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(A) Western blot time-course of PPM1D, <t>p-KAP1,</t> KAP1, and UBR5 in U2OS cells after siRNA mediated knockdown of non-targeting control (NTG), UBR5, PPM1D , or UBR5 and PPM1D and exposure to 0 or 2.5 Gy irradiation. (B) Western blot of PPM1D, p-KAP1, KAP1, and UBR5 in U2OS isogenic cell lines after siRNA mediated knockdown of NTG or UBR5 and exposure to 0 or 2.5 Gy irradiation. (C) Immunofluorescence analysis of p-KAP1 in U2OS parental and isogenic cell lines transfected with NTG or UBR5 siRNA and exposed to 0 or 2.5 Gy irradiation. Representative images are shown on the left, and quantification of the mean fluorescence intensity (MFI) of p-KAP1 is shown on the right. n>230 cells for each condition were analyzed using CellProfiler. (D) Schematic of homology repair (HR) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (E) Schematic of non-homologous end joining (NHEJ) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (F) Representative images of COMET assay performed on U2OS parental or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA, 6 hours after treatment with 0 or 2.5 Gy irradiation. (G) Quantification of tail moments in COMET assay described in (F). n>160 comets for each condition were scored using OpenComet. (H) Schematic of cell growth competition assay. U2OS parental, control, or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA were fluorescently labeled with BFP or RFP, the cells were mixed, exposed to 2.5 Gy irradiation then grown for 72 hours prior to quantification of the RFP:BFP ratio. (I) Normalized quantification of RFP to BFP ratio in the competition assay described in (H). (J) UBR5 truncating mutation frequency in PPM1D truncating mutation absent or present cell lines or primary tumors samples obtained from Depmap or TCGA. (K) UBR5 amplification frequency in PPM1D amplification or truncating mutation patient samples obtained from TCGA. Data are presented as mean ± SEM, n = 4 biological replicates for Figures D and E, and n = 3 biological replicates for Figure I. For Figures D and E, each data point indicates the normalized mean of three internal biological replicates from each independent test. Statistical significance was determined using unpaired two-tailed Student’s t-test for Figures C, D, E, G, and I; using Fisher’s exact test for Figures J and K; ns indicates non- significant ( p >0.05), * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.
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(A) Western blot time-course of PPM1D, <t>p-KAP1,</t> KAP1, and UBR5 in U2OS cells after siRNA mediated knockdown of non-targeting control (NTG), UBR5, PPM1D , or UBR5 and PPM1D and exposure to 0 or 2.5 Gy irradiation. (B) Western blot of PPM1D, p-KAP1, KAP1, and UBR5 in U2OS isogenic cell lines after siRNA mediated knockdown of NTG or UBR5 and exposure to 0 or 2.5 Gy irradiation. (C) Immunofluorescence analysis of p-KAP1 in U2OS parental and isogenic cell lines transfected with NTG or UBR5 siRNA and exposed to 0 or 2.5 Gy irradiation. Representative images are shown on the left, and quantification of the mean fluorescence intensity (MFI) of p-KAP1 is shown on the right. n>230 cells for each condition were analyzed using CellProfiler. (D) Schematic of homology repair (HR) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (E) Schematic of non-homologous end joining (NHEJ) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (F) Representative images of COMET assay performed on U2OS parental or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA, 6 hours after treatment with 0 or 2.5 Gy irradiation. (G) Quantification of tail moments in COMET assay described in (F). n>160 comets for each condition were scored using OpenComet. (H) Schematic of cell growth competition assay. U2OS parental, control, or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA were fluorescently labeled with BFP or RFP, the cells were mixed, exposed to 2.5 Gy irradiation then grown for 72 hours prior to quantification of the RFP:BFP ratio. (I) Normalized quantification of RFP to BFP ratio in the competition assay described in (H). (J) UBR5 truncating mutation frequency in PPM1D truncating mutation absent or present cell lines or primary tumors samples obtained from Depmap or TCGA. (K) UBR5 amplification frequency in PPM1D amplification or truncating mutation patient samples obtained from TCGA. Data are presented as mean ± SEM, n = 4 biological replicates for Figures D and E, and n = 3 biological replicates for Figure I. For Figures D and E, each data point indicates the normalized mean of three internal biological replicates from each independent test. Statistical significance was determined using unpaired two-tailed Student’s t-test for Figures C, D, E, G, and I; using Fisher’s exact test for Figures J and K; ns indicates non- significant ( p >0.05), * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.
