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Supplementary Materials
Figure S1
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Figure S1. Topotecan induced phosphorylation of ATM in A549 and HeLa cell lines.
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(A) A549 and HeLa cells were treated with various concentrations of topotecan (0 to 6
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μg/mL) for 24 h. The expression levels of phospho-ATM were analyzed by
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immunoblotting. (B) A549 and HeLa cell lines were treated with 3 μg/mL topotecan
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from 0 to 36 h; the expression of phospho-ATM was analyzed by immunoblotting. (C)
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A549 and HeLa cells were treated with various concentrations of CDDP (0 to 12
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μg/mL) for 24 h. The expression levels of phospho-ATM were analyzed by
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immunoblotting. (D) A549 and HeLa cell lines were treated with 6 μg/mL CDDP
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from 0 to 36 h; the expression of phospho-ATM was analyzed by immunoblotting.
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Figure S2
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Figure S2. PTEN-WT and PTENS113A were SUMOylated in vitro. HeLa cells were
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transiently transfected with 3×Flag-PTEN-WT or 3×Flag-PTENS113A plasmid,
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together with HA-SUMO1, as indicated. Then, HA-immunoprecipitates (IP) were
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immunoblotted for Flag-PTEN. Standard protein molecular weights (55 kDa and 72
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kDa) are indicated
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Figure S3
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Figure S3. Topotecan induced DNA damage in A549 and HeLa cells. (A) A549 and
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HeLa cells were treated with 3 μg/mL TPT for 12 h or 24 h. Representative
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photomicrograph images showed accumulation of γ-H2AFX as a marker of DNA
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damage (100×). (B) Histogram representing the percentage of γ-H2AFX-positive cells
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in control or TPT-treated group (**P<0.01, t test, n=3, bars represent SEM, Cells
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containingmore than five foci were scored as positive). (C) A549 and HeLa cells were
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treated with 3 μg/mL topotecan for the indicated periods. The expression levels of
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γ-H2AFX protein were analyzed by immunoblotting. (D) A549 cells with PTEN
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shRNA-mediated knockdown were transiently transfected with the 3×Flag-PTEN-WT
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(shPTEN-resistant) or 3×Flag-PTENS113A (shPTEN-resistant) plasmids. 24 h after
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transfection, the cells were treated with or without 3 μg/mL TPT. The expression
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levels of -H2AFX protein were analyzed by immunoblotting.
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Figure S4
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Figure S4. Effect of PTENS113A mutation on cell cycle stage and cell apoptosis. A549
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cells were transiently transfected with the 3×Flag-PTEN-WT or 3×Flag-PTENS113A
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plasmid, and 24 h after transfection, the cells were treated with or without 3 μg/mL
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TPT. (A) TPT induced cell cycle stage was determined by propidium iodide (PI)
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staining. Histogram represents the percentage of cells at different phase (G0/G1, S,
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G2/M) upon TPT treatment (P>0.05, t test, n=3, bars represent SEM). (B) TPT
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induced cell apoptosis was determined by immunoblotting. The expression levels of
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cleaved CASP3 and PARP protein were analyzed. The 17-kDa cleaved CASP3
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subunit is indicated. Bar graphs represent the relative cleaved PARP protein levels
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normalized to that of PARP of different groups. (*P<0.05, t test, n=3).
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Figure S5
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Figure S5. Effect of PTEN rescued expression of PTEN on TPT-induced autophagy
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in A549 cells. (A) A549 cells with PTEN shRNA-mediated knockdown were
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transiently transfected with the 3×Flag-PTEN-WT (shPTEN-resistant) plasmids. Cells
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were treated with or without 3 μg/mL TPT for 24 h. The expression levels of PTEN,
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MAP1LC3/LC3 and SQSTM1/p62 proteins were analyzed by immunoblotting. (B)
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The effect of PTENS113 phosphorylation on autophagy in A549 cells. A549 cells with
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PTEN shRNA-mediated knockdown
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3×Flag-PTEN-WT (shPTEN-resistant) or 3×Flag-PTENS113A (shPTEN-resistant)
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plasmid. The expression levels of the MAP1LC3/LC3 and SQSTM1/p62 proteins
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were analyzed by immunoblotting. (C) A549 cells were treated with or without 3
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μg/mL TPT for 24 h, followed by treatment with 100 mM Bafilomycin A1 (Baf.A1) as
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indicated. The expression level of the MAP1LC3/LC3 and SQSTM1/p62 protein were
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analyzed by immunoblotting.
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were transiently transfected with the
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Figure S6
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Figure S6.
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activation of the p-JUN-SESN2-AMPK pathway in A549 cells. (A) A549 cells with
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PTEN shRNA-mediated knockdown
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3×Flag-PTEN-WT (shPTEN-resistant) or 3×Flag-PTENS113A (shPTEN-resistant)
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plasmid. The cells were treated with or without 3 μg/mL TPT, and the expression
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levels of JUN, p-JUN, SESN2, AMPK, p-AMPK, RPS6KB/p70S6K and
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p-RPS6KB/p70S6K proteins were analyzed by immunoblotting. (B) The cells were
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treated with or without 3 μg/mL TPT and/or JUN siRNA for 24 h, and the expression
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levels
of
Rescue of PTEN expression promoted TPT-induced autophagy via
JUN,
p-JUN,
SESN2,
were transiently transfected with the
AMPK,
p-AMPK,
RPS6KB/p70S6K,
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p-RPS6KB/p70S6K and MAP1LC3/LC3 protein were analyzed by immunoblotting.
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(C) The cells were treated with or without 3 μg/mL TPT and/or AMPK siRNA for 24
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h,
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p-RPS6KB/p70S6K and MAP1LC3/LC3 proteins were analyzed by immunoblotting.
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and
the
expression
levels
of
p-AMPK,
AMPK,
RPS6KB/p70S6K,