B
C
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Figure 1. Establishment of polyQ disease cell models.
(A) Plasmid construction of ΔC-TBP/Q36 and Q79. The ΔC-TBP
constructs with enclosure of 36 or 79 glutamine repeats by conjugating with EGFP at 3’-end were established. (B) The fluorescent images ofΔC-TBP-Q36 or Q79 stable cell line. ΔC-TBP-Q79 cells expressed
more aggregation dots than ΔC-TBP-Q36 cells after treating with DOX (20 µg/mL) for 4 days. (Scale bar = 20 μm) (C) Western blot analysisdetected polyQ-EGFP protein expression. Comparing with different
cloning of stable cell line and detection of recombination protein after cells treated with doxycycline 20 µg/mL for 4 days.62
A
63
B
Figure 2. Screening of autophagy enhancing chemicals
(A) Image of Q79 cells for screening compounds by high-content imaging detection. Lysosome activator were selected by LysoTracker
staining. The nuclei were stained by DAPI (blue) and activated lysosomes stained by LysoTracker (Red). (Scale bar = 100 μm) (B) The candidatechemicals displayed different lysosome staining. Compounds OC-13,
OC-14, YR-21 and YR-72 activated lysosome activity lysosome.Quantitation of LysoTracker fluorescence intensity determination by high-content imaging. (**P < 0.01, unpaired Student's t-test as compared with vehicle control DMSO, N=3)
64
A
B
65
Figure 3. Changes of cell viability and aggregation intensities by candidate compounds.
(A) Cell number counting. Compounds OC-04, OC-10, OC-13, YR-72
and YR-297 maintained cell viabilities in Q79 cells. High-content images were stained DAPI in nucleus and counted. (B) Quantitativeaggregation intensities. Aggregation intensities were decreased by
OC-13, OC-14 and trehalose. (**P < 0.01, unpaired Student's t-test ascompared with vehicle control DMSO, N=3)
66
A
B
67
Figure 4. The variations of autophagy markers.
(A) Western blot analysis detected LC3-II protein. OC-14, YR-21 and
YR-72 induced LC3-II in Q36. OC-13, OC-14, YR-21 and YR-72induced LC3-II in Q79 cells. Cells was treated with candidate chemicals at 20 µM for 48 h. (B) Quantitative densitometry of LC3-II/LC-I in
Western blot. OC-13 and OC-14 specifically induced autophagy in Q79
cells.68
A
B
Figure 5. The candidate chemical OC-13 and cell viability determination
(A) The chemical structure of OC-13. (B) Determination of viable cell number. Q36 and Q79 cells were treated with 5, 10, 20 µM or DMSO
control for 48 h after induction by Dox for 4 days. Cells were trypsinized and collected. And the numbers of viable cells were counted (**P <0.01, one-way ANOVA, compared with vehicle control DMSO).Q36 Q79
69
A
B
70
71
Figure 6. Activation of lysosome by OC-13.
(A) Image of LysoTracker staining. OC-13-activated lysosome was
detected by fluorescence microscopy. Activated lysosomes in Q79 cells were stained by LysoTracker after treatment with OC-13 (5, 10 or 20 µM) or DMSO. (Scale bar = 100 μm) (B) Flow cytometric analysis. The Lysotracker fluorescence was determined by flow cytometric analysis, OC-13 increased LysoTracker (FL1-H) intensity in Q79 cells. The top panel indicated Q36 (top left) or Q79 (top right) cells treatment with 20 µM of OC-13 for 0, 12, 24, or 48 h. The bottom panel indicated Q36 (low left) or Q79 (low right) cells was treated with 5, 10, 20 µM or DMSO control for 48 h. (C) Quantitation of temporal changes of LysoTrackerfluorescence. OC-13 significantly increased LysoTracker fluorescence
intensity in Q79 cells. (*P<0.05, **P<0.01, Student’s t-test, compared with 0 h). (D) Quantitation of LysoTracker fluorescence intensitieswith increasing OC-13 concentrations. OC-13 was increased
LysoTracker fluorescence intensities in Q79 cells in a dose-dependent manner. (*P <0.05, **P <0.01, one-way ANOVA, compared with vehicle control DMSO, N=5).
72
A
B
Q36 Q79
LC3-II/LC3-I ratio
0.0 0.5 1.0 1.5
2.0 DMSO
**
*
*
73
Figure 7. Induction of autophagy markers by OC-13.
