Journal: Science Advances

Article Title: TAp63 determines the fate of oocytes against DNA damage

doi: 10.1126/sciadv.ade1846

Figure Lengend Snippet: ( A to C ) IF assays of γH2AX (A), BAX (B), and cPARP (C) expression in the ovaries from wild-type and Trp63 cKO female mice at 6, 13, 18, and 24 hours after CPA injection. Scale bars, 50 μm. ( D ) Percentage of primordial oocytes with BAX (top) and cPARP (bottom) expression. ( E ) IF assays of BAX (top) and cPARP (bottom) expression in the ovaries from wild-type and Trp63 cKO mice treated with solvent or DOXO for 24 hours. Scale bars, 50 μm. ( F ) Transmission electron microscopy (TEM) images of gross primordial follicles, nucleus, and mitochondria from CD-1 mice at 18 hours after phosphate-buffered saline (PBS), DOXO, and CPA injection. NM, nucleus membrane; Pre-G, pregranulosa cell; N, nucleus; Cyto, cytoplasm; M, mitochondria; C, cristae of mitochondria. ( G ) IF assays of OPA1 expression in the ovaries from wild-type and Trp63 cKO female mice at 6, 13, 18, and 24 hours after CPA injection. Insets show high magnification of primordial follicles with the expression of OPA1. Scale bars, 50 μm. ( H ) Relative intensity of OPA1 in primordial follicles of the ovaries from wild-type and Trp63 cKO female mice at 6, 13, 18, and 24 hours after CPA injection. The numbers of oocytes measured for the intensity of OPA1 were counted. ( I ) Schematic of TAp63-regulated apoptosis in oocytes of primordial follicles.

Article Snippet: The catalog numbers and dilution of primary antibodies were as follows: p63 (D9L7L) (39692s; 1:100), γH2AX (9718S; 1:100), cPARP (9548s; 1:50), p-CHK2 (2197s; 1:50), and OPA1 (D7C1A) (67589s; 1:50) from Cell Signaling Technology; BAX (p-19) (sc-526; 1:50) from Santa Cruz Biotechnology Inc.; and DDX4 (ab270534; 1:100) and FOXL2 (ab246511; 1:50) from Abcam.

Techniques: Expressing, Injection, Solvent, Transmission Assay, Electron Microscopy, Saline, Membrane

Journal: Molecular Cancer Research

Article Title: Constitutive Activation of Caspase-3 and Poly ADP Ribose Polymerase Cleavage in Fanconi Anemia Cells

doi: 10.1158/1541-7786.mcr-09-0373

Figure Lengend Snippet: FIGURE 1. PARP cleavage and upregulation in FA cells. A. Immunoblot showing PARP cleavage in FANCD2-deficient (PD20), FANCD2-proficient (PD20cor), or nonmonoubiquitinable mutant (K561R) cells. B. Immunoblot showing PARP processing in PD20 and PD20-corrected cells treated (+) or untreated (−) with PARP inhibitor 3AB. C. PARP cleavage is increased in primary fibroblasts transfected with FANCD2 siRNA for the corresponding time intervals. Arrows, mobility shift representing PARP phosphorylation. Note the reduction of PARP signal in the sample treated with λ-phosphatase or in the siRNA FANCD2–depleted cells pretreated with PKC inhibitor. D. Immunoblot of the above FANCD2 knockdown samples immunoprecipitated with PARP antibody followed by probing with phosphor-PARP or total PARP antibodies. E. Immunoblots showing PARP cleavage in different FA subgroups treated or untreated with the PARP inhibitor, 3AB.

Article Snippet: In addition to the antibodies used for Western blotting, cleaved PARP (Asp214) antibody (human specific) #9541 (Cell Signaling, Inc.) has been used.

Techniques: Western Blot, Mutagenesis, Transfection, Mobility Shift, Phospho-proteomics, Knockdown, Immunoprecipitation

Journal: Annals of Hematology

Article Title: Inhibition of CDC20 suppresses the development and progression of mantle cell lymphoma through PI3K/AKT pathway

doi: 10.1007/s00277-026-06926-0

Figure Lengend Snippet: Apcin demonstrated anti-tumor efficacy and safety in the Z138 xenograft model. ( A ) Tumor growth curves of the control group and the apcin group. ( B ) Comparison of tumor weight between the two groups. ( C ) Comparison of spleen weight between the two groups. ( D and E ) Representative photographs of harvested tumors ( D ) and spleens ( E ) from both groups. ( F ) Body weight monitoring of mice in the control group and the apcin group. ( G ) Representative HE images (×200 magnification; scale bar, 100 μm) of heart, liver, and kidney tissues of mice in both groups. ( H ) Comparison of relevant parameters in blood biochemistry of mice between the two groups. ( I ) Comparison of relevant parameters in blood routine of mice between the two groups. ( J ) Representative IHC images of CDC20, cleaved PARP and Ki67 staining of tumor tissues in the control group and the apcin group (×200 magnification; scale bar, 100 μm). ( K ) Quantification analysis of CDC20, cleaved PARP and Ki67 expression in the control group and the apcin group calculated by MOD values. ( L ) WB validation of CDC20 and cleaved PARP expression in both groups. ** P < 0.01, *** P < 0.001, ns meant no significance

Article Snippet: Subsequently, sections were incubated overnight at 4 °C with primary antibodies: CDC20 for patients (1:500, 10252-1-AP, Proteintech, China) and mouse tumors (1:200, 10252-1-AP, Proteintech, China), and cleaved PARP (1:200, #94885, CST, USA) and Ki-67 (1:400, #12202, CST, USA) for mouse tumors.

