pp53 ser15 cell signaling  (Cell Signaling Technology Inc)

Journal: Protein & cell

Article Title: CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway.

doi: 10.1007/s13238-020-00699-6

Figure Lengend Snippet: Figure 2. The expression of CAS9 in hiPSCs and hESCs promotes DNA DSB damage. (A) The inducible expression of CAS9 promotes DNA DSB damage responses in hiPSCs after 2 µg/mL Doxy treatment. The relative levels of the phosphorylation of p53 and H2AX are indicated at the bottom. Consistent data were obtained from two independent experiments. (B) The impact of expression levels of CAS9 on DNA DSB damage in hESCs. At the same lentiviral titers, the expression levels of CAS9 in hESCs transduced by standard lentiviral vector are higher than those transduced by the inducible lentiviral vector after 2 µg/mL Doxy treatment. Much lower expression levels of CAS9 can also promote DNA DSB damage in hESCs after the treatment with lower dosages of Doxy. The relative levels of the phosphorylation of p53 and H2AX are indicated at the bottom.

Article Snippet: Antibodies used in this study Antibody Source and catalog Anti-γH2AX Cell signaling (9718S, 80312S) Anti-H2AX Cell signaling (7631S) Anti-p53 Santa Cruze (sc-126) Anti-pp53 Cell signaling (9286S) Anti-CHK1 Cell signaling (2360S) Anti-pCHK1 Cell signaling (12302S) Anti-H3 Abcam (ab1971) Anti-KU86 Santa Cruze (sc-5280), Abcam (ab119935), Cell signaling (2753s) Anti-KU70 Abcam (ab92450) Anti-DNA PKcs Abcam (ab70250) Anti-BRCA1 Cell signaling (14823S) Anti-p-BRCA1 Cell signaling (9009S) Anti-CHK2 Cell signaling (6334S) Anti-pCHK2 Cell signaling (2197S) Anti-CAS9 Cell signaling (14697S), Abcam (ab189380), Novus Biologicals (NBP2-52717) (NBP2-52717) Anti-β-actin Cell signaling (4970S) Anti-Flag Thermo Fisher (MA1-91878) Anti-α-tubulin Santa Cruze (sc-73242) © The Author(s) 2020 357 P ro te in & C e ll D ow nloaded from https://academ ic.oup.com /proteincell/article/11/5/352/6746752 by guest on 21 January 2024 150 kDa 250 kDa 250 kDa 55 kDa 55 kDa 55 kDa 55 kDa 55 kDa 55 kDa 15 kDa 15 kDa 37 kDa Fo ci n um be r o f γ H 2A X *** DAPI H2AX CAS9 Merge pCHK2 (Thr68) CHK2 H2AX γH2AX β-Actin Doxy CHK1 pCHK1 (Ser317) pBRCA1 (Ser1254) CAS9 BRCA1 pS15-p53 p53 R at io o f i nt en si ty * ** ** ** *** *** *** CTL Doxy Ta il le ng th *** *** *** A B C 0 h 24 h 48 h - D ox y + D ox y -D ox y +D ox y0 10 20 30 40 Dox Doxy + Dox0 h 24 h 48 h 0 1 2 pB RA C1 /B RA C1 pC HK 1/ CH K1 pC HK 2/ CH K2 γH 2A X/ H2 AX pp 53 /p 53 3 0 10 20 30 40 CT L D ox y Do x Do xy + D ox P ro te in & C e ll D ow nloaded from https://academ ic.oup.com /proteincell/article/11/5/352/6746752 by guest on 21 January 2024 incubated with secondary antibodies at room temperature for 1 h and detected with Supersignal West Pico or Dura exposure buffer (Thermo Fisher Scientific).