(A) Western blot analysis autophagic marker. OC-13 induced
conversion of LC3-I to LC3-II, level of Beclin 1 and degradation of p62 in Q79 cells. Q36 or Q79 cells were treated with OC-13 (5, 10 or 20 µM) or DMSO control for 48 h. (B) Densitometric analysis of LC3-II/LC3-I
on Western blot. OC-13 induced conversion of LC3-I to LC3-II in
significant increasing manner. (*P <0.05, **P <0.01, one-way ANOVA, compared with vehicle control DMSO, N=5)
74
A
B
Figure 8. Inhibition of autophagy by Baf A1.
(A) Western blot analysis for autophagic flux. OC-13 induced
autophagic flux, degradation of p62 and LC3-II were repressed by Baf A1 in Q79 cells. Q79 cells were pretreated with Baf A1 1 nM and treated with OC-13 (5, 10 or 20 µM) or DMSO control for 48 h. (B)
Densitometric analysis of LC3-II/LC3-I on Western blot. Baf A1
repressed degradation of LC3-II in significant increasing manner. (*P<0.05, **P <0.01, two-way ANOVA, N=5)
D 5 10 20
75
A
B
D 5 10 20
LC3 puncta per 3 cells
0 20 40 60 80
**
*
**
76
Figure 9. Autophagosome formation and aggregates clearance by OC-13.
(A) The confocal microscopy analysis of autophagosome puncta.
OC-13 increased autophagosome in Q79 cells. Q79 cells were treated with OC-13 (5, 10 or 20 µM) or DMSO control for 48 h. Nucleus was stained with DAPI (blue), Q79-EGFP (green) was expressed and autophagosome was stained by LC3 antibody and secondary antibody conjugated with TRITC (red) in Q79 cells. (Scale bar = 10 µm) (B) Counting of LC3puncta.
OC-13 induced formation of autophagosome with increasing OC-13 concentrations. (*P <0.05, **P <0.01, one-way ANOVA, compared with vehicle control DMSO, N=3)77
A.
B.
Figure 10. Elimination of Q79 aggregation dots by OC-13.
(A) Fluorescence microscopy analysis for Q79 aggregation dots.
OC-13 decreased aggregation dots (arrows) in Q79 cells. Q79 cells were treated with OC-13 (5, 10, 20 µM) or DMSO control for 48 h. (scale bar= 20 µm) (B) Counting of aggregation dots in Q79 cells. The dots numbers were counted in the 500 EGFP positive cells at each
concentration. (**P < 0.01, one-way ANOVA as compared with vehicle control DMSO, N=7)
78
A
B
C
Q36 Q79
Insoluble polyQ-EGFP ratio
0.0 0.5 1.0 1.5 2.0
DMSO
** **
**
79
Figure 11. Elimination of the insoluble EGFP by OC-13.
(A) Western blot analysis and filter trap assay for Q79 insoluble EGFP. OC-13 decreased insoluble EGFP and increased soluble EGFP in
Q79 cells byWestern blot analysis and filter trap assay. Protein lysates of the Q36 and Q79 cells was collected by treating with 5, 10, or 20 µM of OC-13 for 48 h. In filter trap assay,the insoluble pellets were collected and lysed in sodium dodecyl sulfate buffer. (B) Densitometric analysisof Q79 insoluble EGFP. OC-13 induced elimination of Q79 insoluble
EGFP in significant increasing manner. (C) Densitometric analysis ofQ79 soluble EGFP. OC-13 increased Q79 soluble EGFP in significant
increasing manner. (*P <0.05, **P < 0.01, one-way ANOVA as compared with vehicle control DMSO, N=5)Q36 Q79
80
A
Figure 12. Activation of JNK pathway.
(A) Western blot analysis for OC-13-induced signaling pathway in Q79. OC-13 induced phosphorylation of Akt, S6k and JNK in Q79 cells.
Protein lysates of the Q36 and Q79 cells was collected by treating with 5, 10, or 20 µM of OC-13 for 48 h.
81
A
B
W.O.I Insulin 3-MA Baf A1 Sp600125MG-132 Number of aggregation cells /500 EGFP+ cells
0 20 40 60 80 100
120 DMSO
OC-13 20 M
** *
*
82
Figure 13. Elimination of Q79 aggregation by JNK pathway and autophagy.
(A) Fluorescence microscopy analysis for Q79 aggregation dots.
