Journal: Scientific Reports

Article Title: TRPV4-dependent induction of a novel mammalian cold-inducible protein SRSF5 as well as CIRP and RBM3

doi: 10.1038/s41598-017-02473-x

Figure Lengend Snippet: Regulatory mechanisms of SRSF5 induction. ( a and b ) Fibroblasts from wild-type (+/+) or knockout (−/−) mice were analyzed for expression of CIRP and RBM3 by western blot (representative of 2 independent experiments) ( a ). CIRP-RBM3 double knockout (DKO) fibroblasts were cultured at 37 °C or 32 °C for 8 or 24 h, and analyzed by western blot. Relative band intensities after normalization to ACTIN expression are shown below the panel (representative of 2 independent experiments) ( b ). ( c ) B22 cells were cultured at 37 °C or 32 °C for indicated times, and analyzed by western blot (representative of 3 independent experiments). ( d ) U-2 OS cells were incubated with isotonic regular media (0%) or hypotonic media containing 10 or 20% (volume/volume) of H 2 O at 37 °C or 32 °C in the presence of 0 or 30 μM RN1734 for 8 h, and analyzed by western blot (representative of 3 independent experiments). ( e ) U-2 OS cells were cultured at 37 °C or 32 °C in the presence of indicated concentrations of RN1734 for 24 h, and analyzed by western blot (representative of 3 independent experiments). ( f ) U-2 OS cells were cultured at 37 °C or 32 °C in the absence (−) or presence (+) of 30 μM RN1734 together with 20 nM doxorubicin (Doxo) for 8 h, and analyzed by western blot (representative of 4 independent experiments). Full-length blots are presented in Supplementary Fig. .

Article Snippet: Inc., North York, ON), Fura-2-AM (Molecular Probes, Carlsbad, CA), gadolinium chloride (Nacalai Tesque), GSK1016790A (Sigma-Aldrich), HC067047 (Tocris Bioscience), RN1734 (Tocris Bioscience), RN1747 (Tocris Bioscience), and ruthenium red (Tocris Bioscience).

Techniques: Knock-Out, Expressing, Western Blot, Double Knockout, Cell Culture, Incubation

Journal: Scientific Reports

Article Title: TRPV4-dependent induction of a novel mammalian cold-inducible protein SRSF5 as well as CIRP and RBM3

doi: 10.1038/s41598-017-02473-x

Figure Lengend Snippet: TRPV4 ion channel activity and induction of CIPs. ( a and b ) U-2 OS cells were cultured at 37 °C or 32 °C for 24 h in the presence of indicated doses of gadolinium chloride (Gd 3+ ) ( a ) or ruthenium red (RR) ( b ), and analyzed by western blot (representatives of 3 independent experiments each). ( c ) U-2 OS transfectants transiently expressing vector alone or shRNA against TRPV4 (shTRPV4) were cultured at 37 °C or 32 °C for 16 h, and analyzed by western blot (representative of 5 independent experiments). ( d,e,f and g ) U-2 OS cells were cultured at 37 °C or 32 °C for 24 h in the presence of indicated doses of RN1747 ( d ), GSK1016790A ( e ), A23187 ( f ), or BAPTA-AM ( g ), and analyzed by western blot (representatives of 3 independent experiments each). ( h ) Quantification of intracellular Ca 2+ concentrations by Fura-2 in U-2 OS cells. Cells were cultured at 37 °C, 35 °C or 32 °C for 24 h in the presence or absence of 30 μM RN1734 (data indicate mean ± SEM; n = 6). Statistical significance was determined by Student’s t -test. **, P < 0.01. ( i ) U-2 OS cells were cultured at 37 °C or 35 °C for indicated times, and analyzed by western blot (representative of 4 independent experiments). Full-length blots are presented in Supplementary Fig. .

Article Snippet: Inc., North York, ON), Fura-2-AM (Molecular Probes, Carlsbad, CA), gadolinium chloride (Nacalai Tesque), GSK1016790A (Sigma-Aldrich), HC067047 (Tocris Bioscience), RN1734 (Tocris Bioscience), RN1747 (Tocris Bioscience), and ruthenium red (Tocris Bioscience).

Techniques: Activity Assay, Cell Culture, Western Blot, Expressing, Plasmid Preparation, shRNA

Journal: International Journal of Molecular Sciences

Article Title: Weak Ultraviolet B Enhances the Mislocalization of Claudin-1 Mediated by Nitric Oxide and Peroxynitrite Production in Human Keratinocyte-Derived HaCaT Cells

doi: 10.3390/ijms21197138

Figure Lengend Snippet: Involvement of transient receptor potential type vanilloid 1 (TRPV1) in the UVB-induced production of RNS. ( A,B ) After we exposed cells to weak UVB, the cells were incubated for 3 h in the absence (Veh) and presence of 10 μM AMG9810 (AMG), 10 μM RN1734 (RN), or 2 mM glycoletherdiaminetetraacetic acid (EGTA). ( C ) Cells were incubated for 3 h in the absence (Cont) and presence of olvanil (Olv). The cells were incubated with 5 μM Fluo-8 AM, 5 μM DAF-2DA, or 5 μM NiSPY-3 for 30 min. The fluorescence intensities of Fluo-8, DAF-2, and NiSPY-3 were measured using a plate reader. n = 8. ** p < 0.01 vs. Cont. ## p < 0.01 and NS p > 0.05 vs. Veh.

Article Snippet: AMG9810, olvanil, and RN1734 were from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan).

Techniques: Incubation, Fluorescence