Journal: Cancer letters

Article Title: Targeting UHRF1-dependent DNA repair selectively sensitizes KRAS mutant lung cancer to chemotherapy

doi: 10.1016/j.canlet.2020.08.008

Figure Lengend Snippet: Chemical screen of DNA damage response inhibitors in KRAS mutant lung cancer cell. (A) Representative Western blot results. VE822 is a known ATR inhibitor. (B) Screening results. Each dot represents one compound. The value represents pCHK1/CHK1 ratio normalized to that of CPT only and are presented in log2 scale. Zero represents that of CPT. (C) A549 cells were pre-, co- or 1 h post-treated with 500 nM AT2, AT3 or TP4 with 500 nM CPT for 4 h, and protein levels of pCHK1 and CHK1 were analyzed. Ponceau S staining indicates equal protein loading. (D) A549 cells were pre-treated with 500 nM AT2 for 2 h, added 500 nM CPT for 1, 2 and 8 h, and levels of pCHK1 and CHK1 were examined. (E) A549 cells were pre-treated with different concentrations of AT2 for 2 h, added 500 nM CPT for another 4 h, and protein expression was examined using indicated antibodies. Lower: levels of pCHK1 normalized to total CHK1 from 5 independent experiments were used to determine the IC50 of AT2. (F) A549 cells were transfected with siRNA control or targeting the α1 subunit of Na/K-ATPase for 48 h, treated with 500 nM AT2 for 2 h, added 200 nM CPT for another 4 h, and protein expression was examined. Relative pCHK1 levels are shown above.

Article Snippet: Chemical library screen The chemical library contains 874 compounds from both the Institute of Traditional Chinese Medicine and the DNA damage and repair library from TargetMol (#L3900) ( Supplementary Table S1 ).

Techniques: Mutagenesis, Western Blot, Staining, Expressing, Transfection

Journal: bioRxiv

Article Title: SUMOylation of PTEN promotes DNA end resection through directly dephosphorylating 53BP1 in homologous recombination repair

doi: 10.1101/2023.02.06.527258

Figure Lengend Snippet: PTEN promotes HR repair through facilitating DNA end resection. ( A ) Immunoblot of pS4/8-RPA32 of HeLa-pLKO and -shPTEN cells treated with 20 Gy and recovery for indicated time. ( B ) Immunoblot of pS4/8-RPA32 of DU145-PTEN WT and -PTEN −/− cells treated with 10 Gy and recovered for indicated time. ( C ) Immunoblot of pS4/8-RPA32 of DU145-PTEN −/− cells stably re-expressed PTEN WT , K254R and K266R cells after treated with 10 Gy. ( D ) Immunoblot of chromatin associated RPA32 and RAD51 in DU145 cells after treated with Zeocin (200 μg/mL) for 1 h and recovered for indicated time. ( E ) RAD51 foci were quantified in DU145 cells treated with 5 Gy and recovery for 6 h and then presented with dot graph (n(Vector)=223, n(WT)=135, n(C124S)=221, n(G129E)=187, n(K254R)=244, n(K266R)=167). Representative images of RAD51 foci were shown at right panel. scale bar, 20 μm. ( F ) HR efficiency were detected in U2OS-DR-GFP-shPTEN cells stably re-expressing PTEN WT , PTEN C124S , PTEN G129E , PTEN K254R and PTEN K266R (n=4 for each group). Inset: immunoblot of PTEN in U2OS-DR-GFP-shPTEN cells stably re-expressing indicated PTEN mutants. Left panel: quantification of HR efficiency shown as bar graph. Right panel: representative images of FACS. Unpaired Student’s t-test was used (**p< 0.01, ***p< 0.001) and data were shown as mean or mean±s.d. Figure 1-source data 1. Western blots of Figure 1.

Article Snippet: For detection of PTEN SUMOylation during DNA damage repair, 293T cells were transfected with His-SUMO1 and indicated plasmids for 48 h. CPT (#S1288, Selleck) and Zeocin (#60216ES80, Yeasen) were used to induce DNA damage for 1 h. cells were harvested at indicated time, 10% cells were used as input.

Techniques: Western Blot, Stable Transfection, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: SUMOylation of PTEN promotes DNA end resection through directly dephosphorylating 53BP1 in homologous recombination repair

doi: 10.1101/2023.02.06.527258

Figure Lengend Snippet: DNA damage promotes PTEN chromatin loading by inducing its SUMOylation. ( A ) 293T cells transfected with His-SUMO1 and Flag-PTEN were treated with Zeocin (200 μg/mL) for 1h and recovered for indicated time. His-SUMO1 conjugates were pulled down with Ni 2+ -NTA beads and SUMOylated PTEN were analyzed with immunoblot. ( B ) 293T cells transfected with His-SUMO1 and Flag-PTEN were treated with CPT (20 μM) for 1h and recovered for indicated time. His-SUMO1 conjugates were pulled down with Ni 2+ -NTA beads and SUMOylated PTEN were analyzed with immunoblot. ( C ) 293T cells transfected with His-SUMO1 and Flag-PTEN WT , C124S , G129E , K254R , K266R were treated with Zeocin (200 μg/mL) for 1h and recovered for 4 h, Ni 2+ -NTA pulldown were used to analyze PTEN SUMOylation. ( D ) Images of staining of PTEN in DU145-shPTEN cells stably re-expressed PTEN-WT, K254R and K266R. ( E ) Nuclear-Cytosol separation was performed in DU145-PTEN −/− cells stably re-expressing PTEN WT , C124S , G129E , K254R and K266R treated with Zeocin (400 μg/mL) for 1 h and recovery for 4 h. Localization of PTEN was detected with immunoblot. ( F ) Immunoblot of chromatin associated PTEN in DU145 cells treated with Zeocin (400 μg/mL) for 1 h and recovery for indicated time. ( G ) Chromatin protein separation were performed at harsh condition in DU145-PTEN −/− cells stably re-expressing PTEN WT , K254R and K266R after treatment with 10 Gy. PTEN tightly associated with chromatin was detected with immunoblot. Figure 2-source data 1. Western blots of Figure 2.

