CYNA protected against glutamate-induced neurotoxicity in PC12 cells and cerebellar granule neurons

TNIP1 played a role in modulating the proliferation of human keratinocytes and exaggerated IMQ-induced psoriasis-like dermatitis in mice. These findings may constitute an attractive target for therapeutic interventions for psoriasis. 2 / 18 TNIP1 Regulates the Proliferation of Keratinocytes Materials and Methods The experimental protocol was established according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of Southwest Hospital of the Third Military Medical University in Chongqing, China. Written informed consent was obtained from individual participants. Written informed consent was obtained from the guardians on behalf of the children enrolled in this study. All animal studies were approved by the animal ethics committee of the Third Military Medical University according to Dutch legislation on animal experiments. Surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Cell cultures HaCaT, a spontaneously immortalized human keratinocyte cell line, was purchased from Cell Resource Center. Cells were cultured in RPMI 1640 with 10% fetal bovine serum under a humidified atmosphere containing 5% CO2 at 37C. PHKs were isolated from normal foreskin discarded during circumcision of two patients as previously described. Briefly, an epidermal cell suspension was obtained by incubating dispase-separated epidermal sheets in 0.25% trypsin PR-619 biological activity solution at 37C for 15 min. Cells were cultured on tissue culture plates coated with Collagen IV in the presence of defined keratinocyte serum-free media at 37C in humidified atmosphere with 5% CO2. Pan-Cytokeratin AE1/AE3 was positive after PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698000 PHKs were exposed to 10% FBS for 24 h, as confirmed by immunofluorescence staining. The experiments on PHKs were randomly conducted at passages 24. Immunofluorescence staining and confocal microscopy HaCaT cells or PHKs were first fixed in acetone, permeabilized with 0.25% Triton for 10 min, blocked with 1% BSA for 30 min, and then incubated with mice anti-human TNIP1 monoclonal antibody or mouse monoclonal anti-Pan-Cytokeratin AE1/AE3 antibody, respectively. FITC-labeled goat anti-mice IgG was used to detect bound antibodies. 40-6-Diamidino-2-phenylindole was used for nuclear counterstaining. PHKs were incubated in RPMI 1640 containing 10% FBS for 24 h before fixing. The cells were observed by confocal microscopy. A negative control was included. Small interfering RNA transfection The C/EBP-targeting oligonucleotide was designed based on the full-length human C/EBP cDNA sequence . Three sequences targeting different regions of the C/EBP gene were designed. The first sequence, which matches the sequence located at nucleotides 14481466 of the C/EBP cDNA, was the most effective and was used to knock down endogenous C/EBP in the following experiments. A nonsilencing-siRNA was used as a negative control. The transfection process was performed according to the Lipofectamine 2000 instructions. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19697345 Briefly, 24 h before transfection, cells were grown in six-well plates and the media was changed with 800 L growth media supplemented with serum, but without antibiotics. In addition, 6 L of 20 M siRNA or 4 L of Lipofectamine 2000 was added to 100 L of 1640 media without serum and incubated for 5 min at room temperature separately before mixing. The mixture was incubated for 20 min at room temperature before it was added to the cells, and the final 3 / 18 TNIP1 Regulates the Prolife