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(A) Western blot time-course of PPM1D, <t>p-KAP1,</t> KAP1, and UBR5 in U2OS cells after siRNA mediated knockdown of non-targeting control (NTG), UBR5, PPM1D , or UBR5 and PPM1D and exposure to 0 or 2.5 Gy irradiation. (B) Western blot of PPM1D, p-KAP1, KAP1, and UBR5 in U2OS isogenic cell lines after siRNA mediated knockdown of NTG or UBR5 and exposure to 0 or 2.5 Gy irradiation. (C) Immunofluorescence analysis of p-KAP1 in U2OS parental and isogenic cell lines transfected with NTG or UBR5 siRNA and exposed to 0 or 2.5 Gy irradiation. Representative images are shown on the left, and quantification of the mean fluorescence intensity (MFI) of p-KAP1 is shown on the right. n>230 cells for each condition were analyzed using CellProfiler. (D) Schematic of homology repair (HR) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (E) Schematic of non-homologous end joining (NHEJ) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (F) Representative images of COMET assay performed on U2OS parental or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA, 6 hours after treatment with 0 or 2.5 Gy irradiation. (G) Quantification of tail moments in COMET assay described in (F). n>160 comets for each condition were scored using OpenComet. (H) Schematic of cell growth competition assay. U2OS parental, control, or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA were fluorescently labeled with BFP or RFP, the cells were mixed, exposed to 2.5 Gy irradiation then grown for 72 hours prior to quantification of the RFP:BFP ratio. (I) Normalized quantification of RFP to BFP ratio in the competition assay described in (H). (J) UBR5 truncating mutation frequency in PPM1D truncating mutation absent or present cell lines or primary tumors samples obtained from Depmap or TCGA. (K) UBR5 amplification frequency in PPM1D amplification or truncating mutation patient samples obtained from TCGA. Data are presented as mean ± SEM, n = 4 biological replicates for Figures D and E, and n = 3 biological replicates for Figure I. For Figures D and E, each data point indicates the normalized mean of three internal biological replicates from each independent test. Statistical significance was determined using unpaired two-tailed Student’s t-test for Figures C, D, E, G, and I; using Fisher’s exact test for Figures J and K; ns indicates non- significant ( p >0.05), * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.
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(A) Western blot time-course of PPM1D, <t>p-KAP1,</t> KAP1, and UBR5 in U2OS cells after siRNA mediated knockdown of non-targeting control (NTG), UBR5, PPM1D , or UBR5 and PPM1D and exposure to 0 or 2.5 Gy irradiation. (B) Western blot of PPM1D, p-KAP1, KAP1, and UBR5 in U2OS isogenic cell lines after siRNA mediated knockdown of NTG or UBR5 and exposure to 0 or 2.5 Gy irradiation. (C) Immunofluorescence analysis of p-KAP1 in U2OS parental and isogenic cell lines transfected with NTG or UBR5 siRNA and exposed to 0 or 2.5 Gy irradiation. Representative images are shown on the left, and quantification of the mean fluorescence intensity (MFI) of p-KAP1 is shown on the right. n>230 cells for each condition were analyzed using CellProfiler. (D) Schematic of homology repair (HR) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (E) Schematic of non-homologous end joining (NHEJ) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (F) Representative images of COMET assay performed on U2OS parental or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA, 6 hours after treatment with 0 or 2.5 Gy irradiation. (G) Quantification of tail moments in COMET assay described in (F). n>160 comets for each condition were scored using OpenComet. (H) Schematic of cell growth competition assay. U2OS parental, control, or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA were fluorescently labeled with BFP or RFP, the cells were mixed, exposed to 2.5 Gy irradiation then grown for 72 hours prior to quantification of the RFP:BFP ratio. (I) Normalized quantification of RFP to BFP ratio in the competition assay described in (H). (J) UBR5 truncating mutation frequency in PPM1D truncating mutation absent or present cell lines or primary tumors samples obtained from Depmap or TCGA. (K) UBR5 amplification frequency in PPM1D amplification or truncating mutation patient samples obtained from TCGA. Data are presented as mean ± SEM, n = 4 biological replicates for Figures D and E, and n = 3 biological replicates for Figure I. For Figures D and E, each data point indicates the normalized mean of three internal biological replicates from each independent test. Statistical significance was determined using unpaired two-tailed Student’s t-test for Figures C, D, E, G, and I; using Fisher’s exact test for Figures J and K; ns indicates non- significant ( p >0.05), * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.
