Journal: Biochemical pharmacology

Article Title: The synthetic bryostatin analog Merle 23 dissects distinct mechanisms of bryostatin activity in the LNCaP human prostate cancer cell line.

doi: 10.1016/j.bcp.2011.03.018

Figure Lengend Snippet: Fig. 3. Evaluation of the role of individual PKC isoforms in the biological effects of PMA, bryostatin 1 and Merle 23 using PKC inhibitors or siRNA. (A) The effect of PKC inhibitors Go¨6976 (2 mM) and Go¨6983 (2 mM) on cell growth in the presence of vehicle (DMSO) or 100 nM PMA, bryostatin 1 or Merle 23 was determined by the Incucyte as described for Fig. 2A. Values represent the mean S.E.M. of thtee independent experiments. PMA versus PMA + Go¨6983, p = 0.0091; PMA versus PMA + Go¨6976, p = 0.44. (B) The effect of different siRNAs on cell growth. Cells were transfected with the indicated siRNAs as described in Section 2 and were treated 48 h later with DMSO as control or 300 nM PMA, bryostatin 1 or Merle 23 for 48–60 h. Confluency was determined by the Incucyte. Values represent the mean S.E.M. of seven independent experiments. Co = control, non-treated cells; scr = scrambled siRNA; a, d, e si = siRNA against PKC alpha, delta and epsilon isoforms, respectively. p values for Merle 23 control versus DMSO, PMA and bryostatin1 control are 0.0015, 0.0046 and 0.010, respectively; p values for delta siRNA versus control for PMA and Merle 23 are p < 0.0001 and 0.014; all p values for PMA and Merle 23 between control and alpha or epsilon siRNAs are >0.5. (C) The effect of PKC inhibitors Go¨6976 (2 mM) and Go¨6983 (2 mM) on TNF-alpha secretion induced by 10 nM PMA, bryostatin 1 or Merle 23. Secretion of TNF-alpha into the supernatants was measured by ELISA 24 h after treatment. Values represent the mean S.E.M. of three independent experiments. PMA versus PMA + Go¨6976, p = 0.0005. (D) The effect of different siRNAs on TNF-alpha secretion. Cells transfected with the indicated siRNAs as described in Section 2 were treated 48 h later with DMSO as control or 10 nM PMA, 100 nM bryostatin 1 or 100 nM Merle 23 for 24 h. TNF-alpha secreted into the supernatant was measured by ELISA. Values represent the mean S.E.M. of three independent experiments. For abbreviations see legend above. PMA versus PMA + alpha siRNA, p = 0.0006.

Article Snippet: The primary antibodies against PKC alpha (C-20), delta (C-20), epsilon (C-15), beta (C-16 and C-18), eta (31), theta (C-18 and 1C2), p65 (F-6), and cFos (H-125) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Control, Enzyme-linked Immunosorbent Assay

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 1. (A) Hematoxylin and eosin staining and immunohistochemical staining for PKCα and claudin-1 in normal pancreatic ducts, well- and poorly differentiated pancreatic carcinomas. Bar: 50 μm. (B and C) Western blotting for pPKCα, panPKCα (PKCα), Snail, claudin-1, -4, -7 and occludin in normal pancreatic duct epithelial cells (hTERT-HPDEs) and pancreatic cancer cell lines HPAC, HPAF-II, BXPC-3 and PANC-1. Snail (1): exposed to X-ray film for 1 min, Snail (2): exposed to X-ray film for 20 min. The corresponding expression levels of B and C are shown as bar graphs. n = 3, *P < 0.05 and **P < 0.01 versus hTERT-HPDEs.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Staining, Immunohistochemical staining, Western Blot, Expressing

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 2. (A) Western blotting for Snail, claudin-1, -4, -7 and occludin in PANC-1 cells after treatment with 0–2 μg/ml Gö6976 for 24 h. (B) Real-time PCR for Snail, claudin-1 and occludin in PANC-1 cells after treatment with 0–2 μg/ml Gö6976 for 24 h. (C) Western blotting for claudin-1, -4, -7 and occludin in hTERT- HPDEs after treatment with 1 and 2 μg/ml Gö6976 for 24 h. (D) Real-time PCR for claudin-1, -4, -7 and occludin in hTERT-HPDEs after treatment with 1 μg/ ml Gö6976 for 24 h. (E) TER values in hTERT-HPDEs after treatment with 1 μg/ml Gö6976 for 24 h. iPKCα: PKCα inhibitor Gö6976. n = 3, *P < 0.05 and **P < 0.01 versus control (0 μg/ml).

