Figure S3

Figure S3. Error bars correspond to mean??SEM (units. The final hitlist compounds were selected after evaluating for binding by combining the consensus scoring function CScore (consensus score? ?3), Surflex-Dock total score ( ?8), and Lipinskis rule-of-five filter. Similarity-based virtual screening was conducted using flexible ligand superpositioning algorithm FlexS implanted in Sybyl [26]. The X-ray pose of PF-470871(PDB ID: 3WE4) was used as the template molecule. A higher similarity score represented a greater similarity of a tested molecule to the template molecule (maximum score is 10.0). Immunoblotting The cells were lysed in Pro-Prep (iNtRON Biotechnology, Korea) and centrifuged at 13,000?rpm for 18?min. For immunoblotting, proteins of each sample were separated through SDS-polyacrylamide gel electrophoresis (PAGE). The proteins were transferred to polyvinylidene difluoride (PVDF) membranes with a semi-dry transfer Rabbit Polyclonal to DAPK3 apparatus (Bio-Rad, Hercules, CA). The membranes were incubated overnight with the indicated primary antibodies, then incubated with horseradish peroxidase-conjugated secondary antibodies for 1?h (Abcam). The signals were detected through chemiluminescence reagents (AbClon, Korea) and quantified with ImageJ program. Immunofluorescence For the ectopic expression of the LC3B vectors, HeLa and SiHa cells were transfected with GFP-LC3B vectors using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA), according to the manufacturers instructions. After 24?h, Amidopyrine cells were fixed in 4$ paraformaldehyde and then GFP Amidopyrine signal from ectopically expressed LC3B was observed using Amidopyrine confocal microscope (Olympus FV-1000 confocal laser scanning microscope) with an Apochromat 60 objective. Quantitative real-time PCR (qPCR) RNA extracts were prepared as previously described [27]. To extract total RNA, cells were lysed in Easy-Blue reagent (iNtRON Biotechnology). Then, 1?g of total RNA was reversely transcribed into cDNA using a Reverse Transcription kit (Promega, USA). Quantitative real-time PCR was performed using KAPATM SYBR FAST qPCR (KAPABIOSYSTEMS) with the CFX96? or Chromo4? real-time PCR detector (Bio-Rad). The relative mRNA levels were normalised to the mRNA levels for each reaction. The qPCR primer sequences used are as follow: forward, 5-GAGTCAACGGATTTGGTCGT-3; reverse, 5-TTGATTTTGGAGGGATCTCG-3; forward, 5-GGACACCATCAGGCTCTTCC-3; reverse, 5-GAAGCC GAAGTCAGCGATCT-3; forward, 5-AGCAACTCTGGATGGGATTG-3; reverse, 5-CACTGCAGAGGTGTTTCCAA-3; forward, 5-AACCTCAGCCGAAGACTGAA-3; reverse, 5-GACGTTGAGCTGAGTGTCCA-3; forward, 5-ACCCAGAAGAAGCTGAACGA-3; reverse, 5-AGACAGAGGGCAGGATAGCA-3; forward, 5-GGCAGTAGAGCGAACACGAA-3; reverse, 5-GGGAAGGAGCAAAGGACTGA-3; forward, 5-CCCAGGACAGAAAGGACCTG-3; reverse, 5-AACCAATCTGAACCCGTTGG-3; forward, 5-TCTACTTTGCCAGCAAACTGG-3; reverse, 5-TGTCCAGCCCATGATGGTTCT-3; forward, 5-AGAGCTGGAAGTCGAGTGT-3; reverse, 5-GCACCTTCACATTCCTCTC-3; forward, 5-GACCTCAACGCACAGTA-3; reverse, 5-CTAATTGGGCTCCATCT-3; forward, 5-TGCGAGAACGACATCAACAT-3; reverse, 5-TCCCGGCAAAAACAAATAAG-3; forward, 5-CACCGAGACACCACTGGAGG-3; reverse, 5-GAGAAGATCAGCCGGCGTTT-3; forward, 5-TTTCCTCTCCAGACTGACAAACTGTT-3; reverse, 5-TAGAACTGAGCTGCAGCTGTAAA-3. Cell viability assay HeLa and SiHa cells were plated in 6 well plates at a density of 6??105 and 2??105 cells Amidopyrine per well, respectively. Cells were treated with DMSO or RAME (40, 80?M) for 24 and 48?h before the cells were counted. For cell counting, cells trypsinized using Trypsin EDTA were counted using a haemocytometer. In vitro kinase assay In vitro kinase assay was performed as previously described [27]. Briefly, recombinant S6K1 (R&D systems, Minneapolis, MN; 896-KS), GST-S6 (Abnova, Taipei city, Taiwan; H00006194-P01), and H2B (BioLabs, MA, USA; M2505S) were used. The reactions were performed in the presence of 100?M adenosine triphosphate (ATP) and kinase reaction buffer [25?mM Tris-HCl (pH?7.5), 5?mM -glycerophosphate, 2?mM dithiothreitol (DTT), 0.1?mM Na3VO4, 10?mM MgCl2] at 37?C for 45?min. The reactions were stopped with 5 Laemmli loading buffer and then subjected to immunoblot analysis. Clonogenic assay For clonogenic assays, HeLa and SiHa cells were seeded in 1??103 cells per well of a 6-well plate and cultured in complete media for 10~20?days. Cells were fixed with glutaraldehyde (6%), stained with 0.5% crystal violet, and photographed using a digital scanner. All experiments were performed at least three times. Representative experiments are shown. Statistical analysis Statistical significance was analysed using the Students and and and em 14C3-3 /em ) increased only after RA treatment, but not after RAME treatment (Fig. ?(Fig.7e;7e; Additional file 1: Figure S6B). The apoptotic marker, cleaved PARP-1, also increased after combination treatment (Fig. ?(Fig.7a).7a). Moreover, clonogenic assay data showed that co-treatment with RAME enhanced the inhibitory effects of cisplatin against colony formation in both HeLa and SiHa cells (Fig. ?(Fig.7f).7f). Lastly, we measured the cell viability of cisplatin-treated SiHa cells by co-treating with RAME at different concentrations. The IC50 values of.