Clonogenic survival in relation to <i>MSH3</i> knockdown in HCT116+3+5 and SW480 cells treated with SN-38, oxaliplatin or 5-FU.

<p>A, D, The effect of suppression of <i>MSH3</i>, using an inducible shRNA system (doxycycline 1 µg/ml, DOX+), upon chemosensitivity of cell lines to treatment with SN-38 (A) or oxaliplatin (D) [48 h] relative to control cells (DOX-). B,C,E,F, HCT116+3+5 and SW480 cells lines with constitutive knockdown of <i>MSH3</i> by shRNA were treated with SN-38 (B, C) or oxaliplatin (E, F) and clonogenic survival was determined relative to cells with control shRNA. G,H,I, HCT116+3+5 cells with <i>MSH3</i> suppression (DOX+) or gene knockdown using shRNA were treated with 5-FU for 48 h and clonogenic survival was determined compared to control cells (DOX- or non targeted shRNA). Data are presented as mean ± standard errors <i>vs</i> control cells from at least 3 independent experiments. Statistical significance was determined by a two-sided Student's t test (* P<0.05; † P<0.01).</p

Effect of chemotherapeutic agents on DNA damage and apoptosis in <i>MSH3</i>-proficient and –deficient cells.

<p>A, Analysis of pH2AX and pChk2 expression and PARP cleavage in <i>MSH3</i>–deficient HCT116+3+5 cells (left) and SW480 cells (right) with constitutive shRNA knockdown of <i>MSH3</i>. Cells were treated with SN-38 or oxaliplatin for 48h. B, C, Immunofluorescence staining to detect the formation of 53BP1 nuclear foci in irradiated (2Gy) HCT116+3+5 cells with control shRNA (left) versus shRNA suppression of <i>MSH3</i> (right). The percentage of cells with >10 53BP1 nuclear foci was determined at the indicated time-points. At least 100 total cells were counted in each slide. D, Analysis of caspase-3/7 activity in HCT116+3+5 cells with and without <i>MSH3</i> knockdown that were treated with 5-FU, SN-38 or oxaliplatin (48 h). E, Analysis of pH2AX and pChk2 expression and PARP cleavage in 5-FU-treated (48h) HCT116+3+5 cells (left) that are <i>MSH3</i>–deficient and in SW480 cells (right) with constitutive shRNA knockdown of MSH3. Data represent mean ± standard errors from 3 independent experiments. Statistical significance was determined by a two-sided Student's t test (* P<0.05; † P<0.01).</p

MSH3 protein expression in HCT116-derived clones and SW480 cells.

<p>A, Western blot analysis of MSH3, MLH1, and tubulin in HCT116, HCT116+3, and HCT116+3+5 cells. B, MSH3 expression is controlled by a Tet-on shRNA system in HCT116+3+5 cells cultured in medium with and without 1 µg/ml doxycycline. C, D MSH3 or MLH1 expression was suppressed using constitutive <i>MSH3</i> or <i>MLH1</i> knockdown by shRNA in HCT116+3+5 cells. E, MSH3 expression was also suppressed using constitutive <i>MSH3</i> shRNA in SW480 cells.</p

Effect of chemotherapeutic agents on DNA damage and apoptosis in HCT116, HCT116+3 and HCT116+3+5 cells.

<p>A, B, Analysis of pH2AX and pChk2 expression and PARP cleavage in HCT116, HCT116+3, and HCT116+3+5 cells treated with 5-FU, SN-38 or oxaliplatin for 48 h and compared to untreated control cells. C, Analysis of caspase-3/7 activity in HCT116, HCT116+3 and HCT116+3+5 cells treated with 5-FU, SN-38 or oxaliplatin (48 h). Data are shown as mean ± standard errors from 3 independent experiments. Statistical significance was determined by a two-sided Student's t test (* P<0.05; † P<0.01).</p

<i>MSH3</i>-deficient (vs proficient) cells are more sensitive to SN-38 and oxaliplatin in contrast to 5-FU.