Autophagy and JNK inhibitor repressed OC-13-mediated elimination of aggregation dots (arrows) in Q79 cells. Q79 cells were pretreated with 200 nM insulin, 20 µM 3-MA, 1 nM Baf A1, 10 µM Sp600125 or MG-132 for 24 h and treated with OC-13 20 µM or DMSO control for 48 h.
(scale bar = 20 µm) (B) Counting of aggregation dots in Q79 cells.
Elimination of aggregation dots was repressed by autophagy and JNK inhibitor in significant increasing manner. The dots numbers were
counted in the 500 EGFP positive cells at each concentration. (*P <0.05,
**P < 0.01, two-way ANOVA, N=5)
83
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B
Figure 14. Elimination of the insoluble EGFP as affected by inhibitors.
(A) Western blot analysis for Q79 insoluble EGFP. Autophagy and
JNK inhibitor repressed OC-13-mediated elimination of insoluble EGFP in Q79 cells byWestern blot analysis. Protein lysates of the Q79 was collected by pretreating with inhibitor and treating with 20 µM of OC-13 for 48 h. (B) Densitometric analysis of Q79 insoluble EGFP.Autophagy and JNK inhibitor repressed OC-13-mediated elimination of Q79 insoluble EGFP in significant increasing manner. (*P <0.05, **P <
0.01, two-way ANOVA, N=5)
W.O.I Insulin 3-MA Baf A1 Sp600125MG-132
Insoluble polyQ-EGFP ratio
84
A
B
W.O.I Insulin 3-MA Baf A1 Sp600125MG-132
LC3 puncta per 3 cells
0 20 40 60 80
DMSO OC-13 20 M
** *
*
*
85
Figure 15. The increase of autophagosomes by JNK pathway.
(A) Confocal microscopy analysis of autophagosome puncta.
Autophagy and JNK inhibitor repressed OC-13-induced autophagosome in Q79 cells. Q79 cells were pretreated inhibitor and treated with 20 µM OC-13 or DMSO control for 48 h. Nucleus was stained with DAPI (blue), Q79-EGFP (green) was expressed and autophagosome was stained by LC3 antibody and 2° antibody connection with TRITC (red) in Q79 cells.
(Scale bar = 10 µm) (B) Counting of LC3 puncta. Autophagy and JNK inhibitor repressed OC-13-induced formation of autophagosome in significant increasing manner. (*P <0.05, **P <0.01, two-way ANOVA, N=3)
86
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Cytoplasm Nucleus
Number of Nucleic polyQ aggrgation /500 GFP+ cell 0 10 20 30 40
0 5 10 20 M
****
** **
**
87
C
D
cytoplasm nucleus
Insoluble polyQ
0.0 0.5 1.0 1.5 2.0
0 5 10 20M
** **
**
*
88
Figure 16. Elimination of nucleus Q79 aggregation.
(A) Confocal microscopy analysis for Q79 aggregation dots. OC-13
decreased nucleus aggregation dots in Q79 cells. Q79 cells were treated with OC-13 (5, 10 or 20 µM) or DMSO control for 48 h. Nucleus was stained with DAPI (blue), Q79-EGFP (green) was expressed andlysosome was stained by LysoTracker (red) in Q79 cells. (Scale bar = 10 µm) (B) Counting of nucleus aggregation dots. OC-13 decreased nucleus and cytoplasmic aggregation dots in Q79 cells by significant increasing manner.The nucleus or cytoplasmic dot numbers were counted in the 500 EGFP positive cells at each concentration. (C) Western blot
analysis for nucleus insoluble EGFP. OC-13 induced elimination of
insoluble nucleus EGFP in Q79 cells byWestern blot analysis. Both nucleus and cytoplasmic protein lysates of the Q79 was collected after treating with 5, 10 or 20 µM of OC-13 for 48 h. (D) Densitometricanalysis of Q79 insoluble EGFP. OC-13 eliminated of nucleus insoluble
EGFP as the concentrations were increased. (*P <0.05, **P < 0.01, one-way ANOVA as compared with vehicle control DMSO, N=5)89
A
B
W.O.I 3-MA Baf A1 sp600125 MG-132
% of nucleus aggrgation cells
0 10 20 30 40 50 60 70
DMSO OC-13 20 M
** *
90
Figure 17. Clearance of nucleus Q79 aggregation by autophagy and JNK pathway.