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(A) Western blot time-course of PPM1D, <t>p-KAP1,</t> KAP1, and UBR5 in U2OS cells after siRNA mediated knockdown of non-targeting control (NTG), UBR5, PPM1D , or UBR5 and PPM1D and exposure to 0 or 2.5 Gy irradiation. (B) Western blot of PPM1D, p-KAP1, KAP1, and UBR5 in U2OS isogenic cell lines after siRNA mediated knockdown of NTG or UBR5 and exposure to 0 or 2.5 Gy irradiation. (C) Immunofluorescence analysis of p-KAP1 in U2OS parental and isogenic cell lines transfected with NTG or UBR5 siRNA and exposed to 0 or 2.5 Gy irradiation. Representative images are shown on the left, and quantification of the mean fluorescence intensity (MFI) of p-KAP1 is shown on the right. n>230 cells for each condition were analyzed using CellProfiler. (D) Schematic of homology repair (HR) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (E) Schematic of non-homologous end joining (NHEJ) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (F) Representative images of COMET assay performed on U2OS parental or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA, 6 hours after treatment with 0 or 2.5 Gy irradiation. (G) Quantification of tail moments in COMET assay described in (F). n>160 comets for each condition were scored using OpenComet. (H) Schematic of cell growth competition assay. U2OS parental, control, or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA were fluorescently labeled with BFP or RFP, the cells were mixed, exposed to 2.5 Gy irradiation then grown for 72 hours prior to quantification of the RFP:BFP ratio. (I) Normalized quantification of RFP to BFP ratio in the competition assay described in (H). (J) UBR5 truncating mutation frequency in PPM1D truncating mutation absent or present cell lines or primary tumors samples obtained from Depmap or TCGA. (K) UBR5 amplification frequency in PPM1D amplification or truncating mutation patient samples obtained from TCGA. Data are presented as mean ± SEM, n = 4 biological replicates for Figures D and E, and n = 3 biological replicates for Figure I. For Figures D and E, each data point indicates the normalized mean of three internal biological replicates from each independent test. Statistical significance was determined using unpaired two-tailed Student’s t-test for Figures C, D, E, G, and I; using Fisher’s exact test for Figures J and K; ns indicates non- significant ( p >0.05), * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.
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(A) Western blot time-course of PPM1D, <t>p-KAP1,</t> KAP1, and UBR5 in U2OS cells after siRNA mediated knockdown of non-targeting control (NTG), UBR5, PPM1D , or UBR5 and PPM1D and exposure to 0 or 2.5 Gy irradiation. (B) Western blot of PPM1D, p-KAP1, KAP1, and UBR5 in U2OS isogenic cell lines after siRNA mediated knockdown of NTG or UBR5 and exposure to 0 or 2.5 Gy irradiation. (C) Immunofluorescence analysis of p-KAP1 in U2OS parental and isogenic cell lines transfected with NTG or UBR5 siRNA and exposed to 0 or 2.5 Gy irradiation. Representative images are shown on the left, and quantification of the mean fluorescence intensity (MFI) of p-KAP1 is shown on the right. n>230 cells for each condition were analyzed using CellProfiler. (D) Schematic of homology repair (HR) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (E) Schematic of non-homologous end joining (NHEJ) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (F) Representative images of COMET assay performed on U2OS parental or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA, 6 hours after treatment with 0 or 2.5 Gy irradiation. (G) Quantification of tail moments in COMET assay described in (F). n>160 comets for each condition were scored using OpenComet. (H) Schematic of cell growth competition assay. U2OS parental, control, or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA were fluorescently labeled with BFP or RFP, the cells were mixed, exposed to 2.5 Gy irradiation then grown for 72 hours prior to quantification of the RFP:BFP ratio. (I) Normalized quantification of RFP to BFP ratio in the competition assay described in (H). (J) UBR5 truncating mutation frequency in PPM1D truncating mutation absent or present cell lines or primary tumors samples obtained from Depmap or TCGA. (K) UBR5 amplification frequency in PPM1D amplification or truncating mutation patient samples obtained from TCGA. Data are presented as mean ± SEM, n = 4 biological replicates for Figures D and E, and n = 3 biological replicates for Figure I. For Figures D and E, each data point indicates the normalized mean of three internal biological replicates from each independent test. Statistical significance was determined using unpaired two-tailed Student’s t-test for Figures C, D, E, G, and I; using Fisher’s exact test for Figures J and K; ns indicates non- significant ( p >0.05), * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.
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( A ) HCT116 SIRT2 WT and KO cells were treated with 6 Gy IR, harvested after 4 hours, run on SDS-PAGE, and probed with the indicated antibodies. The image shown is representative of data from 3 separate experiments conducted independently. ( B ) Quantitation of IR-induced phosphorylation of <t>KAP1</t> and CHK2 relative to total KAP1 and CHK2 protein levels. Data are represented as the mean ± SD from 3 independent experiments. ** P < 0.01 and *** P < 0.001, by 2-tail Student’s t test. ( C ) HCT116 SIRT2 KO cells expressing SFB-MRE11 WT, K393Q, and K393R were treated with or without 6 Gy IR, harvested after 4 hours, run on SDS-PAGE, and probed with the indicated antibodies. Shown is a representative image of 3 independent experiments. ( D and E ) Quantitation of phosphorylation of KAP1 and CHK2 relative to total KAP1 and CHK2 protein levels. Data are presented as the mean ± SD of 3 independent experiments. Statistical significance was determined using 1-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. **** P < 0.0001.
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Image Search Results


