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: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: DEPP1 enhances oxaliplatin resistance in gastric cancer cells in vitro . Effects of oxaliplatin and fluorouracil supplementation on DEPP1 protein levels in (A) MKN45 and (B) HGC27 cells, respectively. Validation of DEPP1 overexpression in (C) MKN45 and (D) HGC27 cells. (E) Impact of DEPP1 overexpression on MKN45 cell proliferation, assessed using EdU flow cytometry. (F) Influence of DEPP1 overexpression on MKN45 proliferation following a 24-h treatment with 10 µM oxaliplatin. (G) Effects of ectopic DEPP1 expression on oxaliplatin-induced apoptosis (20 µM) in MKN45 cells, as measured by Annexin V/PI flow cytometry. (H) Analysis of full-length and cleaved PARP protein levels following forced DEPP1 expression in (H) MKN45 and (J) HGC27 cells. *P<0.05 and ***P<0.001 DEPP1, decidual protein induced by progesterone. ns, not significant.

Article Snippet: The membranes were then incubated with 5% skimmed milk at room temperature for 1 h. Following this, the membranes were probed with primary antibodies against DEPP1 (1:1,000; cat. no. 25833-1-AP; Proteintech Group, Inc.), PARP (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), cleaved PARP (1:500; cat. no. HY- P80448 ; MedChemExpress), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), or β-actin (1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) at 4°C overnight, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (33101ES60, 1:5,000, Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG (1:5,000; cat. no. BL001A; Biosharp Life Sciences) at room temperature for 1 h. Finally, proteins were visualized using an Omni-ECLTMPico Light Chemiluminescence Kit (cat. no. SQ202L; Epizyme, Inc.).

Techniques: In Vitro, Biomarker Discovery, Over Expression, Flow Cytometry, Expressing