Sirolimus

cKO mice and littermates (n = 5 mice/goup) were tested for seizure behavior during the night, because mice are nocturnal animals and more active at night. Spontaneous seizure and seizures induced by the ringing of a clock every 1 hour were video-recorded for 5 days. The behaviors of the mice were scored by two independent observers, who were blinded to their genotype. In the rapamycin treatment experiments, behavior analysis was performed after 3 weeks of oral sirolimus (Selleck) in distilled water.

CFPAC-1 cells were seeded on glass coverslips in 12-well plates containing 1 ml of culture medium per well. At 30% confluency, cell media was replaced by low serum medium containing 2% FBS. After 8 days of compound treatment, cells were fixed with 4% Formaldehyde (Merck), permeabilized with 0.1% Triton X100 (Merck) in DPBS, blocked with 1% BSA (Applichem) in DPBS, and incubated with 1:1000 dilution of anti-acetylated tubulin antibody or 1:500 dilution of anti-IFT88 antibody (Cat. No. 13967–1-AP, Proteintech) for 1 h, followed by incubation with 1:1000 dilution of fluorescent secondary antibody for 1 h. Nuclei were counterstained with DAPI (Vector Laboratories, Vectashield (Cat. No. H-1500). Images of primary cilia were captured by acquiring Z-stacks using either a Bio-Rad Radiance or Nikon C2 Eclipse Ti-E confocal laser scanning microscope by 40X or 60X oil immersion lenses. All drugs selected for confocal reconfirmation experiments were purchased from Sigma, except for Almotriptan Malate and Sirolimus which were obtained from Selleckchem. Gefitinib was from Invivogen, Cefprozil Monohydrate from Abcam and Imexon from MicroSource Discovery Systems Inc.

Antibodies used in these experiments included the following: anti-RND2 (13844–1-AP, Proteintech, USA), anti-Rho7/Rnd2 (GXT56070, GeneTex, USA), anti-p-p38 (#4511, Cell Signaling Technology, USA), anti-p38 (#9212, Cell Signaling Technology, USA), anti-cleaved-caspase3 (ab32042, Abcam, UK), anti-caspase3 (NB100-56708SS, Novus, USA), anti-BAX (50599–2-Ig, Proteintech, USA), anti-GAPDH (#5174, Cell Signaling Technology), anti-P62 (M162–3, Medical Biological Laboratories, Japan), anti-Beclin1 (11306–1-AP, Proteintech, USA), anti-LC3B (GB11124, Servicebio, China), anti-DYKDDDK/Flag-tag (ANT102, Antgene, China), and anti-His-tag (D291–3, Medical Biological Laboratories, Japan). The autophagy inhibitor wortmannin (3-MA) and the autophagy activator rapamycin (Sirolimus) (S1039, USA) were purchased from Selleck (S2758, USA).

Tacrolimus (FK506) (Selleckchem), pimecrolimus (Selleckchem), sirolimus (Selleckchem), and ionomycin (Alomone labs) were all dissolved in DMSO (Sigma Aldrich). Activity of the compounds on VZV gB/gH-gL mediated cell fusion were evaluated by stable reporter fusion assay as described previously [28 (link)]. Briefly, CHO-DSP1 or MeWo-DSP1 cells transfected with equal quantities of pCAGGS-gB, pME18S-gH[TL], and pcDNA3.1-gL plasmids using Lipofectamine 2000 were harvested at 6 hrs post-transfection, and mixed with MeWo-DSP2 cells in the presence of various concentrations of the compounds prepared in two-fold serial dilutions, ranging from 10 μM to 1.25 μM. Co-culture of cells were seeded into Nunc MicroWell 96-well Optical-Bottom Plates (ThermoFisher) and incubated for 48 hrs. The activity of Renilla luciferase was read immediately after adding substrate h-Coelenterazine (Nanolight Technology). Transfection with only vehicle plasmids pcDNA3.1 (+) and pME18S, or pcDNA3.1 (+), pME18S and pCAGGS-gB served as negative control. Cell viability was measured using CellTiter-Glo Luminescent substrate (Promega). Luminescence signal was recorded using Synergy H1 Hybrid Multi-Mode Reader (BioTek). Experiments were performed at least in triplicate.

Tsc2^flox/flox mice and Mitf-M-Cre mice were generated as described previously[9 (link)]. Melanocyte-specific Tsc2 knockout mice were generated by breeding Tsc2^flox/flox mice with Mitf-M-Cre mice. Both lines maintained a C57BL/6 inbred background. The controls were littermates, either without cre or in a few cases, Mitf-M-cre;Tsc2^flox/-. For rapamycin treatment, sirolimus was purchased from Selleck (Osaka, Japan) and dissolved in distilled water for oral administration at 2.285 mg/kg/day for 3 weeks (n = 5 mice/goup). All animal experiments were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, and the experimental protocol used in this study was approved by the Committee for Animal Experiments at Osaka University (Osaka, Japan).