3.4 Autophagy protects DADS-，SACand DTS-induced ap...
Figure 4 Knockdown of ATG5 expression inhibits the induction of autophagy by SAC，DADSand DTS
(A)Three shRNAs targeting ATG5 were transfected into RAW264.7 cells using lentivirus.Cells transfected with Pll3.7-puro vector served as the negative control(NC).The total cell extracts were subject to western blot analysis using antibodies against ATG5 or GAPDH.(B)ATG5-knockdown RAW264.7 cells were incubated with 1μg/m L SAC，1μg/m L DADS，or 0.1μg/mL DTS for 4h.The cell extracts were subject to western blot analysis using antibodies against LC3.GAPDH served as a loading control.(C)RAW264.7 cells and ATG5-knockdown RAW264.7 cells were incubated with or without 1μg/mL DADS，1μg/m L SAC or 0.1μg/m L DTS for 4h.The cells were stained with LC3 antibodies and analyzed by confocal microscopy.(D)The number of autophagosomes in the cells from(C)was counted.At least 30 cells were counted，and at least three replicates were performed.Data are presented as the mean±SD.There was no significant change in autophagosome number compared with control cells.(E)Extracts from cells treated as in(B)were subject to western blot analysis using antibodies against SQSTM1/p62 or GAPDH.(F)The ratio of SQSTM1/GAPDH from the samples in(E)was determined by IOD using Image Pro-Plus 6.0 software；at least three replicates were performed.(https://www.daowen.com)
Figure 5 DADS，SACand DTSinduce apoptosis by increasing autophagic flux
Treatment with 1μg/mL DADS，1μg/mL SAC or 0.1μg/mL DTS increased both the total apoptotic rate and Caspase 3 activity in RAW264.7 cells，and the total apoptotic rate and Caspase 3 activity were further increased after knockdown of ATG5 expression(Figure 5A-C，E).Similar results were observed for early apoptotic rate.The primary macrophages were treated with 1μg/mL SAC，1μg/mL DADS，0.1μg/mL DTS，5 mM 3-MA，1μg/mL SAC plus 5 mM 3-MA，1μg/mL DADS plus 5 mM 3-MA，or 0.1μg/mL DTS plus 5 mM 3-MA for 4 h，and the activity of Caspase 3 was tested.Our data showed that treatment with 1μg/mL DADS，1μg/mL SAC or 0.1μg/mL DTS increased the activity of Caspase 3 in primary macrophage cells，and the activity of Caspase 3 was further increased after the cells were treated with 3-MA(Figure 5F)However，there were no significant differences in late apoptosis between untreated cells and cells treated with 1μg/mL DADS，1μg/mL SAC or 0.1μg/mL DTS(Figure 5D).(A)The RAW264.7 cells transfected with Pll3.7-puro vector(NC)or ATG5-knockdown RAW264.7 cells were incubated with or without 1μg/mL DADS，1μg/mL SACor 0.1μg/mL DTS for 4 h and stained with Annexin V and PI for 10min.Then the cells were analyzed by flow cytometry.At least three replicates were performed.For cells treated as in(A)，the percentages of total apoptotic cells(B)，early apoptotic cells(C)and late apoptotic cells(D)were determined.Significant differences between the NC group and cells treated with 1μg/mL DADS，1μg/mL SAC or 0.1μg/mL DTS were determined by one-way ANOVA.*P＜0.05，**P＜0.01，***P＜0.001.At least three replicates were performed.(E)The Caspase 3 activity of cells treated as in(A).Data are presented as the mean±SD；*P＜0.05，**P＜0.01 indicate significant differences for comparisons with ATG5knockdown RAW264.7 cells treated with 1μg/mL DADS，1μg/mL SACor 0.1μg/mL DTS.##P＜0.01 indicates significant differences for comparisons with control cells.At least three replicates were performed.(F)The Caspase 3 activity of primary macrophages treated with 1μg/mL SAC，1μg/mL DADS，0.1μg/mL DTS，5 mM 3-MA，1μg/mL SAC plus 5 mM 3-MA，1μg/mL DADS plus 5 mM 3-MA，or 0.1μg/mL DTS plus 5 mM 3-MA for 4h.Data are presented as the mean±SD；**P＜0.01 indicate significant differences for comparisons with the cells treated with 1μg/mL DADS，1μg/mL SAC or 0.1μg/mL DTS.##P＜0.01 indicates significant differences for comparisons with control cells.At least three replicates were performed.