Fig 1.
TIPRL interacts with the protein phosphatase 4 complex.
(A) 293T cells transfected with LPC FLAG vector (FLAG-TIPRL (-)) or LPC FLAG-TIPRL (FLAG-TIPRL (+)) were immunoprecipitated with FLAG conjugated beads followed by immunoblotting with the indicated antibodies. (B) TIPRL was immunoprecipitated from HeLa cells followed by immunoblotting with the indicated antibodies to assess endogenous interaction with TIPRL.
Fig 2.
TIPRL inhibits PP4 phosphatase activity and complex assembly.
(A) HeLa cells were transiently transfected with LPC FLAG (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL). 24hrs after transfection, cells were lysed and immunoprecipitated with PP4-C antibody, upon which phosphatase activity was measured. Data represent ± standard deviation of the mean of three independent experiments. (B&C) HeLa cells were transfected with a scramble (siSCR) or TIPRL siRNA (siTIPRL). 48hrs after transfection, cell lysates were immunoprecipitated with PP4-C (B) or PP4R2 (C) antibody and phosphatase activity was measured. Data represent ± standard deviation of the mean of three independent experiments. (D) HeLa cells transfected with LPC FLAG-TIPRL (FLAG-TIPRL) were immunoprecipitated with the indicated antibodies and phosphatase activity was measured. (E) HeLa cells were transfected with a scramble (siSCR) or TIPRL siRNA (siTIPRL) and immunoprecipitated with PP4R2 antibody followed by immunoblotting with the indicated antibodies. (F) HeLa cells were transiently transfected with LPC FLAG (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL) and immunoprecipitated with PP4-C antibody followed by immunoblotting with the indicated antibodies. Long and short refer to the film exposure time. (G) 3T3 cells were treated with DMSO (CONT) or 5μM CPT for 1hr. Cells were lysed and immunoprecipitated with TIPRL followed by immunoblotting with the indicated antibodies. Band intensity was quantified using Image ***p<0.001, Student’s t test.
Fig 3.
TIPRL promotes γ-H2AX phosphorylation in response to DNA damage.
(A) HeLa cells were transiently transfected with vector control (VEC) or FLAG-TIPRL. 24hrs after transfection, cells were treated with CPT (2.5μM) or (B) doxorubicin (DOXO, 2μg/ml) for the indicated time points. Cells were lysed and immunoblots were probed with the indicated antibodies. (C) HeLa cells were transiently transfected with vector control (VEC) or FLAG-TIPRL. 24hrs after transfection, cells were treated with CPT (2.5μM) for 1.5h followed by washout for 6h. Cells were lysed and immunoblots were probed with the indicated antibodies. (D) 3T3 cells stably expressing LPC FLAG vector control (VEC) or LPC FLAG- TIPRL (TIPRL) were treated with 5μM CPT for 1.5hrs. Cells were fixed and stained for both DAPI and γ-H2AX and (E) images were quantified. Data represent ± standard deviation of the mean of three fields**p<0.01, Student’s t test.
Fig 4.
Knockdown of TIPRL inhibits γ-H2AX phosphorylation upon DNA damage.
(A) HeLa cells were transfected with a scramble (siSCR) or TIPRL siRNA (siTIPRL). 48hrs after transfection, cells were treated with 2.5μM CPT for 1.5hrs. Cell lysates were prepared and immunoblotting was performed using the indicated antibodies. (B) 3T3 MEFs infected with retrovirus containing a short hairpin (sh) against TIPRL (shTIPRL) or scrambled shRNA (shSCR) were treated with 2.5μM CPT for 1.5hrs. The drug was washed out of the cells and fresh media was added back for the indicated amount of time. Cell lysates were prepared and immunoblots were probed with the indicated antibodies. (C) 3T3 MEFs expressing a short hairpin (sh) against TIPRL (shTIPRL) or scrambled shRNA (shSCR) were treated with 5μM CPT for 1hr and stained with both DAPI and anti-γ-H2AX (D) followed by quantification of the images. Data represent ± standard deviation of the mean of three fields. *p<0.01, Student’s t test.
Fig 5.
Overexpression of TIPRL promotes cell death in response to genotoxic stress.
(A) 3T3 MEFs stably expressing LPC FLAG vector control (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL) were lysed and immunoblotted with the indicated antibodies. (B) Cells were treated with 10μM CPT or 2μg/ml doxorubicin for 24hrs. Viability was measured by propidium iodide exclusion. Data represent ± standard deviation of the mean of three independent experiments. (C) 3T3 MEFs stably expressing LPC FLAG vector control (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL) were treated with the indicated concentration of doxorubicin (DOXO) for 24hrs. Cell viability was measured by MTS assay. (D) 3T3 cells stably expressing LPC FLAG vector control (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL) were treated with the indicated concentration of doxorubicin (DOXO) for 24hrs. Cells were lysed and immunoblotted with the indicated antibodies. (E) 3T3 MEFs stably expressing LPC FLAG vector control (VEC) or LPC FLAG-TIPRL (FLAG-TIPRL) were treated with the indicated concentration of CPT for 24hrs. Cells were lysed and immunoblotted with the indicated antibodies. **p<0.005, ***p<0.001, Student’s t test.
Fig 6.
Knockdown of TIPRL protects from cell death in response to genotoxic stress.
(A) 3T3 MEFs expressing a short hairpin against scramble (shSCR) or TIPRL (shTIPRL) were immunoblotted with the indicated antibodies. (B) 3T3 MEFs expressing a short hairpin against scramble (shSCR) or TIPRL (shTIPRL) were treated with 10μM CPT or (2μg/ml) doxorubicin (DOXO) for 24hrs. Cell viability was measured using propidium iodide exclusion. Data represent ± standard deviation of the mean of three independent experiments. (C) 3T3 MEFs expressing a short hairpin against scramble (shSCR) or TIPRL (shTIPRL) were treated with the indicated amounts of doxorubicin (DOXO) for 24hrs. Viability was determined by MTS assay. (D) 3T3 MEFs expressing a short hairpin against scramble (shSCR) or TIPRL (shTIPRL) were treated with the indicated amounts of CPT or (E) doxorubicin (DOXO) for 24hrs and PARP and caspase 3 cleavages were evaluated by immunoblotting. ***p<0.001, Student’s t test.