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: Frontiers in Oncology

Article Title: Targeting BET Proteins With a PROTAC Molecule Elicits Potent Anticancer Activity in HCC Cells

doi: 10.3389/fonc.2019.01471

Figure Lengend Snippet: BETd-260 triggers massive apoptosis in HCC cells. (A) HepG2 and BEL-7402 cell lines were treated with BETd-260, HJB-97 or JQ1 as indicated for 48 h. Apoptosis was examined by propidium iodide (PI)/Annexin-V staining in combination with flow cytometry assay. (B) Average percentages of apoptosis from three independent experiments were plotted in the graphs. (C) HepG2 and BEL-7402 cell lines were treated for 24 h. Treated cells were lysed and the protein levels of full-length PARP (fl-PARP), cleaved PARP (cl-PARP), pro-Caspase3 (pro-Casp3), and cleaved caspase-3 (cl-Casp3) were examined by western blot analysis. Actin was used as a loading control. (D) HepG2 and (E) BEL-7402 cell lines were transfected with siRNA against caspases-9,−8, for 24 h. The transfected effectiveness was examined by western blotting (inserts). The transfected cells were treated by BETd-260 at 100 nmol/L for another 48 h. The cell death was determined by trypan blue assay and cell death percentages were plotted. (F) HepG2 and BEL-7402 cell lines were treated for 24 h. Treated cells were cell fractionated into cytosolic and mitochondria-enriched membranes fractions, and the protein level of cytochrome c was examined by western blot analysis. Actin was used as a loading control for cytosolic fraction and COX IV was used as a loading control for mitochondrial enriched membranes fraction. Data are representative of three independent experiments. ** p < 0.01.

Article Snippet: The following antibodies were used for IHC: BRD2 (IHC-00612), BRD4 (HC-00396), BAD (A302-384A) from Bethyl Laboratories (Shanghai, China); BRD3 (ab264420) from Abcam (Shanghai, China); cleaved PARP (Asp214) (#32563), activated caspase-3 (#9664), and Ki-67 (8D5) (9449) from Cell Signaling Technology (CST, Shanghai, China), Anti-Mcl-1 (MAB828) from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Staining, Flow Cytometry, Western Blot, Control, Transfection

Journal: Frontiers in Oncology

Article Title: Targeting BET Proteins With a PROTAC Molecule Elicits Potent Anticancer Activity in HCC Cells

doi: 10.3389/fonc.2019.01471

Figure Lengend Snippet: BETd-260 induces BET degradation, triggers apoptosis, and inhibits proliferation in HCC xenograft tissue in mice. BALB/c mice bearing HepG2 and BEL-7402 xenograft tumors were treated by a single intravenous dose of 5 mg/kg BETd-260 (BETd) for 24 h or vehicle (Veh). (A) Two to three mice were sacrificed and tumor tissue was harvested. The expression of BRD2, BRD3, and BRD4, Mcl-1, Bad, activated caspase-3, cleaved PARP, and Ki 67 was examined by immunohistochemistry staining. Representative photographs were presented. (B) The percentages of HCC tumor cells positively stained with BRD2, BRD3, and BRD4, Mcl-1, Bad, cleaved caspase-3 (c-Casp3), cleaved PARP (c-PARP), and Ki 67 were quantified under microscopy, and plotted. Data are representative of three independent experiments. ** p < 0.01.

Article Snippet: The following antibodies were used for IHC: BRD2 (IHC-00612), BRD4 (HC-00396), BAD (A302-384A) from Bethyl Laboratories (Shanghai, China); BRD3 (ab264420) from Abcam (Shanghai, China); cleaved PARP (Asp214) (#32563), activated caspase-3 (#9664), and Ki-67 (8D5) (9449) from Cell Signaling Technology (CST, Shanghai, China), Anti-Mcl-1 (MAB828) from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Expressing, Immunohistochemistry, Staining, Microscopy