vitro parylation  (Thermo Fisher)

Journal: Cell Research

Article Title: Destabilization of linker histone H1.2 is essential for ATM activation and DNA damage repair

doi: 10.1038/s41422-018-0048-0

Figure Lengend Snippet: H1.2 PARylation permits its displacement from chromatin upon DNA damage. a HeLa cells were transfected with GFP-H1.2 and treated with 20 μM Ku55933 or 2 μM Ku57788 for 4 h or 5 μM PJ34 for 1 h followed by laser micro-irradiation. Images were taken every 10 s for 5 min and quantifications of the IR path signal intensity were shown and ~15 IR paths from 10 separate cells were calculated. The data represent the mean ± SD. Scale bars, 10 μm. b HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for the indicated time. Chromatin was fractionated and analyzed by immunoblotting. c Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and chromatin was fractionated and analyzed by immunoblotting. d HeLa cells were transfected with FLAG-H1.2 and treated with 40 μM etoposide for 15 min with or without 5 μM PJ34 for 1 h. Cell extracts were immunoprecipitated with FLAG-conjugated M2 beads. e Recombinant HIS-H1.2 was subjected to in vitro PARylation assay in the presence of NAD + or 10 μM PJ34, as indicated. f HeLa cells were transfected with wild-type or S188A mutated FLAG-H1.2 and treated with 40 μM etoposide for 15 min with or without 5 μM PJ34 for 1 h, as indicated. Cells were extracted and immunoprecipitated with FLAG-conjugated M2 beads. g Recombinant wild-type, S188A mutated or C1-deleted (ΔC1) HIS-H1.2 were subjected to in vitro PARylation assay. h HeLa cells were transfected with wild-type, ΔC1 or S188A mutated GFP-H1.2 and subjected to laser micro-irradiation. Images were taken every 20 s for 5 min and representative images were shown. Quantifications were calculated as in a . The data represent the mean ± SD. Scale bars, 10 μm

Article Snippet: HIS-H1.2 was subjected to in vitro PARylation at room temperature for 30 min or the indicated time in a reaction buffer (50 mM Tris·HCl pH 8, 25 mM MgCl 2 , 50 mM NaCl) supplemented with 200 μM NAD + , activated DNA and PARP1 enzyme (Thermo Fisher, or immunoprecipitated from HET293T cells).

Techniques: Transfection, Irradiation, Western Blot, Immunoprecipitation, Recombinant, In Vitro

Journal: Cell Research

Article Title: Destabilization of linker histone H1.2 is essential for ATM activation and DNA damage repair

doi: 10.1038/s41422-018-0048-0

Figure Lengend Snippet: PARylation of H1.2 is essential for ATM activation. a Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and analyzed by immunoblotting. b HeLa cells were treated with 40 μM etoposide for the indicated time with or without exposure to 5 μM PJ34 1 h before etoposide treatment and analyzed by immunoblotting. c Two clones of PARP1 stable knockdown (shPARP1 #1 and #3) and control (shCtr) HeLa cells were treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. d shPARP1 (1#) and shCtr HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. e HCT116 cells were transfected with the indicated plasmids and treated with 40 μM etoposide for the indicated times and analyzed by immunoblotting. f HeLa cells were transfected with wild-type or S188A mutated GFP-H1.2, treated with 40 μM etoposide for 2 h and the fluorescence intensity of phospho-ATM S1981 in the untransfected cells was normalized to 1. The arrows indicate representative cells. The data represent the mean ± SD. Scale bars, 10 μm. g Recombinant HIS-H1.2 was incubated for 30 min at 37 °C with PARP1 with or without NAD + for in vitro PARylation assay (Incubation 1, Inc. 1). H1.2 was eluted and used for in vitro phosphorylation assay (Incubation 2, Inc. 2). An N-terminal GST-p53 (1–99 aa) peptide was used as the substrate. h Recombinant GST-H1.2 was incubated with PARP1 with or without NAD + for in vitro PARylation assay. GST alone and PARylated GST-H1.2 were then incubated with HIS-MRE11 for GST-pulldown assay. * indicates specific protein bands. i HeLa cells were transfected with the indicated plasmids and treated with 40 μM etoposide for 1 h or 5 μM PJ34 for 1 h. Whole cell extractions were prepared and subjected to Co-IP assay with FLAG-conjugated M2 beads

Article Snippet: HIS-H1.2 was subjected to in vitro PARylation at room temperature for 30 min or the indicated time in a reaction buffer (50 mM Tris·HCl pH 8, 25 mM MgCl 2 , 50 mM NaCl) supplemented with 200 μM NAD + , activated DNA and PARP1 enzyme (Thermo Fisher, or immunoprecipitated from HET293T cells).