(A) Counting of nucleus aggregation dots. Autophagy and JNK
inhibitor suppressed OC-13-mediated clearance of nucleus and cytoplasmic aggregation dots in Q79 cells by significant increasing manner.The nucleus or cytoplasmic dots numbers were counted in the 500 EGFP positive cells at each concentration. (*P <0.05, **P < 0.01, two-way ANOVA as compared with vehicle control DMSO, N=5) (B)Western blot analysis for nucleus insoluble EGFP. Autophagy and
JNK inhibitor suppressed OC-13-induced elimination of nucleus and cytoplasmic insoluble EGFP in Q79 cells byWestern blot analysis.Nucleus and cytoplasmic protein lysates of the Q79 cells was collected by pretreating with 10 µM of 3-MA for 24 h and treating with 20 µM of OC-13 for 48 h.
91
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Figure 18. Establishment of Httex1 Q25 and Q97 cell models.
(A) Plasmid construction of Httex1-Q25 and Q97. The polyQ in
N-terminal huntingtin fragments contained various repeats of huntingtin exon. Httex1-Q25 and Q97 constructs joined with GFP at 3’-end. (B)Image of cells with transient transfection of Httex1-Q25 and Q97.
Cells transfected with Httex1-Q97 contained more visible aggregation dots (arrows) than those with Httex1-Q25 after 24 h. (scale bar = 20 μm)
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93
C
Figure 19. Increasing of autolysosome by OC-13 in Httex1-Q25 and Q97-transfected cells.
(A) Gating the GFP positive cells.
The major populations in SSC/FSC and GFP positive population were gated in cells transfected withHttex1-Q25 and - Q97. (B) Flow cytometric analysis. Lysotracker fluorescence
was determined by flow cytometric analysis, OC-13 increasedLysoTracker (FL1-H) intensity in Httex1-Q97-transfected cells. Both transfected cells were treated with OC-13 (5, 10 or 20 µM) or DMSO control for 12 h. (C) Quantitative LysoTracker fluorescence
intensities. OC-13 increased LysoTracker fluorescence intensities in Httex1-Q97-transfected cells. (*P <0.05, **P <0.01, one-way ANOVA,
compared with vehicle control DMSO, N=5).Httex1-Q25 Httex1-Q97
94
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B
95
Figure 20. Activation of lysosomes by OC-13 in Httex1-Q97 transfected cells.
(A) Image of lysosome puncta in Httex1-Q97-transfected cells. OC-13
induced activation of lysosome by fluorescence microscopy.Httex1-Q97
transfected cells were treated with 5, 10 or 20 µM of OC-13 or DMSO control for 12 h and stained with LysoTracker. Nucleus was stained by DAPI (blue) and activation of lysosomes were stained by LysoTracker (Red). (Scale bar = 100 μm) (B) Quantitative of lysosome intensity.OC-13 induced LysoTracker fluorescence intensities in Httex1-Q97-transfected cells. The LysoTracker fluorescence intensities were quantized by imageJ in fluorescence microscopy image. (**P < 0.01, one-way ANOVA as compared with vehicle control DMSO, N=3)
96
A
Figure 21. Formation of autophagosome in Httex1-Q97 transfected cells.
(A) Confocal assay for autophagosome. Formation of autophagosome
were increased after treatment with 5, 10 or 20 µM of OC-13 and DMSO control for 12 h in Httex1-Q97 transfected cells. Httex1-Q97 transfected cells were treated with 5, 10 or 20 µM of OC-13 or DMSO control for 12 h. Nucleus was stained with DAPI (blue), Httex1-Q97-GFP (green) was expressed and autophagosome was stained by LC3 antibody and 2°antibody connection with TRITC (red) in Q79 cells. (Scale bar = 10 µm)
97
A
B
98
Figure 22. Induction of autophagic markers in Httex1-Q97 transfected cells by OC-13.
(A) Western blot analysis autophagic marker. OC-13 induced
conversion of LC3-I to LC3-II and degradation of p62 in Httex1-Q97 transfected cells. Httex1-Q25 and Q97 transfected cells were treated with OC-13 or DMSO control for 12 h. (B) Densitometric analysis ofII/I on Western blot. OC-13 induced conversion of I to
LC3-II in significant increasing manner. (*P <0.05, **P <0.01, one-way ANOVA, compared with vehicle control DMSO, N=5)99
A
B
100
C
Figure 23. Maintenance of cell viability in Httex1-Q97 transfected cells.