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 3. Western blotting for pMAPK and panMAPK (MAPK) in PANC-1 cells (A) and hTERT-HPDEs (B) after treatment with 0–2 μg/ml Gö6976 for 24 h. Western blotting for claudin-1, occludin, pMAPK, MAPK and Snail in PANC-1 cells (C) and hTERT-HPDEs (D) pretreated with 20 μM MAPK/ERK inhibitor U0126 before treatment with 1 μg/ml Gö6976 for 24 h. Western blotting for claudin-1 in PANC-1 cells (E) and hTERT-HPDEs (F) pretreated with 20 μM MAPK/ERK inhibitor U0126, 10 μM p38 MAPK inhibitor SB203580 (SB), 10 μM PI3K inhibitor LY294002 (LY), 10 μM panPKC inhibitor GF109203X (GF), 10 μM JNK inhibitor SP600125 (SP) and 0.1 μM NF-κB inhibitor IMD-0354 (IMD) before treatment with 1 μg/ml PKCα inhibitor Gö6976 for 24 h. iPKCα: PKCα inhibitor Gö6976.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 4. (A) Western blotting for Snail, pPKCα, panPKCα (PKCα), pMAPK, panMAPK (MAPK), claudin-1, -4, -7 and occludin in PANC-1 cells after treatment with 100 ng/ml TGF-β1 for 24 and 48 h. (B) Western blotting for claudin-1, -4, -7, occludin, Snail, PKCα, pMAPK and MAPK in hTERT-HPDEs after treatment with 20 ng/ml TGF-β1 for 24 and 48 h. (C) Western blotting for Snail and claudin-1 in PANC-1 cells treated with siRNAs of Snail. (D) Western blotting for Snail and claudin-1 in PANC-1 cells treated with siRNAs of Snail and 100 ng/ml TGF-β1 for 48 h. Control cells in Figure 4C and D are transfected with a scrambled siRNA. KD: knockdown.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot, Control, Transfection, Knockdown

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 5. (A) Western blotting for Snail, claudin-1, -4, -7, occludin, pMAPK and panMAPK (MAPK) in PANC-1 cells pretreated with 1 μg/ml Gö6976 before treatment with 100 ng/ml TGF-β1 for 24 h. (B) Western blotting for claudin-1, -4, -7, occludin, Snail, panPKCα (PKCα), pMAPK and MAPK in hTERT-HPDEs pretreated with 1 μg/ml Gö6976 before treatment with 20 ng/ml TGF-β1 for 24 h. (C) Western blotting for Snail, claudin-1, occludin, pPKCα, PKCα, pMAPK and MAPK in PANC-1 cells with or without 1 μg/ml Gö6976 under hypoxia for 24 h. (D) Western blotting for claudin-1, -4, -7, occludin, pMAPK, MAPK and Snail in hTERT-HPDEs with or without 1 μg/ml Gö6976 under hypoxia for 24 h. iPKCα: PKCα inhibitor Gö6976.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot

Journal: Carcinogenesis

Article Title: Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer.

doi: 10.1093/carcin/bgt057

Figure Lengend Snippet: Fig. 6. (A) Western blotting for Snail, pPKCα, panPKCα (PKCα), claudin-1, -4, -7 and occludin in HPAC cells after treatment with 100 μg/ml TGF-β1 for 24 and 48 h. (B) Western blotting for Snail, pPKCα, PKCα, claudin-1, -4, -7 and occludin in HPAC cells pretreated with 1 μg/ml Gö6976 before treatment with 100 ng/ml TGF-β1 for 24 h. (C) TER values in HPAC cells pretreated with 100 ng/ml TGF-β1 for 24 h with or without 1 μg/ml Gö6976. (D) Fence function examined by diffusion of labeled BODIPY-sphingomyelin into HPAC cells pretreated with 100 ng/ml TGF-β1 for 24 h with or without 1 μg/ml Gö6976. In HPAC cells treated with TGF-β1, the probe strongly labels the basolateral surface and appears to penetrate the cells (arrowheads), whereas the probe is effectively retained in the apical domain of the control cells and the cells were treated with TGF-β1 and Gö6976. Bar: 20 μm. iPKCα: PKCα inhibitor Gö6976.

Article Snippet: Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. at U niversity of Illinois at U rbana-C ham paign on M arch 16, 2015 http://carcin.oxfordjournals.org/ D ow nloaded from from Cell Signaling (Beverly, MA).

