Figure 1.
Beclin 1 suppression enhances radiation-induced DNA damage and cell death.
A, B, HT-29 (A) or DLD1 (B) cells were transfected with Beclin 1 vs control siRNA and treated with radiation (RT; 4 Gy) alone or combined with 5-FU (2 µM). Results of the MTS (24 h) and clonogenic survival assays are shown. Data are presented as mean ± standard deviation for experiments performed in triplicate. Statistical significance was determined by a two-sided Student’s t test and defined as *P<0.05. C, Cells with Beclin 1 or control siRNA were irradiated (4 Gy) and then were probed for expression of LC3I-II, γH2Ax, and cleaved caspase-3 by immunoblotting at 24 h. Protein bands are quantified and relative intensity was labelled underneath the corresponding blot. Only LC3II was quantified for LC3 protein. D, Time course of the effect of radiation on pATM expression in cells with Beclin 1 siRNA vs control siRNA. E, Effect of Beclin 1 siRNA on autophagic flux in cells that were treated with RT (4 Gy) and/or 5-FU (4 µM).
Figure 2.
Knockdown of Beclin 1, UVRAG, and ATG5 increase radiation-induced 53BP1, but not RAD51, nuclear foci.
A, B, Immunofluorescence staining for 53BP1 (A) or RAD51 (B) was performed in HT-29 cells with knockdown of Beclin 1, UVRAG or ATG5 and exposed to radiation (2 Gy) at the indicated times. The percentage of cells with >10 nuclear foci expressing either 53BP1 or RAD51 was calculated and plotted as shown. 53BP1 and RAD51 are markers of nonhomologous end joining and homologous recombination, respectively. DAPI was utilized to counterstain the nucleus. Data are presented as mean ± standard deviation for experiments performed in triplicate. Statistical significance was determined by a two-sided Student’s t test and defined as *P<0.05.
Figure 3.
Autophagy inhibition by USP10 shRNA or spautin-1 enhances irradiation-induced DNA damage and cell death.
A, Cells with USP10 vs control shRNA were irradiated and analyzed for expression of USP10, LC3I-II, γH2Ax and cleaved caspase-3 at 24 h by immunoblotting (left), or for clonogenic survival (right). B, Cells were treated with irradiation alone or combined with spautin-1 (10 µM) and expression of the indicated proteins were analyzed at 24 h by immunoblotting. C, D, Cells were treated with spautin-1 (10 µM) alone or combined with RT (4 Gy) (C) or RT ± 5-FU (2 µM) (D). In these cells, annexin V+PI− labeling at 24 h (C) and clonogenic survival (D) were analyzed. Data are presented as mean ± standard deviation compared to controls for triplicate experiments. **P<0.01.
Figure 4.
Suppression of Beclin 1, UVRAG or both sensitize cells to DNA damage and apoptosis.
A, B, HT-29 and DLD1 cells were transfected with Beclin 1 (A) or UVRAG (B) vs control siRNA. Cells were irradiated and the expression of the indicated proteins was analyzed at 24 h post-radiation by immunoblotting. C, Time course of the effect of radiation on pATM expression in cells with UVRAG vs control siRNA. D, Effect of radiation on DNA damage and apoptosis markers in cells with dual knockdown of Beclin 1 and UVRAG vs UVRAG siRNA alone (24 h). Densitometry was performed and normalized against tubulin.
Figure 5.
UVRAG ΔCCD reduces binding to Beclin 1 and promotes DNA double strand breaks.
A, Immunoprecipitation of UVRAG followed by probing for Beclin 1 was performed in HT-29 cell lysates (4 h) following radiation (4 Gy) vs untreated cells. Normal IgG was utilized as a control for antibody specificity. Both short (SE) and longer (LE) exposures are shown for UVRAG. B, HT-29 cells overexpressing UVRAG wild-type (wt) or a deletion mutant at its coil-coil domain (ΔCCD), both labeled with a three-tandem-tag [3tag: s-tag, 2XFLAG and streptavidin binding protein (SBP)], were subjected to immunoprecipitation for FLAG. Precipitated proteins were probed using antibodies against Beclin 1, UVRAG or FLAG. Normal IgG was utilized as a control. C, Cell lysates from irradiated (4 Gy) cells were probed for LC3I-II and γH2Ax at 24 h post-irradiation by immunoblotting. Stable UVRAG wt or ΔCCD mutant cells were utilized here and in Fig. 5B. D, Cells with wt UVRAG or the UVRAG ΔCCD mutant vs empty vector control were treated with vehicle or radiation, and long-term clonogenic survival was determined. The data were normalized relative to untreated cells for each cell phenotype. Data are presented as mean ± standard deviation for experiments performed in triplicate. Statistical significance was determined by a two-sided Student’s t test and defined as *P<0.05.
Figure 6.
Beclin 1 or UVRAG suppression induces centrosome amplification.
A, Effect of knockdown of LC3 (left) or ATG5 (right) on markers of DSBs (γH2Ax), apoptosis (caspase-3), and autophagy (LC3I-II conversion) in HT-29 and/or DLD1 cells exposed to radiation vs control 24 h post-irradiation. B, Centrosome number was determined by immunofluorescence in Beclin 1, UVRAG, or ATG5 knockdown cells (HT-29 or HeLa) treated with radiation (4, 8 Gy) vs control. Cells were then stained for γ-tubulin (red color) and representative images are shown (left). The percentage of cells with more than two centrosomes (multi-centrosomes) was counted and plotted (right). Data are presented as mean ± standard deviation for experiments performed in triplicate. Statistical significance was determined by a two-sided Student’s t test and defined as *P<0.05 as compared to the control cells.