(A) Image of death cells in Httex1-Q97 transfected cells. Death cells
were indicated by PI staining (arrows) in Httex1-Q97 transfected cells.Httex1-Q97 expression cells were stained with PI 0.5 µg/mL. (Scale bar = 20 µm) (B) Flow cytometric analysis. To Gate the GFP positive cells, ratio of PI positive cells was determined by flow cytometric analysis. OC-13 decreased PI positive cells (M1) in Httex1-Q25 and Httex1-Q97
transfected cells. Httex1-Q25 (left) or Q97 (right) transfected cells were treated with 5, 10 or 20 µM of OC-13 or DMSO control for 12 h. (C)
Quantitative of ratio of PI positive cells in increasing of OC-13 concentrations. OC-13 was decreased PI positive cells in Httex1-Q25
and Httex1-Q97 transfected cells. (*P <0.05, one-way ANOVA,compared with vehicle control DMSO, N=5).
Httex1-Q25 Httex1-Q97
101
A
Figure 24. Activation of JNK, Akt and S6K in Httex1-Q97 transfected cells.
Western blot analysis for autophagy signaling pathway.
Phosphorylation of JNK, Akt and S6K were induced by OC-13.
Httex1-Q97 transfected cells were treated with OC-13 (5, 10 or 20 µM) or
DMSO control for 12 h.102
A
B
Number of aggregation cells /500 GFP+ cells 0 20 40 60 80
D 5 10 20
OC-13
**
103
Figure 25. Elimination of aggregations in Httex1-Q97 transfected cells.
(A) Fluorescence microscopy analysis for Httex1-Q97 aggregation dots. OC-13 decreased aggregation dots (arrows) in Httex1-Q97
transfected cells. Httex1-Q97 transfected cells were treated with 5, 10 or 20 of µM OC-13 or DMSO control for 12 h. (scale bar = 20 µm) (B)
Counting of aggregation dots in Httex1-Q97 transfected cells. OC-13
decreased Httex1-Q97 aggregation dots in significant decreasing manner.The dots numbers were counted in the 500 GFP positive cells at each concentration. (**P < 0.01, one-way ANOVA as compared with vehicle control DMSO, N=5)
104
A
B
C
105
Figure 26. Elimination of the insoluble Httex1-Q97 aggregates by OC-13.
(A) Western blot analysis for insoluble from protein. In interface of
stacking gel, Httex1-Q97 insoluble from protein was decreased after by OC-13. Httex1-Q97 transfected cells were treated with 5, 10 or 20 µM of OC-13 and DMSO control for 12 h. (B) Filter retardationassay. The insoluble Httex1-Q97 was decreased by OC-13 in
filter retardation assay. The insoluble pellets were collected and lysed in sodium dodecyl sulfate buffer. (C) Quantitation of insoluble frompolyQ-EGFP densitometry. The insoluble form polyQ-EGFP were
decreased by OC-13 in a dose-dependent manner with significant difference. (**P < 0.01, one-way ANOVA as compared with vehicle control DMSO, N=3)106
A
B
Number of aggregation cells /500 GFP+ cells 0 20 40 60 80 100
**
OC-13
3-MA
- -
+ +-
+-
+107
Figure 27. Clearance inhibition of aggregation.
(A) Fluorescence microscopy analysis for Httex1-Q97 aggregation dots. Autophagic inhibitor blocked OC-13-mediated clearances of
aggregation dots (arrows) in Httex1-Q97 transfected cells. Httex1-Q97 transfected cells were pretreated inhibitor 3-MA at 10 µM for 1 h and treated with OC-13 at 20 µM or DMSO control for 12 h. (scale bar = 20 µm) (B) Counting of aggregation dots inHttex1-Q97 transfected cells.
3-MA inhibits OC-13-induced clearance of Httex1-Q97 aggregation dots in significant decreasing manner. The dots numbers were counted in the 500 GFP positive cells at each concentration. (**P < 0.01, two-way ANOVA as compared with vehicle control DMSO, N=5)
108
A
B
Insoluble polyQ-EGFP Ratio
0.0 0.4 0.8 1.2 1.6
OC-13
**
3-MA
- -
+ +-
+-
+109
Figure 28. Clearance inhibition of insoluble Httex1-Q97.