Article Snippet: For detection of PTEN SUMOylation during DNA damage repair, 293T cells were transfected with His-SUMO1 and indicated plasmids for 48 h. CPT (#S1288, Selleck) and Zeocin (#60216ES80, Yeasen) were used to induce DNA damage for 1 h. cells were harvested at indicated time, 10% cells were used as input.

Techniques: Transfection, Western Blot, Staining, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: SUMOylation of PTEN promotes DNA end resection through directly dephosphorylating 53BP1 in homologous recombination repair

doi: 10.1101/2023.02.06.527258

Figure Lengend Snippet: (A) Denatured Co-IP were performed to analyze SUMOylation of PTEN in 293T cells transfected with Flag-SUMO1 and HA-PTEN after treatment with Zeocin (400 μg/mL). (B) PTEN localization was detected with immunoblot after Nuclear-Cytosol separation in DU145-PTEN −/− cells stably re-expressing PTEN mutants after treatment with CPT (20 μM) for 1 h and recovery for 4 h. (C) 293T senp−/− cells transfected with PCNA-RFP and EGFP-PTEN were treated with laser micro-irradiation. Images were taken before and 10 min post laser micro-irradiation. PCNA-RFP were used as indicator of DNA damage location. (D) Chromatin loading of PTEN was detedcted with immunoblot in DU145-PTEN −/− cells stably re-expressing PTEN mutants after treatment with CPT (20 μM) for 1 h and recovery for indicated time. Figure 2-figure supplement 1-source data 1. Western blots of Figure 2-figure supplement 1.

Article Snippet: For detection of PTEN SUMOylation during DNA damage repair, 293T cells were transfected with His-SUMO1 and indicated plasmids for 48 h. CPT (#S1288, Selleck) and Zeocin (#60216ES80, Yeasen) were used to induce DNA damage for 1 h. cells were harvested at indicated time, 10% cells were used as input.

Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot, Stable Transfection, Expressing, Irradiation

Journal: bioRxiv

Article Title: SUMOylation of PTEN promotes DNA end resection through directly dephosphorylating 53BP1 in homologous recombination repair

doi: 10.1101/2023.02.06.527258

Figure Lengend Snippet: Blocking PTEN SUMOylation pathway sensitizes tumor cells to DNA damage reagents. ( A-C ) DU145-PTEN −/− and PTEN WT cells were treated with or without different doses of Cisplatin for 3 days, Zeocin for 2 days or CPT for 2 days and then cultured for another 7-10 days. Colony number was counted and presented as bar graph (n=3 for each group). ( D ) DU145-PTEN −/− , PTEN WT , PTEN K254R and PTEN K266R cells were treated with or without TAK981 (100 nM) for 3 days and cultured for another 7-10 days. Total intensity of stained colony was measured by ImageJ and then normalized to DU145-PTEN −/− group without TAK981 treatment (n=3 for each group). ( E-G ) DU145-PTEN −/− , PTEN WT , PTEN K254R and PTEN K266R cells were treated with Cisplatin (1.5 μM) for 3 days, Zeocin (10 μg/mL) for 2 days or CPT (100 nM) for 2 days or combination with TAK981 (100 nM) and cultured for another 7-10 days. Colony number was counted and presented as bar graph (n=3 for each group). ( H ) DU145-PTEN −/− and PTEN WT cells were treated with different doses of CPP-p14ARF(2-13) for 3 days and cultured for another 7-10 days (n=3 for each group). ( I-K ) DU145-PTEN −/− and PTEN WT cells were treated with combination of Cisplatin (1.5 μM for 3 days), Zeocin (10 μg/mL for 2 days) or CPT (100 nM for 2 days) and different doses of CPP-p14ARF(2-13). Colony number was counted after cultured for another 7-10 days (n=3 for each group). ( L ) A schematic model to show the molecular mechanism of PTEN SUMOylation in HR repair. DNA damage induced phosphorylation of p14ARF enhanced its interaction with PTEN and which subsequently promoted SUMOylation of PTEN. SUMOylated PTEN was recognized and recruited into DNA breaks by BRCA1 and then directly dephosphorylated 53BP1 which helped release of its downstream effectors and DNA end resection. 500 cells were seeded in 12-well plate for all colony assays except ( C ) in which 1000 cells were seeded at the beginning. Unpaired Student’s t-test was used (*p< 0.05, **p< 0.01, ***p< 0.001) and data are shown as mean±s.d from three biological replicates.

Article Snippet: For detection of PTEN SUMOylation during DNA damage repair, 293T cells were transfected with His-SUMO1 and indicated plasmids for 48 h. CPT (#S1288, Selleck) and Zeocin (#60216ES80, Yeasen) were used to induce DNA damage for 1 h. cells were harvested at indicated time, 10% cells were used as input.

Techniques: Blocking Assay, Cell Culture, Staining, Phospho-proteomics