Journal: Gynecologic oncology

Article Title: MEK inhibition overcomes resistance to EphA2-targeted therapy in uterine cancer.

doi: 10.1016/j.ygyno.2021.08.003

Figure Lengend Snippet: Fig. 4. Effects of dasatinib, trametinib, and dasatinib plus trametinib on MAPK pathway and downstream targets. (A) Heatmap of reverse phase protein array (RPPA) analysis of dasatinib- resistant HEC-1A, Ishikawa, and RL-95 cells before and after treatment with dasatinib alone, trametinib alone, or dasatinib plus trametinib for 16 h. Data represent the means of triplicate measurements. (B) RPPA analysis of the normalized expression levels of pMAPK, BIM, JAG1, and c-MYC in dasatinib-resistant HEC-1A, Ishikawa, and RL-95 cells before and after treatment. Data represent the means of triplicate measurements. (C) Pathway analysis of target modulation in dasatinib-resistant RL-95 cells before and after treatment with dasatinib alone or with dasatinib plus trametinib, as detected by NetWalker pathway analysis. (D) Western blot analysis of BIM, c-MYC, cleaved PARP, HES1 and HEYL in RL-95 cells treated with 100 nM dasatinib alone, 1 μM ALWII alone, 100 nM trametinib alone, dasatinib plus trametinib, or ALWII plus trametinib for 48 h. β-actin was used as a loading control.

Article Snippet: Antibodies against p-MEK (Ser217/Ser221), #3958; p-p44/42 MAPK (Thr202/Tyr204), #9101; c-MYC (#5605); and cleaved PARP (#5625) were obtained from Cell Signaling Technology (Danvers, MA).

Techniques: Protein Array, Expressing, Western Blot, Control

Journal: International journal of molecular sciences

Article Title: Fibrinogen-Like Protein 1 Modulates Sorafenib Resistance in Human Hepatocellular Carcinoma Cells.

doi: 10.3390/ijms22105330

Figure Lengend Snippet: Figure 3. Effect of sorafenib on cell death and cell proliferation factors in high FGL1- and low FGL1-expressing hepatocellular carcinoma (HCC) cell lines. (A): Protein expression was analyzed by western blotting 48 h after treatment with 10 µM sorafenib. Expression levels of PCNA, p-ERK/ERK, LC3-II, and cleaved PARP1 quantified in Huh7 (B), Hep3B (C), SNU387 (D), and SNU475 (E) cell lines. Data are presented as the mean ± standard deviation (SD) (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001, significantly different from 0 µM sorafenib-treated cells.

Article Snippet: 2021, 22, 5330 10 of 12 dilution; Cell Signaling Technologies), anti-LC3-II (1:3000 dilution; Novus Biologicals, Littleton, CO, USA), anti-cleaved PARP1 (1:1000 dilution; Cell Signaling Technologies), anti-Beclin-1 (1:1000 dilution; Cell Signaling Technologies), and anti-β-actin (1:2000 dilution; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Western Blot, Standard Deviation

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