(A) Western blot analysis for insoluble Httex1-Q97. OC-13-mediated
clearance of Httex1-Q97 insoluble from protein was repressed byautophagy inhibitor. Httex1-Q97 transfected cells pretreated 10 µM 3-MA 1 h after treatment with 20 µM OC-13 and DMSO control for 12 h. (B)
Quantitation of insoluble from polyQ-EGFP Densitometry. 3-MA
significantly repressed OC-13-mediated clearance of Httex1-Q97insoluble from protein. (**P < 0.01, two-way ANOVA as compared with vehicle control DMSO, N=3)
110
A
B
W.O.I Insulin 3-MA Baf A1 Sp600125MG-132 Number of aggregation cells /500 EGFP+ cells
0 20 40 60 80
DMSO OC-13 20 M
** *
*
111
Figure 29. Elimination of Httex1 aggregation dots by JNK pathway and autophagy.
(A) Fluorescence microscopy analysis for Q79 aggregation dots.
Autophagy and JNK inhibitor repressed OC-13-mediated elimination of aggregation dots (arrows) in Httex1-Q97 transfected cells. Httex1-Q97 transfected cells were pretreated with 200 nM insulin, 20 µM 3-MA, 1 nM Baf A1, 10 µM Sp600125 or MG-132 for 1 h and treated with OC-13 at 20 µM or DMSO control for 12 h. (scale bar = 20 µm) (B) Counting
of aggregation dots in Httex1-Q97 transfected cells. Elimination of
aggregation dots was repressed by autophagy and JNK inhibitor insignificant increasing manner. The dots numbers were counted in the 500 GFP positive cells at each concentration. (*P <0.05, **P < 0.01, two-way ANOVA as compared with vehicle control DMSO, N=5)
112
A
Figure 30. The increase of autophagosomes by JNK pathway in Httex1-Q97 transfected cells.
(A) Confocal microscopy analysis of autophagosome puncta.
Autophagy and JNK inhibitor repressed OC-13-induced autophagosome in Httex1-Q97 transfected cells. Httex1-Q97 transfected cells were pretreated inhibitor and treated with OC-13 at 20 µM or DMSO control for 48 h. Nucleus was stained with DAPI (blue), Q79-EGFP (green) was expressed and autophagosome was stained by LC3 antibody and
secondary antibody connection with TRITC (red) in Httex1-Q97 transfected cells. (Scale bar = 10 µm)
113
Figure 31. Summary of OC-13 induced polyQ clearance by autophagy.
OC-13 induced autophagic marker and flux to reduce nucleus and cytoplasm polyQ aggregation by JNK activation in Httex1- and TBP-polyQ expression neuroblastoma SK-N-SH.
114
115
D
Figure 32. Inhibition of cell proliferation in human non-small cell lung cancer cells.
(A) The chemical structure of the compound BTO (B) MTT assay for cell viability. BTO inhibited the cell viability of A549 and H460 cells as
indicated. A549, H1299 and H460 were treated with BTO (2, 5 or 10 μM) or DMSO control.The relative cell viabilities were converted from the absorbance of drug-treated cells against that of DMSO control. (C)Colony formation assay for cell proliferation. BTO inhibited cell
proliferation in A549, H1299 and H460 colonies. 500 cells were growth in fresh medium for 15 days after cells treated with BTO. (D)Quantitative analysis of colony formation assay. BTO significantly
inhibited proliferation cell in NSCLC by dose-dependent manner. The number of colonies, defined as more than 50 cells per colony, was counted. (*P <0.05, **P < 0.01, one-way ANOVA as compared with vehicle control DMSO, N=5)A549 H1299 H460
116
A
B
117
118
Figure 33. Activation of apoptosis by BTO.
(A) Flow cytometry for apoptosis assay.
BTO induced apoptosis in A549 and H460.A549, H1299 and H460 cells were treated with BTO for 48 h and assayed by double staining with Annexin V-FITC/PI. Thepercentage of the upper right quadrant indicated late apoptosis and the lower right early apoptosis. (B) Quantitative analysis for apoptosis
population. Population cells of early (dark) and late (light) apoptosis
were quantized byflow cytometry. (*P <0.05, one-way ANOVA as compared with vehicle control DMSO, N=5)119
A
Figure 34. Activation of autophagy and apoptosis by BTO.