Techniques: Activation Assay, Western Blot, Clone Assay, Knockdown, Control, Transfection, Fluorescence, Recombinant, Incubation, In Vitro, Phospho-proteomics, GST Pulldown Assay, Co-Immunoprecipitation Assay

Journal: Advanced Science

Article Title: Nudix Hydrolase 13 Impairs the Initiation of Colorectal Cancer by Inhibiting PKM1 ADP‐Ribosylation

doi: 10.1002/advs.202410058

Figure Lengend Snippet: PARP1‐induced PKM1 ADP‐ribosylation signals for ubiquitination and degradation. A) In vivo PARylation assay was performed in SW480 cells transfected with PKM1‐Myc. B) Control and NUDT13‐overexpressing SW480 cells pre‐treated with DMSO or Olaparib (10 µ m ) were exposed to 50µg mL −1 CHX for the indicated time. Quantification of PKM1 by densitometry. C) Immunoblot analysis of PKM1 ubiquitination levels in NUDT13‐overexpressing SW480 cells transfected with NUDT13 siRNA, and treated with or without Olaparib (10 µ m ) for 48h. (D and E) The interaction between PKM1 and PARP1 was detected by Co‐IP D) and in vitro pull‐down assays E). F) In vitro PARylation assay to detect the PARylation of purified PKM1 catalyzed by recombinant human PARP1. G) Immunoblot analysis of PKM1 PARylation levels in SW480 cells treated with Olaparib (10 µ m ) for 48h, or transfected with NUDT13‐Flag plasmids. H) Immunoblot analysis of PKM1 PARylation levels in 293T cells transfected with WT or mutant PKM1. The amounts of PKM1 levels in different groups were adjusted by MG132 treatment. I) In vitro PARylation assay to detect the PARylation of purified PKM1. J) CHX chase assays were conducted to assess the stabilities of PKM1 WT and A4 mutant. All results mentioned above were obtained from 3 or more independent experiments. Data are presented as mean ± SD; P value was calculated by two‐way ANOVA (B and J).

Article Snippet: For the de‐PARylation assay, in vitro PARylation reactions were stopped by the addition of 1 μ m Olaparib (S1060, Selleck, USA), then added different concentrations of purified NUDT13 and adjusted the Mg 2+ concentration to 15 m m by MgCl 2 .

Techniques: Ubiquitin Proteomics, In Vivo, Transfection, Control, Western Blot, Co-Immunoprecipitation Assay, In Vitro, Purification, Recombinant, Mutagenesis

Journal: Advanced Science

Article Title: Nudix Hydrolase 13 Impairs the Initiation of Colorectal Cancer by Inhibiting PKM1 ADP‐Ribosylation

doi: 10.1002/advs.202410058

Figure Lengend Snippet: NUDT13 suppresses PKM1 ADP‐ribosylation to stabilize PKM1 protein. A) Immunoblot analysis of PKM1 PARylation levels after incubation of PARylated PKM1 with control or indicated concentrations of recombinant hNUDT13. B) Sequence alignment of the Nudix box motif of NUDT13 among different species. Highly conserved amino acids are shown in red. Mutant sites are shown in green. C) Co‐IP assay in 293T cells co‐transfected with PKM1‐Myc and NUDT13‐Flag WT or EQ mutant plasmids. D) Immunoblot analysis of PKM1 PARylation and ubiquitination levels in SW480 cells transfected with NUDT13‐Flag or EQ mutant plasmids. E) SW480 cells transfected with control or EQ mutant plasmids were exposed to 50µg/mL CHX for the indicated time. Quantification of PKM1 by densitometry. F) Immunoblot analysis of the PKM1 levels in DLD‐1 cells transfected with NUDT13‐Flag WT or EQ mutant plasmids. G) The doubling time of EQ mutant proficient DLD‐1 cells under normoxic or hypoxic conditions, as measured by CCK8. H) Top: the OCR of DLD‐1 and SW480 cells transfected with EQ mutant plasmids in response to oligomycin, FCCP, and rotenone/antimycin A. Bottom: bar graphs depicting the basal OCR (left) and the maximal OCR (right) of DLD‐1 and SW480 cells. I) Xenograft tumors formed in BALB/c nude mice ( n = 7). Subcutaneous tumors were measured by volume and weight. J) Top: experimental scheme of Olaparib treatment schedule in the Nudt13 VillKO ‐AOM‐DSS mouse model. Olaparib was injected intraperitoneally every two days during the fresh drinking water period. Tumor burden was assessed at 55 days post‐AOM (red arrows). Bottom: diagram of the Nudt13 flox and Villin‐CreERT2 alleles. K) Macroscopic tumor burden formed in colons was counted in control and Nudt13 flox mice, with or without Olaparib treatment ( n = 5). Gross images of the distal colons are shown, and the red arrowhead indicates macroscopic tumors. Scale bars, 5 mm. L) Hematoxylin and eosin (H&E) and anti‐PKM1 staining of colon tumor sections from control and Nudt13 flox mice, with or without Olaparib treatment. Scale bars, 2 mm or 50 µm. All results mentioned above were obtained from 3 or more independent experiments. Data are presented as mean ± SD; P values were calculated by two‐way ANOVA (E and I) and Student's t‐ test (G, H, I, and K).

