Journal: Cell Death & Disease

Article Title: PKC signaling prevents irradiation-induced apoptosis of primary human fibroblasts

doi: 10.1038/cddis.2013.15

Figure Lengend Snippet: Inhibition or downregulation of PKC reduces prosurvival signaling in NHFs. Phosphorylation of PKC ζ/λ and Bad ( a ) or PKC β II and ERK ( b ) in response to IR. Western blot analysis of irradiated MRC-5 cells after recovery for 0.5–2 or 2–4 h following IR. Total PKC ζ/λ and Bad ( a ) or total PKC δ and ERK ( b ) were used as loading control. ( c ) Phosphorylation of CREB and ATF-1 is downstream of PKC activation in response to IR. Western blot analysis of irradiated MRC-5 cells (4 h recovery) that were treated either with the PKC-specific inhibitor Ro-318220 (5 μ M, added before irradiation) or with the ATM inhibitor KU-55933 (10 μ M, added before irradiation). Tubulin was used as loading control. ( d ) Phosphorylation of Bad is downstream of PKC activation in response to IR. Western blot analysis of irradiated MRC-5 cells (4 h recovery) that were exposed to the PKC-specific inhibitor GF109203X (5 μ M, for 2 h) or siPKC pan. ( e ) Phosphorylation of CREB, ATF-1 and Bad is downstream of PKC activation in response to IR. Western blot analysis of irradiated IMR-90 cells (4 h recovery) that were treated with GF109203X or Ro-318220 (2.5 μ M, 2 h pre-incubation before irradiation). Tubulin was used as loading control. ( f ) Pharmacological inhibition of PKC reduces senescence associated β -galactosidase ( β -gal) staining. SA- β -gal staining of irradiated MRC-5 in presence of PKC inhibitor (GF109203X, 5 μ M, 72 h recovery) with untreated (U) as control. ( g ) Pharmacological inhibition of PKC (GF109203X, 5 μ M) leads to ARTD1 cleavage, Bcl-2 downregulation and Bad upregulation 48 h after IR. ( h ) Knockdown of PKC leads to increased p53 stabilization and reduced p16 protein levels as well as increased ARTD1 cleavage in response to 10 and 40 Gy at 48 h after IR. Western blot analysis of irradiated MRC-5, transfected either with siPKC pan or scrambled siRNA as control for 3 days before IR. Tubulin was used as loading control. ARTD1, ADP-ribosyltransferase diphtheria toxin-like 1; ATF, cyclic AMP-dependent transcription factor 1; ATM, ataxia–telangiectasia mutated; Bad, Bcl2 antagonist of cell death; Bcl-2, B-cell lymphoma 2; CREB, cAMP response element-binding protein; DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated kinase; Et, etoposide; PKC, protein kinase C

Article Snippet: Antibodies used for western blotting were anti-ATM (GeneTex, Irvine, CA, USA), anti-ATM Phospho (pS1981) (Epitomics-an Abcam Company, Burlingame, CA, USA), anti-Bad (CST), anti-Bad Phospho (pS136) (Cell Signaling Technology (CST), Danvers, MA, USA), anti-Chk1 (CST), anti-Chk1 Phospho (pS345) (1 : 500; CST), anti-Chk2 Phospho (pT68) (CST), anti-CREB (CST), anti-CREB Phospho (pS133)/anti-ATF-1 (phospho) (CST), anti-histone H2A.X Phospho (pS139) (Millipore, Billerica, MA, USA), anti-Hsp27 (CST), anti-Hsp27 Phospho (pS78) (1 : 500; CST), anti-PARP1 (Santa Cruz Biotechnology), anti-PARP1 cleaved (1 : 500; CST), anti-p16 (1 : 500; Santa Cruz Biotechnology), anti-p21 (Santa Cruz Biotechnology), anti-p38 (CST), anti-p38 Phospho (pThr180/Tyr182), anti-p44/42 Erk1/2 (CST), anti-p44/42 Erk1/2 Phospho (pThr202/Tyr204) (CST), anti-p53 (Santa Cruz Biotechnology), anti-Rb (1 : 500; Epitomics), anti-Rb (pS780) (CST), anti-tubulin (1 : 10 000; Sigma-Aldrich, St Louis, MO, USA), anti-PKC β II Phospho (pT641) (CST), anti-PKC δ (CST), anti-PKC ζ/λ (CST) and anti-PKC ζ/λ Phospho (pT410/pT403) (CST).