(A) Western blot analysis for autophagy and apoptosis. OC-13
induced apoptosis marker cleavage PARP, Bax and cytochrome c and reduced anti-apoptosis maker Akt and Bcl-2 in A549 and H460 cells.Autophagy maker Beclin 1 was induced by BTO in A549 and H460 cells.
Protein lysates of the A549, H1299 and H460 cells was collected by treating with 2, 5 or 10 µM of BTO for 48 h.Numbers underneath signified relative intensities compared with that of DMSO vehicle control.
120
A
B
121
Figure 35. Apoptosis and autophagy in adenocarcinoma.
(A) Western blot analysis for apoptosis. OC-13 induced apoptosis
marker cleavage PARP, Bax and cytochrome c and reduced anti-apoptosis maker Akt and Bcl-2 in A549 and H460 cells. Autophagy maker Beclin 1 was induced by BTO in A549 and H460 cells. Protein lysates of the A549 and H460 cells was collected by treating with 2, 5 or 10 µM of BTO for 48 h.Numbers underneath signify relative intensities compared with that of DMSO vehicle control treatment at 12 h122
A
B
123
Figure 36. Formation of autophagosome and autolysosome by BTO.
(A-B) Confocal microscopy analysis for apoptosis BTO induced
formation of autophagosome (Green) and autolysosome (Orange) in A549 cells. A549 cells were treated with BTO at 2, 5, 10 µM or DMSO control for 48 h and 10 µM for 12, 24, 48 h. Nucleus was stained with DAPI (blue), autophagosome was indicated by GFP-LC3 (green) expression and lysosome was stained LysoTracker in A549. (Scale bar = 10 µm)
124
A
B
D BTO
Ratio of autolysosome
0 2 4 6 8 10 12
w/o 3-MA
* 3-MA
125
Figure 37. Repression of autophagic flux by 3-MA.
(A) Confocal microscopy analysis for autophagy. Autophagic inhibitor
3-MA repressed formation of autophagosome (Green) and autolysosome (orange) in A549. A549 cells were pretreated with 10 µM 3-MA and treated with BTO at 10 µM or DMSO control for 48 h. The nucleus was stained with DAPI (blue), autophagosome was indicated by GFP-LC3 (green) expression and lysosome was stained LysoTracker in A549.(Scale bar = 10 µm) (B) Quantitation of autolysosome puncta.
Formation of autolysosome (Orange) was induced OC-13 and suppressed by 3-MA in significant increasing manner. Counting of autolysosome was indicated by colocalization with LC3-GFP puncta and LysoTracker. (*P
<0.05, two-way ANOVA, compared with vehicle control DMSO, N=3)
126
127
Figure 38. Inhibition of BTO-mediated apoptosis by 3-MA.
(A) Flow cytometry for apoptosis assay. 3-MA inhibited
BTO-induced apoptosis in A549.A549 and H1299 cells were pretreated 3-MA for 24 h and treated with BTO for 48 h and assayed by double staining withAnnexin V-FITC/PI. The percentage of the upper right quadrant indicated late apoptosis and the lower right early apoptosis. (B) Quantitative
analysis for apoptosis population. Population cells of early (dark) and
late (light) apoptosis were quantized byflow cytometry. (*P <0.05, two-way ANOVA, compared with vehicle control DMSO, N=3)128
A
Figure 39. BTO-induced autophagic apoptosis in A549.
(A) Western blot analysis for autophagic inhibitor. 3-MA repressed
OC-13-mediated apoptosis marker, anti-apoptosis maker and autophagy maker in A549 and H460 cells. Protein lysates of the A549 and H460 cells was collected by treating with 10 µM of BTO for 48 h. Numbers underneath signify relative intensities compared with that of DMSO vehicle control treatment of each cell line.129
A
B
130
Figure 40. Increase of ROS intensity by BTO.
(A) Flow cytometric analysis for ROS. ROS was increased by treatment
with BTO by flow cytometric analysis. Cellular ROS was detected by10 µM H2DCFDA for 1 h after treating with BTO (2, 5 or 10 µM) for 12, 24 or 48 h. Positive control was indicated by H2O2 and negative control was indicated by NAC. (B) Quantitative analysis of ROS intensities. BTO were induced ROS intensity by time and dose-dependent manner. Mean of ROS intensity was quantized by flow cytometric. (*P<0.05, **P <0.01, one-way ANOVA as compared with DMSO for 12 h, N=3)
131
A
B
control BTO
Fold of ROS intensity changes
0.0 0.5 1.0 1.5
2.0 w/o NAC NAC
*
132
C
Figure 41. Inhibition of autophagy by NAC.