Article Snippet: For the de‐PARylation assay, in vitro PARylation reactions were stopped by the addition of 1 μ m Olaparib (S1060, Selleck, USA), then added different concentrations of purified NUDT13 and adjusted the Mg 2+ concentration to 15 m m by MgCl 2 .

Techniques: Western Blot, Incubation, Control, Recombinant, Sequencing, Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Ubiquitin Proteomics, Injection, Staining

Journal: Advanced Science

Article Title: Nudix Hydrolase 13 Impairs the Initiation of Colorectal Cancer by Inhibiting PKM1 ADP‐Ribosylation

doi: 10.1002/advs.202410058

Figure Lengend Snippet: NUDT13 230–252 AA elicits tumor‐suppressive effects. A) Immunoblot analysis of PKM1 levels in DLD‐1 and SW480 cells transfected with NUDT13 truncated plasmids. B) Crystal structures of the predicted docking interfaces between PKM1 (green) and NUDT13 (red). C) Co‐IP assay in 293T cells co‐transfected with PKM1‐Myc and NUDT13‐Flag WT, or △230–252 plasmids. D) Coomassie blue staining showed the direct interaction between N13 peptide and purified PKM1, but not N13 peptide and PKM2, by in vitro pull‐down assay. E) Immunoblot analysis of PKM1 PARylation levels in DLD‐1 cells treated with different doses of N13‐iRGD for 48 h. F) Immunoblot analysis of PKM1 protein levels in SW480 cells treated with the indicated doses of fusion peptides for 48h. G) The doubling time of SW480 and DLD‐1 cells treated with indicated peptides under normoxic or hypoxic conditions, as measured by CCK8. H) The OCR of DLD‐1 and SW480 cells treated with indicated peptides in response to oligomycin, FCCP, and rotenone/antimycin A. I) Experimental scheme of the peptide treatment in Apc Min/+ ‐DSS mouse model. J) Macroscopic tumor burden formed in colons was counted ( n = 4). The red arrowhead indicates macroscopic tumors. K) H&E and anti‐PKM1 staining of colon tumor sections from mice treated with control peptide, N13‐iRGD, or EQ‐iRGD. Scale bars, 2 mm or 100 µm. L) Experimental scheme of the peptide treatment in subcutaneous xenograft mouse model. M) Xenograft tumors formed in BALB/c nude mice ( n = 6). Subcutaneous tumors were measured by volume (left) and weight (right). N) Schematic model illustrating the role of the NUDT13‐PKM1 axis in the regulation of CRC initiation. All results mentioned above were obtained from 3 or more independent experiments. Data are presented as mean ± SD; P values were calculated by Student's t‐ test (G, H, J, and M) and two‐way ANOVA (M).

Article Snippet: For the de‐PARylation assay, in vitro PARylation reactions were stopped by the addition of 1 μ m Olaparib (S1060, Selleck, USA), then added different concentrations of purified NUDT13 and adjusted the Mg 2+ concentration to 15 m m by MgCl 2 .

Techniques: Western Blot, Transfection, Co-Immunoprecipitation Assay, Staining, Purification, In Vitro, Pull Down Assay, Control