Techniques: Control, Comparison, Staining, Expressing, Biomarker Discovery

Journal: Oncogene

Article Title: TKT-PARP1 axis induces radioresistance by promoting DNA double-strand break repair in hepatocellular carcinoma

doi: 10.1038/s41388-023-02935-9

Figure Lengend Snippet: A The correlation analysis between TKT and γH2AX expression using the published proteomic and phosphoproteomics dataset of 159 HCC patients from Fudan University. B The expression levels of γH2AX in TKT high and low tissues from 6 patients with HCC were compared using WB. C Representative MIF staining micrographs showing TKT (low or high) and γH2AX expression in an HCC microarray. Scale bar, 200 μm (TKT, γH2AX, DAPI and Merged) and 20 μm (Enlarged). D The Hep3B (Left) and HCCLM3 (Right) cells were collected without treatment, or at 2 h, 6 h, 12 h and 24 h post 4 Gy X-Ray treatment with siCtrl or siTKT transfection. Cell lysates were immunoblotted with the indicated antibodies. M, protein ladder. E Representative fluorescence images (green, γH2AX; blue, DAPI) and quantification of γH2AX immunostaining in cells at the indicated time after X-Ray treatment with or without TKT depletion. The correlation analysis between TKT and RAD51 ( F ), PARP1 ( G ), XRCC6 ( H ), XRCC4 ( I ) expression in HCC tissues by Spearman correlation analysis from TCGA database. Adapted from GEPIA: http://gepia.cancer-pku.cn/ . TPM, transcripts per million.

Article Snippet: Antibodies against β-actin (AC038), PARP1 (A0942), Lamin B (A16909), FLAG (AE092), γ-H2AX (Ser139) (AP0099), GAPDH (AC001), HIS (AE028) were from ABclonal, while antibody against PAR (ALX-804-220-R100) was from Enzo Life Sciences.

Techniques: Expressing, Phospho-proteomics, Staining, Microarray, Transfection, Fluorescence, Immunostaining

Journal: Oncogene

Article Title: TKT-PARP1 axis induces radioresistance by promoting DNA double-strand break repair in hepatocellular carcinoma

doi: 10.1038/s41388-023-02935-9

Figure Lengend Snippet: A Coomassie staining of the TKT complex separated by SDS–PAGE. Hep3B cells stably expressing TKT-Flag were used for immunoprecipitation with an anti-Flag or anti-IgG antibody. TKT and TKT-interacting proteins, including PARP1, are indicated. The gel pieces containing regions of interest were analyzed by mass spectrometry. The list shows the peptides and coverage of TKT and PARP1 from the mass spectrometry. B Co-IP and WB analysis of the interaction between TKT and PARP1 in TKT-Flag and PARP1-His co-transfected Hep3B cells at different time points after X-Ray treatment. C Immunoblotting of indicated proteins in the in vitro pull-down assay of PARP1-His and TKT-Flag. D Coomassie staining of recombinant TKT, TKT-GFP and PARP1 protein (Left) and immunoblotting detection of PARylated TKT and PARP1 with the recombinant TKT, TKT-GFP and PARP1 protein (Right). E Co-IP and WB analysis of PARylated TKT in HCCLM3 and Hep3B cells in response to X-Ray. F , G Immunoblotting detection of PARylated TKT and PARP1 with or without PARP1 in an in vitro PARylation assay. H The activity detection of TKT and PARylated TKT by Transketolase Activity Assay Kit. Statistical analyses are presented as the mean ± s.d. from three independent experiments. * p < 0.05 of Student’s t test. The R5P abundance in DMSO or PARG inhibitor ( I ), PARP1 inhibitor ( J ), X-Ray ( K ) treated Hep3B cells. Statistical analyses are presented as the mean±s.d. from three independent experiments. * p < 0.05; ** p < 0.01; **** p < 0.0001 of Student’s t test. L , M Immunoblotting detection of PARylation in siCtrl or siTKT transfected HepG2 and HCCLM3 cells at different time points after X-Ray treatment.

Article Snippet: Antibodies against β-actin (AC038), PARP1 (A0942), Lamin B (A16909), FLAG (AE092), γ-H2AX (Ser139) (AP0099), GAPDH (AC001), HIS (AE028) were from ABclonal, while antibody against PAR (ALX-804-220-R100) was from Enzo Life Sciences.

Techniques: Staining, SDS Page, Stable Transfection, Expressing, Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Transfection, Western Blot, In Vitro, Pull Down Assay, Recombinant, Activity Assay