(A) Flow cytometric analysis for anti-ROS. NAC inhibited
BTO-induced ROS. Cellular ROS was detected by staining with10 µM of H2DCFDA for 1 h after pretreating 5 mM of NAC for 1 h and treating with BTO (2, 5 or 10 µM) for 12, 24 or 48 h. Mean of ROS intensity was quantized by flow cytometric. (*P<0.05, one-way ANOVA as compared with PBS, N=3) (B) Confocal microscopy analysis for autophagyinhibition by ROS. ROS inhibitor NAC repressed formation of
autophagosome (green) and autolysosome (orange) in A549. A549 cells were pretreated with 5 mM of NAC for 1 h and treated with 10 µM of BTO or DMSO control for 48 h. Nucleus was stained with DAPI (blue), autophagosome was indicated by GFP-LC3 (green) expression and lysosome was stained LysoTracker (red) in A549. (Scale bar = 10 µm)
(C) MTT assay for ROS repression by NAC. NAC suppressed
BTO-mediated inhibition of cell viability. A549 cells were pretreated with 5 mM of NAC for 1 h and treated with BTO (2, 5 or 10 µM) or DMSO for 48 h. (*P<0.05, two-way ANOVA as compared with PBS, N=3)DMSO 2 5 10
133
A
B
134
C
Figure 42. Inhibition of tumor growth in nude mice xenografts.
(A) Determination of tumor size. Tumor growth was inhibited by BTO
(1 mg/kg/mouse). BTO were injected subcutaneously every 3 or 4 days for four weeks. (B) The excised tumor xenografts of A549. BTOdecreased excised tumor xenografts of A549. (C)
Quantitative analysis for volume of excised tumor. The decreased tumors volume by BTO
treatment as excised from nude mice.(*P <0.05, **P < 0.01, unpaired Student's t-test as compared with PBS, N=3)PBS BTO
135
A
B
C
136
Figure 43. Increase of apoptosis in nude mice xenografts.
(A) Determination of body weight of mice. The average body weight of
mice showed no significant change as compared with PBS and BTO treatment. (B) Western blot analysis for tumor apoptosis. Protein lysates of the extracted tumors were analyzed by Western blot, BTO induced apoptosis and reduced proliferation markers. (C) H&E stainingof tumor. The tumor sections reduced cell densities by inducing apoptotic
body (white arrows) after BTO treatment. (Scale bar = 50 μm)137
A
B
138
Figure 44. Decrease of proliferation and increase of autophagy in tumor.
(A) Confocal microscopy analysis for proliferation. The proliferation
marker PCNA was decreased by treatment with BTO. Nucleus was stained with DAPI (blue) and PCNA stained by PCNA antibody and secondary antibody connection with FITC (green) in tumor tissues of xenografts tumor. (Scale bar = 50 µm). The areas of inset were amplified to the right.(Scale bar = 20 µm)(B) Confocal microscopy analysis for
autophagy. Autophagy marker LC3 puncta was increased by treatment
with BTO. Nucleus was stained with DAPI (blue), LC3 was stained by LC3 antibody and secondary antibody connection with FITC (green) in xenografts tumor.(Scale bar = 50 µm). The areas of inset were amplified to the right. (Scale bar = 20 µm)139
A
B
140
Figure 45. Promotion of apoptosis marker in tumors.
(A) Confocal microscopy analysis for p53. Tumor suppressor marker
p53 was increased and transported to nucleus by treatment with BTO.Nucleus was stained with DAPI (blue), p53 was stained by p53 antibody and secondary antibody connection with TRITC (red) in xenografts
tumor. The areas of inset were amplified to the right. (Scale bar = 50 µm).
The areas of inset were amplified to the right. (Scale bar = 20 µm) (B)
Confocal microscopy analysis for apoptosis. TUNEL positive cells
were increased by treatment with BTO. Nucleus was stained with DAPI (blue) and apoptosis cells were stained by TUNEL (green) assay in xenografts tumor (scale bar = 50 µm). The areas of inset were amplified to the right. (Scale bar = 20 µm)141
Figure 46. Summary of BTO induced autophagy.
BTO induced autophagic marker and flux to promote apoptosis and reduce proliferation by increasing of ROS level in A549 non-small cell lung cancer.
142
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