Techniques: Expressing, Phospho-proteomics, Plasmid Preparation

Journal: Protein & cell

Article Title: CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway.

doi: 10.1007/s13238-020-00699-6

Figure Lengend Snippet: Figure 3. The expression of CAS9 in human fibroblasts promotes DNA DSB damage and activates DNA damage response pathways. (A) The expression of CAS9 in human fibroblasts activates DNA damage responses. The expression of CAS9 was induced with 2 µg/mL Doxy treatment. The relative levels of phosphorylation of BRCA1, CHK1, CHK2 and p53 are indicated at the bottom. n = 3. Data are presented as mean value ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (B) The expression of CAS9 increased the number of γH2AX foci in human fibroblasts. CTL or CAS9, human fibroblasts with CAS9 inducible expression cassette plated on chamber slides were treated with or without 2 µg/mL doxycycline for 3 days. The expression of CAS9 and γH2AX foci was revealed by immunoflourescence analysis. Representative images are shown. Scale bar, 10 µm. Unpaired t test. n = 20. Data are presented as mean values ± SD. ***P < 0.001. (C) CAS9 induces DNA DSB damage in human fibroblasts. CTL, human fibroblasts with lentiviral empty vector were treated with 2 µg/mL doxycycline for three days; Doxy, Dox, Doxy + Dox, human fibroblasts with lentiviral CAS9 inducible expression vector were treated with 2 µg/mL doxycycline for 3 days or 0.5 µmol/L Dox for 2 h or 2 µg/mL doxycycline for three days + 0.5 µmol/L Dox for 2 h, respectively. Representative images are shown. n = 40. Unpaired t test. Data are presented as mean value ± SD. **P < 0.01.

Article Snippet: Antibodies used in this study Antibody Source and catalog Anti-γH2AX Cell signaling (9718S, 80312S) Anti-H2AX Cell signaling (7631S) Anti-p53 Santa Cruze (sc-126) Anti-pp53 Cell signaling (9286S) Anti-CHK1 Cell signaling (2360S) Anti-pCHK1 Cell signaling (12302S) Anti-H3 Abcam (ab1971) Anti-KU86 Santa Cruze (sc-5280), Abcam (ab119935), Cell signaling (2753s) Anti-KU70 Abcam (ab92450) Anti-DNA PKcs Abcam (ab70250) Anti-BRCA1 Cell signaling (14823S) Anti-p-BRCA1 Cell signaling (9009S) Anti-CHK2 Cell signaling (6334S) Anti-pCHK2 Cell signaling (2197S) Anti-CAS9 Cell signaling (14697S), Abcam (ab189380), Novus Biologicals (NBP2-52717) (NBP2-52717) Anti-β-actin Cell signaling (4970S) Anti-Flag Thermo Fisher (MA1-91878) Anti-α-tubulin Santa Cruze (sc-73242) © The Author(s) 2020 357 P ro te in & C e ll D ow nloaded from https://academ ic.oup.com /proteincell/article/11/5/352/6746752 by guest on 21 January 2024 150 kDa 250 kDa 250 kDa 55 kDa 55 kDa 55 kDa 55 kDa 55 kDa 55 kDa 15 kDa 15 kDa 37 kDa Fo ci n um be r o f γ H 2A X *** DAPI H2AX CAS9 Merge pCHK2 (Thr68) CHK2 H2AX γH2AX β-Actin Doxy CHK1 pCHK1 (Ser317) pBRCA1 (Ser1254) CAS9 BRCA1 pS15-p53 p53 R at io o f i nt en si ty * ** ** ** *** *** *** CTL Doxy Ta il le ng th *** *** *** A B C 0 h 24 h 48 h - D ox y + D ox y -D ox y +D ox y0 10 20 30 40 Dox Doxy + Dox0 h 24 h 48 h 0 1 2 pB RA C1 /B RA C1 pC HK 1/ CH K1 pC HK 2/ CH K2 γH 2A X/ H2 AX pp 53 /p 53 3 0 10 20 30 40 CT L D ox y Do x Do xy + D ox P ro te in & C e ll D ow nloaded from https://academ ic.oup.com /proteincell/article/11/5/352/6746752 by guest on 21 January 2024 incubated with secondary antibodies at room temperature for 1 h and detected with Supersignal West Pico or Dura exposure buffer (Thermo Fisher Scientific).

Techniques: Expressing, Phospho-proteomics, Plasmid Preparation