(A) Western blot time-course of PPM1D, p-KAP1, KAP1, and UBR5 in U2OS cells after siRNA mediated knockdown of non-targeting control (NTG), UBR5, PPM1D , or UBR5 and PPM1D and exposure to 0 or 2.5 Gy irradiation. (B) Western blot of PPM1D, p-KAP1, KAP1, and UBR5 in U2OS isogenic cell lines after siRNA mediated knockdown of NTG or UBR5 and exposure to 0 or 2.5 Gy irradiation. (C) Immunofluorescence analysis of p-KAP1 in U2OS parental and isogenic cell lines transfected with NTG or UBR5 siRNA and exposed to 0 or 2.5 Gy irradiation. Representative images are shown on the left, and quantification of the mean fluorescence intensity (MFI) of p-KAP1 is shown on the right. n>230 cells for each condition were analyzed using CellProfiler. (D) Schematic of homology repair (HR) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (E) Schematic of non-homologous end joining (NHEJ) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (F) Representative images of COMET assay performed on U2OS parental or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA, 6 hours after treatment with 0 or 2.5 Gy irradiation. (G) Quantification of tail moments in COMET assay described in (F). n>160 comets for each condition were scored using OpenComet. (H) Schematic of cell growth competition assay. U2OS parental, control, or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA were fluorescently labeled with BFP or RFP, the cells were mixed, exposed to 2.5 Gy irradiation then grown for 72 hours prior to quantification of the RFP:BFP ratio. (I) Normalized quantification of RFP to BFP ratio in the competition assay described in (H). (J) UBR5 truncating mutation frequency in PPM1D truncating mutation absent or present cell lines or primary tumors samples obtained from Depmap or TCGA. (K) UBR5 amplification frequency in PPM1D amplification or truncating mutation patient samples obtained from TCGA. Data are presented as mean ± SEM, n = 4 biological replicates for Figures D and E, and n = 3 biological replicates for Figure I. For Figures D and E, each data point indicates the normalized mean of three internal biological replicates from each independent test. Statistical significance was determined using unpaired two-tailed Student’s t-test for Figures C, D, E, G, and I; using Fisher’s exact test for Figures J and K; ns indicates non- significant ( p >0.05), * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Journal: bioRxiv

