In this study, we noted that HDAC6-flag (a Class II HDAC) or HDAC8-flag (a Class I HDAC) significantly reduced WMJ-J-09-induced -tubulin acetylation. Immunofluorescence Microscopy To determine tubulin distribution, FaDu cells were seeded on glass cover slips for 24 h. Cells were treated with WMJ-J-09, paclitaxel or colchicine for another 24 h. Cells were then washed twice with PBS and fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. After permeabilization for 30 min in 0.1% Triton X-100 in PBS, FaDu cells were washed twice and incubated with 1% BSA in PBS for another 1 h. To observe tubulin distribution, cells were reacted with rabbit anti–tubulin antibody (Cell Signaling Technology, Danvers, MA, United States) (1:100 dilution in PBS) for 16 h at 4C. Slides were washed twice and incubated with FITC-conjugated goat anti-rabbit IgG for another 1 h. Slides were mounted with DAPI containing mounting solution (SlowFad Gold, Thermo Fisher Scientific, Waltham, MA, United States) and then observed under a confocal microscope (Zeiss, LSM 410). Green fluorescence indicated -tubulin, and blue fluorescence (derived from DAPI) represented nuclei. Reverse-Transcription-Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) FaDu cells with or without treatments were harvested and total RNA Asoprisnil was isolated for complementary DNA (cDNA) synthesis as described previously (Chuang et al., 2017). Real time PCR was performed with the GoTaq qPCR Master Mix (Promega, Madison, Asoprisnil WI, United States) using StepOne Real-Time PCR systems (Applied Biosystems, Grand Island, NY, United States). The cycling conditions were: hot-start activation at 95C for 2 min, followed by 40 cycles of denaturation at 95C for 15 s, annealing/extension at 60C for 60 s, respectively. Primer pairs for the transcripts of survivin and GAPDH are: survivin sense, 5-gcc ttt cct taa agg cca Asoprisnil tc-3; survivin anti-sense, 5-aac cct tcc cag act cca ct-3; GAPDH sense, 5-gtc agt ggt gg acct gac ct-3; GAPDH anti-sense, 5-agg ggt cta cat ggc aac tg-3. Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH publication no. 85-23, revised 1996). The protocols described below were also approved by the Taipei Medical University Laboratory Animal Care and Use Committee (Permit Number: LAC-2015-0215). Mouse Asoprisnil Xenograft Model Animal studies are reported in accordance with hDx-1 the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). The xenograft model with nudenu/nu mice as described previously (Yen et al., 2016) was employed to determine WMJ-J-09s anti-tumor effects. Four-week old male nudenu/nu mice with body weight about 25 g were obtained from BioLasco (Taipei, Taiwan) and used for the experiment presented in Figure ?Figure88. All the mice were housed (three mice per cage) in clean specific pathogen free (SPF) rooms (standard 12-h light/12-h dark cycle at 22C) in Laboratory Animal Center of Taipei Medical University, and maintained on standard chow and autoclaved water. All mice were randomly allocated to individually ventilated cage (IVC) by vivarium staff, upon transfer from BioLASCO into the animal housing room. All mice purchased from BioLASCO were acclimatized in the animal housing room for 7 days prior to starting experiments. FaDu cells were harvested and resuspended in PBS, and 5 106 cells in a volume of 250 l were injected subcutaneously into the flank of each mouse. Once the tumor reached approximately 150 mm3, animals were randomized into the control group (six mice) and the treatment group (six mice), which received WMJ-J-09 20 mg/kg/day. The treatment was administered intraperitoneally once daily for 23 days. Tumors were measured every day by a digital caliper. Tumor volume was calculated using the formula V (mm3) =?[ab2]??0.52, where is the length and is the width of the tumor (Chang et al., 2015). The body weights of the nude mice were examined daily within 23 days treatment of vehicle or WMJ-8-B. At the end of treatment, animals were sacrificed by carbon dioxide euthanasia and tumors were removed and weighed. The study conforms to the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1996) and was approved by the Taipei Medical University Animal Care and Use Committee. Open in a separate window FIGURE 8 WMJ-J-09.