<p>A–C, MTS assay in HCT116 (<i>MLH1−/MSH3−</i>), HCT116+3 (<i>MLH1+/MSH3−</i>), and HCT116+3+5 (<i>MLH1+/MSH3+</i>) cells treated with SN-38 (A), oxaliplatin (B) or 5-FU (C). D–F, Clonogenic survival of these same cell lines treated for 48 h with SN-38 (D), oxaliplatin (E), or 5-FU (F). Data are presented as mean ± standard errors <i>vs</i> controls for at least 3 independent experiments. Statistical significance was determined by a two-sided Student's t test (* P<0.05; † P<0.01).</p

<i>Beclin 1</i> suppression enhances radiation-induced DNA damage and cell death.

<p><i>A, B,</i> HT-29 (A) or DLD1 (B) cells were transfected with <i>Beclin 1</i> 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 *<i>P</i><0.05. <i>C</i>, Cells with <i>Beclin 1</i> 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. <i>D,</i> Time course of the effect of radiation on pATM expression in cells with <i>Beclin 1</i> siRNA vs control siRNA. <i>E</i>, Effect of <i>Beclin 1</i> siRNA on autophagic flux in cells that were treated with RT (4 Gy) and/or 5-FU (4 µM).</p

Knockdown of <i>Beclin 1, UVRAG, and ATG5</i> increase radiation-induced 53BP1, but not RAD51, nuclear foci.

<p><i>A, B,</i> Immunofluorescence staining for 53BP1 (A) or RAD51 (B) was performed in HT-29 cells with knockdown of <i>Beclin 1</i>, <i>UVRAG</i> or <i>ATG5</i> 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 *<i>P</i><0.05.</p

<i>Beclin 1</i> suppression enhances radiation-induced DNA damage and cell death.

<p><i>A, B,</i> HT-29 (A) or DLD1 (B) cells were transfected with <i>Beclin 1</i> 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 *<i>P</i><0.05. <i>C</i>, Cells with <i>Beclin 1</i> 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. <i>D,</i> Time course of the effect of radiation on pATM expression in cells with <i>Beclin 1</i> siRNA vs control siRNA. <i>E</i>, Effect of <i>Beclin 1</i> siRNA on autophagic flux in cells that were treated with RT (4 Gy) and/or 5-FU (4 µM).</p

<i>UVRAG ΔCCD</i> reduces binding to Beclin 1 and promotes DNA double strand breaks.

<p><i>A,</i> Immunoprecipitation of UVRAG followed by probing for Beclin 1 was performed in HT-29 cell lysates (4 h) following radiation (4 Gy) <i>vs</i> untreated cells. Normal IgG was utilized as a control for antibody specificity. Both short (SE) and longer (LE) exposures are shown for UVRAG. <i>B,</i> HT-29 cells overexpressing <i>UVRAG</i> 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. <i>C,</i> Cell lysates from irradiated (4 Gy) cells were probed for LC3I-II and γH2Ax at 24 h post-irradiation by immunoblotting. Stable <i>UVRAG</i> wt or <i>ΔCCD</i> mutant cells were utilized here and in Fig. 5B. <i>D</i>, Cells with wt <i>UVRAG</i> or the <i>UVRAG ΔCCD</i> 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.</p

Suppression of <i>Beclin 1, UVRAG</i> or both sensitize cells to DNA damage and apoptosis.

<p><i>A, B,</i> HT-29 and DLD1 cells were transfected with <i>Beclin 1</i> (A) or <i>UVRAG</i> (B) <i>vs</i> control siRNA. Cells were irradiated and the expression of the indicated proteins was analyzed at 24 h post-radiation by immunoblotting. <i>C,</i> Time course of the effect of radiation on pATM expression in cells with <i>UVRAG vs</i> control siRNA. <i>D,</i> Effect of radiation on DNA damage and apoptosis markers in cells with dual knockdown of <i>Beclin 1</i> and <i>UVRAG</i> vs <i>UVRAG</i> siRNA alone (24 h). Densitometry was performed and normalized against tubulin.</p