Techniques: Western Blot, Diffusion-based Assay, Labeling, Control

Journal: Endocrinology

Article Title: High glucose potentiates collagen synthesis and bone morphogenetic protein-2-induced early osteoblast gene expression in rat spinal ligament cells.

doi: 10.1210/en.2009-0833

Figure Lengend Snippet: FIG. 4. Involvement of p38, PKC, and ERK1/2 in BMP-2-induced osteogenesis of ligament cells promoted by high glucose (NO, no inhibitor or agonist added; GF, 5000 nM GF109203X; PD, 5000 nM PD98059; SB, 10,000 nM SB203580; PMA, 10,000 nM PMA; P79350, 50,000 nM P79350). A–D, Expression of Runx2, ALP, OP, and COL1a1 (COL I) mRNA in cells; E, ALP activity; F, PINP synthesis (*, P 0.05 vs. NO of respective group). Values represent the mean SD of three independent experiments in triplicate dishes.

Article Snippet: Proteins were electrotransferred to nitrocellulose membranes at 100 V for 1.5 h. They were blocked using 5% milk in Tris-buffered saline [20 mmol/liter Tris (pH 7.5), 500 mmol/liter NaCl, and 0.05% Tween 20] and then hybridized for 1 h with anti-PKC, anti-p38 (Thr180/Tyr182) and its phosphorylation antibody [anti-phospho-PKC, (pan) ( Thr410) (190D10); anti-phospho-p38 (Thr180/Tyr182) (3D7)] (Cell Signaling Technology) followed by incubation for 1 h with horseradish peroxidase-conjugated secondary IgG.

Techniques: Expressing, Activity Assay

Journal: Endocrinology

Article Title: High glucose potentiates collagen synthesis and bone morphogenetic protein-2-induced early osteoblast gene expression in rat spinal ligament cells.

doi: 10.1210/en.2009-0833

Figure Lengend Snippet: FIG. 5. Influence of high glucose on the p38 and PKC pathway in ligament cells. Whole-cell extracts were prepared 30 min after changing the media and analyzed by Western blot. A, Influence of different stimulations on p38 in ligament cells (*, P 0.05 vs. CTR; #, P 0.05); B, influence of BMP-2 plus 25 mM glucose for various times (5–45 min) on p38 in ligament cells (*, P 0.05 vs. each other time point); C, influence of different stimulations on PKC in ligament cells (*, P 0.05 vs. CTR; #, P 0.05); D, influence of BMP-2 plus 25 mM glucose various time (5–45 min) on PKC in ligament cells (*, P 0.05 vs. each other time point) (CTR, control culture; HIGH, 25 mM glucose; BMP, 500 ng/ml BMP-2; BMP HIGH, 500 ng/ml BMP-2 plus 25 mM glucose). Values represent the mean SD of three independent experiments in triplicate dishes. P-p38, Phosphorylation of p38; P-PKC, phosphorylation of PKC.

Article Snippet: Proteins were electrotransferred to nitrocellulose membranes at 100 V for 1.5 h. They were blocked using 5% milk in Tris-buffered saline [20 mmol/liter Tris (pH 7.5), 500 mmol/liter NaCl, and 0.05% Tween 20] and then hybridized for 1 h with anti-PKC, anti-p38 (Thr180/Tyr182) and its phosphorylation antibody [anti-phospho-PKC, (pan) ( Thr410) (190D10); anti-phospho-p38 (Thr180/Tyr182) (3D7)] (Cell Signaling Technology) followed by incubation for 1 h with horseradish peroxidase-conjugated secondary IgG.

Techniques: Western Blot, Control, Phospho-proteomics

Journal: Endocrinology

Article Title: High glucose potentiates collagen synthesis and bone morphogenetic protein-2-induced early osteoblast gene expression in rat spinal ligament cells.

doi: 10.1210/en.2009-0833

Figure Lengend Snippet: FIG. 6. Influence of cellular ROS induced by high glucose on the p38 and PKC pathways in ligament cells. Whole-cell extracts were prepared 30 min after medium was added with 0.1 mM NAC and analyzed by Western blot. A, Influence of NAC on p38 in ligament cells (*, P 0.05 vs. BMP HIGH); B, influence of NAC on PKC in ligament cells (*, P 0.05 vs. BMP HIGH). Values represent the mean SD of three independent experiments with triplicate dishes.

Article Snippet: Proteins were electrotransferred to nitrocellulose membranes at 100 V for 1.5 h. They were blocked using 5% milk in Tris-buffered saline [20 mmol/liter Tris (pH 7.5), 500 mmol/liter NaCl, and 0.05% Tween 20] and then hybridized for 1 h with anti-PKC, anti-p38 (Thr180/Tyr182) and its phosphorylation antibody [anti-phospho-PKC, (pan) ( Thr410) (190D10); anti-phospho-p38 (Thr180/Tyr182) (3D7)] (Cell Signaling Technology) followed by incubation for 1 h with horseradish peroxidase-conjugated secondary IgG.

Techniques: Western Blot