Journal: Oncotarget

Article Title: Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways

doi: 10.18632/oncotarget.25481

Figure Lengend Snippet: (A) Cells were pretreated with EGF (100ng/ml) and harvested after the indicated times. Lysates were collected, and p38 MAPK phosphorylation levels were analyzed by Western blotting using the antiphospho-p38 antibody. The same membrane was striped and re-probed with a different antibody for measuring total-p38 MAPK protein levels. (B) Caki-1 cells were preincubated with 400pM of the secreted form of the Klotho (KL) protein at the indicated times followed by EGF (100ng/ml) stimulation for additional 5 min. Cell lysates were subjected to Western blot analysis as described in (A). Plots indicate means ± S.E.M of three independent experiments.

Article Snippet: Rabbit anti-human polyclonal active p38 MAPK antibody and SB203580 were purchased from Promega (Madison, WI).

Techniques: Phospho-proteomics, Western Blot, Membrane

Journal: Oncotarget

Article Title: Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways

doi: 10.18632/oncotarget.25481

Figure Lengend Snippet: (A) Classical in vitro wound healing assay was performed using near-confluent serum-starved Caki-1 cells grown on collagen type 1. Cells were either pretreated with 400pM Klotho (KL) or 1μM p38 MAPK-specific inhibitor SB203580 as positive control, for 60 min followed by EGF (100ng/ml) treatment. Cell culture images shown here were taken at 0 and 24 h. (B) Plots of quantification of the resultant cell motility values were computed by gap surface area measurements for four selected microscopic fields in each assay condition. The degree of migration is expressed as % wound closure compared with zero time point. The results represent means ± S.E.M of three independent experiments. (C) Wound healing assays performed under 3D settings. Images are microphotographs of Caki-1 cells showing hole-closure of “tissue openings” generated with magnetic pattering as described in the materials and method. Cells were pretreated the same way with either Klotho (KL) or SB203580 and stimulated with EGF as described for the classical wound healing assay. (D) Plots of rate closure of holes for Caki-1 cells as a function of EGF exposure in the presence or absence of KL or SB203580.

Article Snippet: Rabbit anti-human polyclonal active p38 MAPK antibody and SB203580 were purchased from Promega (Madison, WI).

Techniques: In Vitro, Wound Healing Assay, Positive Control, Cell Culture, Migration, Generated

Journal: Basic Research in Cardiology

Article Title: Oxidized low-density lipoprotein (oxLDL) affects load-free cell shortening of cardiomyocytes in a proprotein convertase subtilisin/kexin 9 (PCSK9)-dependent way

doi: 10.1007/s00395-017-0650-1

Figure Lengend Snippet: Participation of MAPK pathways in oxLDL-dependent effects: a cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and SP600125 (10 µM). b Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and PD98059 (10 µM). c Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and SB202190 (10 µM). In a – c a represents p < 0.095 vs. control. d Representative immunoblot of samples from cardiomyocytes exposed to oxLDL (20 µg/ml) and quantified for p38 MAPK expression and phosphorylation of p38 MAPK. e Quantification of the blot shown in d . f Original western blot showing the oxidative modification of tropomyosin by oxLDL (20 mg/ml) in the left and the control blot under reducing conditions (right). g Quantification of oxidative modification of tropomyosin by oxLDL ( n = 3). Exact p values are given

Article Snippet: Anti-PCSK9 (rabbit polyclonal, ab31762, Abcam, Cambridge, UK), anti p38 MAPK (rabbit polyclonal, #506123, Merck KGaA Darmstadt, Germany), and anti phosphorylated p38 MAPK (rabbit polyclonal, #M0800, Sigma, St. Louis, USA) antibodies were used to detect the expression of PCSK9 protein and phosphorylation of p38 MAPK.

Techniques: Western Blot, Expressing, Modification

Journal: Basic Research in Cardiology

Article Title: Oxidized low-density lipoprotein (oxLDL) affects load-free cell shortening of cardiomyocytes in a proprotein convertase subtilisin/kexin 9 (PCSK9)-dependent way

doi: 10.1007/s00395-017-0650-1

Figure Lengend Snippet: Summary figure of our findings: oxidized LDL (oxLDL) activates the oxLDL receptor. Expression of the receptor by cardiomyocytes was shown in this study and silencing of the receptor by siRNA attenuated all subsequent steps. oxLDL induces oxidative stress as indicated by oxidative modifications of tropomyosin (Trp-ox). Oxidative stress activates p38 MAP kinase as indicated by western blot. Inhibition of p38 MAP kinase activation by SB20190 attenuates this effect. Subsequently, oxLDL causes increased expression of PCSK9 as indicated by western blots and inhibition of transcription by actinomycin D (ActD) or translation by cycloheximide (chx) block all subsequent steps. Silencing of PCSK9 upregulation attenuates future steps

Article Snippet: Anti-PCSK9 (rabbit polyclonal, ab31762, Abcam, Cambridge, UK), anti p38 MAPK (rabbit polyclonal, #506123, Merck KGaA Darmstadt, Germany), and anti phosphorylated p38 MAPK (rabbit polyclonal, #M0800, Sigma, St. Louis, USA) antibodies were used to detect the expression of PCSK9 protein and phosphorylation of p38 MAPK.

Techniques: Expressing, Western Blot, Inhibition, Activation Assay, Blocking Assay