Article Title: Distinct Proteasomal Pathways Drive Oncogenic PPM1D Activation

doi: 10.64898/2026.06.01.729344

Figure Lengend Snippet: (A) Western blot time-course of PPM1D, p-KAP1, KAP1, and UBR5 in U2OS cells after siRNA mediated knockdown of non-targeting control (NTG), UBR5, PPM1D , or UBR5 and PPM1D and exposure to 0 or 2.5 Gy irradiation. (B) Western blot of PPM1D, p-KAP1, KAP1, and UBR5 in U2OS isogenic cell lines after siRNA mediated knockdown of NTG or UBR5 and exposure to 0 or 2.5 Gy irradiation. (C) Immunofluorescence analysis of p-KAP1 in U2OS parental and isogenic cell lines transfected with NTG or UBR5 siRNA and exposed to 0 or 2.5 Gy irradiation. Representative images are shown on the left, and quantification of the mean fluorescence intensity (MFI) of p-KAP1 is shown on the right. n>230 cells for each condition were analyzed using CellProfiler. (D) Schematic of homology repair (HR) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (E) Schematic of non-homologous end joining (NHEJ) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (F) Representative images of COMET assay performed on U2OS parental or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA, 6 hours after treatment with 0 or 2.5 Gy irradiation. (G) Quantification of tail moments in COMET assay described in (F). n>160 comets for each condition were scored using OpenComet. (H) Schematic of cell growth competition assay. U2OS parental, control, or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA were fluorescently labeled with BFP or RFP, the cells were mixed, exposed to 2.5 Gy irradiation then grown for 72 hours prior to quantification of the RFP:BFP ratio. (I) Normalized quantification of RFP to BFP ratio in the competition assay described in (H). (J) UBR5 truncating mutation frequency in PPM1D truncating mutation absent or present cell lines or primary tumors samples obtained from Depmap or TCGA. (K) UBR5 amplification frequency in PPM1D amplification or truncating mutation patient samples obtained from TCGA. Data are presented as mean ± SEM, n = 4 biological replicates for Figures D and E, and n = 3 biological replicates for Figure I. For Figures D and E, each data point indicates the normalized mean of three internal biological replicates from each independent test. Statistical significance was determined using unpaired two-tailed Student’s t-test for Figures C, D, E, G, and I; using Fisher’s exact test for Figures J and K; ns indicates non- significant ( p >0.05), * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: The following antibodies for immunoblotting were used in this study: PPM1D (Cell Signaling Technology, 11901), PPM1D (Santa Cruz Biotechnology, sc-376257), GAPDH (Cell Signaling Technology, 2118), Ubiquitin (Santa Cruz Biotechnology, sc-8017), LC3B (Cell Signaling Technology, 2775), COX IV (Cell Signaling Technology, 4844), HA (Cell Signaling Technology, 3724), V5 (Abcam, ab309485), Myc (Cell Signaling Technology, 2272), PSMA2 (Cell Signaling Technology, 2455), UBR5 (Cell Signaling Technology, 65344), p-KAP1 (S824) (Bethyl Laboratories, A300-767A), KAP1 (Cell Signaling Technology, 4124), K11-linked ubiquitin (kindly provided by Dr. Eugene Oh), K48-linked ubiquitin (kindly provided by Dr. Eugene Oh), FLAG (Thermo Fisher Scientific, MA1-91878), PSMD14 (Cell Signaling Technology, 4197), PSME3 (Proteintech, 14907-1-AP), H2B (Cell Signaling Technology, 12364), OTUD5 (Cell Signaling Technology, 20087), TRIP12 (Proteintech, 25303-1-AP), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (Proteintech, SA00001-2), and HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L) (Proteintech, SA00001-1).

Techniques: Western Blot, Knockdown, Control, Irradiation, Immunofluorescence, Transfection, Fluorescence, Non-Homologous End Joining, Single Cell Gel Electrophoresis, Competitive Binding Assay, Labeling, Mutagenesis, Amplification, Two Tailed Test

( A ) HCT116 SIRT2 WT and KO cells were treated with 6 Gy IR, harvested after 4 hours, run on SDS-PAGE, and probed with the indicated antibodies. The image shown is representative of data from 3 separate experiments conducted independently. ( B ) Quantitation of IR-induced phosphorylation of KAP1 and CHK2 relative to total KAP1 and CHK2 protein levels. Data are represented as the mean ± SD from 3 independent experiments. ** P < 0.01 and *** P < 0.001, by 2-tail Student’s t test. ( C ) HCT116 SIRT2 KO cells expressing SFB-MRE11 WT, K393Q, and K393R were treated with or without 6 Gy IR, harvested after 4 hours, run on SDS-PAGE, and probed with the indicated antibodies. Shown is a representative image of 3 independent experiments. ( D and E ) Quantitation of phosphorylation of KAP1 and CHK2 relative to total KAP1 and CHK2 protein levels. Data are presented as the mean ± SD of 3 independent experiments. Statistical significance was determined using 1-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. **** P < 0.0001.

