Journal: Dose-Response

Article Title: Gentiana lutea Root Extract Attenuates Radiation-Induced Damage in Human PBMCs in vitro

doi: 10.1177/15593258261435484

Figure Lengend Snippet: Relative fold change of genes expression involved in DNA repair processes: Ogg1, Parp1 , and Xrcc1 in PBMCs pretreated with 0.25, 0.5, 1 and 2 mg/mL of Gentiana lutea root extract and exposed to 0.5 and 2 Gy irradiation dose

Article Snippet: Expression levels of the target genes were measured by quantitative Real-time PCR on Applied Biosystems Real-Time 7500 system (Applied Biosystems, Inc., Foster City, CA) using TaqMan® gene expression assays: Parp1 (Hs00242302_m1), Ogg1 (Hs00213454_m1), Xrcc1 (Hs00959834_m1).

Techniques: Expressing, Irradiation

Journal: Frontiers in Oncology

Article Title: Niraparib-induced STAT3 inhibition increases its antitumor effects

doi: 10.3389/fonc.2022.966492

Figure Lengend Snippet: NRP inhibits pSTAT3 and enhances cell apoptosis by regulating STAT3 downstream genes. (A) Western blot indicating levels of pSTAT3(Y-705) in MIA PaCa-2, PANC-1, OVCAR8, and PEO1 cells after NRP or DMSO treatment for 24h or 36 hours (PEO1). GAPDH or β-actin served as a loading control. Band intensities from two or three independent experiments were quantified by ImageJ and showed in a bar graph as mean ± SEM (B) Western blot indicating the level of pSTAT3(Y-705) in MIA PaCa-2 cells transfected with PARP1 siRNA for 2 days, followed by 24-hour NRP treatment. (C) Real-time PCR to examine changes of STAT3 downstream gene expression levels in MIA PaCa-2 and OVCAR8 cells with or without NRP treatment. Gene expression was normalized to the housekeeping gene 18S . Unpaired two-tailed Student t-test. The data shown are as mean ± SEM (N=3). *p<0.05, **p<0.001, ***p<0.0001. (D) NRP-induced apoptosis in MIA PaCa-2 and OVCAR8 cells was examined by Annexin V/PI staining and analyzed by flow cytometry 72h after indicated treatments. Unpaired two-tailed Student t-test. Data are shown as mean ± SEM (N=3). *p<0.05, ***p<0.0001. (E) STAT3C overexpression significantly rescued NRP-induced apoptosis. Mock vector or STAT3C-overexpressing MIA PaCa-2 or OVCAR8 cells were treated with NRP for 48h, followed by Annexin V-APC/PI and flow cytometry. Unpaired two-tailed Student t-test. Data are shown as mean ± SEM (N=3). ns, not significant. *p<0.05, ***p<0.0001. Total STAT3 levels in STAT3C-overexpressing cells were examined by Western blot. β-actin served as a loading control.

Article Snippet: MIA PaCa-2 cells were transfected with PARP1 siRNA ( ) or control siRNA (sc-44236, Santa Cruz Biotechnology) for 48 hours using Lipofectamine RNAiMAX Transfection Reagent (#13778, ThermoFisher Scientific) according to manufacturer’s protocol.

Techniques: Western Blot, Control, Transfection, Real-time Polymerase Chain Reaction, Gene Expression, Two Tailed Test, Staining, Flow Cytometry, Over Expression, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: Niraparib-induced STAT3 inhibition increases its antitumor effects

doi: 10.3389/fonc.2022.966492

Figure Lengend Snippet: NRP treatment reduces pSTAT3 and pSRC in OvCa and PDAC patient tumor samples. (A) Western blot analysis of pSTAT3 and pSRC levels in OvCa patient primary tumor cells or ascites cells after 24h NRP treatment. β-actin served as a loading control. (B) Western blot analysis of levels of pSTAT3 and pSRC in PDAC-derived PDX tumor cells or PDAC patient primary tumor cells treated with NRP for 24h. β-actin served as a loading control. (C) Expression of pSTAT3 and pSRC in OvCa patient tumor slices treated with DMSO or NRP for 24h were examined by fluorescent immunohistochemistry and confocal microscopy. Representative images are shown from two OvCa patients. Red, pSTAT3; Green, pSRC; Magenta, pan-Cytokeratin; Blue/Hoechst 33342, nucleus. Cytokeratin-positive cell clusters demonstrate malignant tumor tissue. Scale bars = 20 μm. Histograms show quantification of M.F.I. of pSTAT3 and pSRC normalized to nuclear staining. Quantification was performed using ImageJ software, and at least five fields were quantified for each condition group. Data are presented as mean ± SEM. Unpaired two-tailed Student t-test, *p<0.05, ***p<0.001, ****p<0.001. (D) Niraparib induces tumor cell apoptosis through two mechanisms: Niraparib inhibits PARP, preventing DNA damage repairs in cells with BRCA mutations, thus causing tumor cell synthetic lethality. Our data show that Niraparib also interferes with SRC/STAT3 pathway to increase apoptosis of tumor cells with or without BRCA mutations.

Article Snippet: MIA PaCa-2 cells were transfected with PARP1 siRNA ( ) or control siRNA (sc-44236, Santa Cruz Biotechnology) for 48 hours using Lipofectamine RNAiMAX Transfection Reagent (#13778, ThermoFisher Scientific) according to manufacturer’s protocol.

Techniques: Western Blot, Control, Derivative Assay, Expressing, Immunohistochemistry, Confocal Microscopy, Staining, Software, Two Tailed Test

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