Techniques: Inhibition, Western Blot, Irradiation, Activation Assay, Incubation, Staining, Transfection, Binding Assay

Journal: Cell Death & Disease

Article Title: PKC signaling prevents irradiation-induced apoptosis of primary human fibroblasts

doi: 10.1038/cddis.2013.15

Figure Lengend Snippet: Model of PKC-dependent cellular prosurvival signaling in response to IR. IR-induced PKC signaling orchestrates cell survival via upregulation of Bcl-2 and phosphorylation of Bad and CREB. Hypersensitization with PKC inhibitors (GF109203X, Ro-318220) and genetic knock down (siPKC pan) leads to IR-induced activation of apoptosis mediated by p53 stabilization, upregulation of Bad and ARTD1 cleavage, as well as reduced senescence mediated via decrease in p21 protein levels and reduced β -galactosidase ( β -Gal) activity in response to IR. ARTD1, ADP-ribosyltransferase diphtheria toxin-like 1; Bad, Bcl2 antagonist of cell death; Bcl-2, B-cell lymphoma 2; CREB, cAMP response element-binding protein

Article Snippet: Antibodies used for western blotting were anti-ATM (GeneTex, Irvine, CA, USA), anti-ATM Phospho (pS1981) (Epitomics-an Abcam Company, Burlingame, CA, USA), anti-Bad (CST), anti-Bad Phospho (pS136) (Cell Signaling Technology (CST), Danvers, MA, USA), anti-Chk1 (CST), anti-Chk1 Phospho (pS345) (1 : 500; CST), anti-Chk2 Phospho (pT68) (CST), anti-CREB (CST), anti-CREB Phospho (pS133)/anti-ATF-1 (phospho) (CST), anti-histone H2A.X Phospho (pS139) (Millipore, Billerica, MA, USA), anti-Hsp27 (CST), anti-Hsp27 Phospho (pS78) (1 : 500; CST), anti-PARP1 (Santa Cruz Biotechnology), anti-PARP1 cleaved (1 : 500; CST), anti-p16 (1 : 500; Santa Cruz Biotechnology), anti-p21 (Santa Cruz Biotechnology), anti-p38 (CST), anti-p38 Phospho (pThr180/Tyr182), anti-p44/42 Erk1/2 (CST), anti-p44/42 Erk1/2 Phospho (pThr202/Tyr204) (CST), anti-p53 (Santa Cruz Biotechnology), anti-Rb (1 : 500; Epitomics), anti-Rb (pS780) (CST), anti-tubulin (1 : 10 000; Sigma-Aldrich, St Louis, MO, USA), anti-PKC β II Phospho (pT641) (CST), anti-PKC δ (CST), anti-PKC ζ/λ (CST) and anti-PKC ζ/λ Phospho (pT410/pT403) (CST).

Techniques: Activation Assay, Activity Assay, Binding Assay

Journal: Journal of Nanobiotechnology

Article Title: Prodrug polymeric micelles integrating cancer-associated fibroblasts deactivation and synergistic chemotherapy for gastric cancer

doi: 10.1186/s12951-021-01127-5

Figure Lengend Snippet: PSN38@TPL-nsa antitumor efficiency in the CAFs-containing intraperitoneal tumor model. a Establishment of CAFs-containing intraperitoneal tumor model and schedule of in vivo antitumor experiments. b In vivo bioluminescence imaging of the mice after intravenous injections of PBS, TPL-nsa, PSN38, PSN38 + TPL-nsa and PSN38@TPL-nsa (n = 4). White dotted lines and arrows indicated the tumor nodules. c Quantitative analysis of bioluminescence imaging intensity on Day 12. d Body weight variation in the tumor-bearing mice during the experimental period (n = 4). e Western blotting analysis of PARP, caspase-3 and BAX family proteins of tumors in the different groups at the end of experiments. f Dissected intraperitoneal tumor nodules after humanitarian execution (n = 4). g , h The intraperitoneal tumor weights ( g ) and nodule numbers ( h ) in each group were statistically analyzed. All data are presented as mean ± SD. Unpaired Student’s t-test was used to analyze the data. (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: The specific primary antibodies were used as follows: FAP (Abcam, ab207178, 1:1000), α-SMA (Sigma, A2547, 1:1000), PARP (Proteintech, 66,520, 1:5000), Caspase 3 (Proteintech, 19,677, 1:1000), Bax (Proteintech, 60,267, 1:5000), Cyclin B1 (CST, #4318, 1:1000), Cyclin D1 (CST, #2978, 1:1000), Phospho-NF-κB p65 (Ser536) (CST, #3033, 1:1000), NF-κB p65 (CST, #8242, 1:1000), GAPDH (CST, #97,166, 1:1000).

Techniques: In Vivo, Imaging, Western Blot

Journal: PLoS ONE

Article Title: The Mutyh Base Excision Repair Gene Influences the Inflammatory Response in a Mouse Model of Ulcerative Colitis

doi: 10.1371/journal.pone.0012070

Figure Lengend Snippet: List of genes involved in the inflammatory response and in DNA repair and analyzed for expression.

Article Snippet: , , Mm00500154_m1 , PARP1.

Techniques: Expressing

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