Journal: The Journal of Clinical Investigation

Article Title: MRE11 deacetylation by SIRT2 promotes DNA binding to facilitate DNA end resection and ATM-dependent signaling

doi: 10.1172/JCI186711

Figure Lengend Snippet: ( A ) HCT116 SIRT2 WT and KO cells were treated with 6 Gy IR, harvested after 4 hours, run on SDS-PAGE, and probed with the indicated antibodies. The image shown is representative of data from 3 separate experiments conducted independently. ( B ) Quantitation of IR-induced phosphorylation of KAP1 and CHK2 relative to total KAP1 and CHK2 protein levels. Data are represented as the mean ± SD from 3 independent experiments. ** P < 0.01 and *** P < 0.001, by 2-tail Student’s t test. ( C ) HCT116 SIRT2 KO cells expressing SFB-MRE11 WT, K393Q, and K393R were treated with or without 6 Gy IR, harvested after 4 hours, run on SDS-PAGE, and probed with the indicated antibodies. Shown is a representative image of 3 independent experiments. ( D and E ) Quantitation of phosphorylation of KAP1 and CHK2 relative to total KAP1 and CHK2 protein levels. Data are presented as the mean ± SD of 3 independent experiments. Statistical significance was determined using 1-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. **** P < 0.0001.

Article Snippet: The antibodies used in this study included SIRT2 (MilliporeSigma, 09-843; Santa Cruz Biotechnology, sc-20966), GAPDH (Santa Cruz, sc-47724), Flag (Santa Cruz Biotechnology, sc-51590), GFP (Abcam, Ab6556), pan acetyl-Lys (Santa Cruz Biotechnology, sc-8649), RPA70 (Cell Signaling Technology, 2267), P-CHK2 T68 (Cell Signaling Technology, 2661S), CHK2 (Santa Cruz Biotechnology, sc-7898), P-KAP1 (Invitrogen, Thermo Fisher Scientific, MA1-2023), and KAP1 (Bethyl Laboratories, A300-767A).

Techniques: SDS Page, Quantitation Assay, Phospho-proteomics, Expressing

(Step 1) DNA damage results in DSBs. (Step 2) SIRT2 deacetylates MRE11 at K393 in response to DNA damage. (Step 3) MRE11 K393 deacetylation by SIRT2 facilitates its recruitment and binding to DNA at DSBs. (Step 4) MRE11-RAD50-NBS1 assembles and promotes DNA end resection and ATM-dependent phosphorylation of downstream substrates, including CHK2 and KAP1.

Journal: The Journal of Clinical Investigation

Article Title: MRE11 deacetylation by SIRT2 promotes DNA binding to facilitate DNA end resection and ATM-dependent signaling

doi: 10.1172/JCI186711

Figure Lengend Snippet: (Step 1) DNA damage results in DSBs. (Step 2) SIRT2 deacetylates MRE11 at K393 in response to DNA damage. (Step 3) MRE11 K393 deacetylation by SIRT2 facilitates its recruitment and binding to DNA at DSBs. (Step 4) MRE11-RAD50-NBS1 assembles and promotes DNA end resection and ATM-dependent phosphorylation of downstream substrates, including CHK2 and KAP1.

Article Snippet: The antibodies used in this study included SIRT2 (MilliporeSigma, 09-843; Santa Cruz Biotechnology, sc-20966), GAPDH (Santa Cruz, sc-47724), Flag (Santa Cruz Biotechnology, sc-51590), GFP (Abcam, Ab6556), pan acetyl-Lys (Santa Cruz Biotechnology, sc-8649), RPA70 (Cell Signaling Technology, 2267), P-CHK2 T68 (Cell Signaling Technology, 2661S), CHK2 (Santa Cruz Biotechnology, sc-7898), P-KAP1 (Invitrogen, Thermo Fisher Scientific, MA1-2023), and KAP1 (Bethyl Laboratories, A300-767A).

Techniques: Binding Assay, Phospho-proteomics