot analysis using antibodies against LC3 or GAPDH.(H)The ratio of LC3-Ⅱ/LC3-Ⅰfor each sample in(G)was determined by IOD analysis using Image Pro-Plus 6.0 software；at least three replicates were performed.
ATG5 is a key factor for autophagy.It is activated by ATG7 and forms a complex with ATG12 and ATG16L1.To determine whether ATG5 is required for the induction of autophagy by DADS，SACand DTS，three shRNAs targeting ATG5 were transfected into RAW264.7 cells.The protein levels of ATG5 and the number of LC3 positive puncta were determined in cells before and after treatment with 1μg/mL DADS，1μg/mL SACor 0.1μg/mL DTS.shRNAs ATG5 1 and ATG5 3 were found to inhibit ATG5 gene expression(Figure 4A).DADS，SACand DTSdid not increase the number of autophagosomes or the ratio of LC3-Ⅱ/LC3-Iin ATG5-knockdown cell lines(Figure 4B-D).In addition，the DADS-，SACand DTSinduced degradation of the autophagic substrate SQSTM1/p62 was inhibited after knockdown of ATG5 expression(Figure 4E，F).