M mlv rt kit

Rabbit monoclonal antibodies specific for p85, p-p85, Akt, p-Akt, FAK, and FAK as well as anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit monoclonal antibodies specific for VEGF-C and control IgG were purchased from Abcam (Cambridge, MA, USA). The ELISA kit for VEGF-C was obtained from PerpoTech (Rocky Hill, NJ, USA). The recombinant human VEGF-C was obtained from R&D Systems (Minneapolis, MN, USA). The human chondrosarcoma tissue array was obtained from Biomax (Rockville, MD, USA). The Matrigel was purchased from BD Biosciences (Bedford, MA, USA). The OBRl antisense and missense oligonucleotide (ODN) were purchased from MDBio (Taipei, Taiwan)31 (link). The Trizol, Lipofectamine 2000, MMLV RT kit, miR-27b mimic, miR-27b inhibitor and control miRNA were obtained from Invitrogen (Carlsbad, CA, USA). The siRNAs against p85, Akt1, FAK, and control were obtained from Dharmacon Research (Lafayette, CO, USA). The TaqMan assay kit and TaqMan MicroRNA Reverse Transcription Kit were obtained from Thermo Fisher Scientific (Grand Island, NY, USA). LY294002 and other pharmacological inhibitors were purchased from Sigma-Aldrich (St. Louis, MO, USA)

Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase as well as rabbit monoclonal antibodies (mAbs) specific for WISP-3, p38, p-p38 (Tyr¹⁸²), c-Src, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody specific for p-Src (Tyr⁵²⁷) was purchased from Cell Signaling and Neuroscience (Danvers, MA, USA). A VEGF-A ELISA kit was obtained from PerpoTech (Rocky Hill, NJ, USA). Control IgG and rabbit mAb specific for VEGF-A were obtained from Abcam (Cambridge, MA, USA). Control miRNA, miR-452 mimic, MMLV RT kit, Trizol and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). The TaqMan MicroRNA Reverse Transcription kit and the TaqMan assay kit were obtained from Thermo Fisher Scientific (Grand Island, NY, USA). c-Src, p38 and control siRNA were purchased from Dharmacon Research (Lafayette, CO, USA). PP2 and SB203580 were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Total RNA from mesothelioma cell lines differently treated or not was extracted by using Trizol Reagent following manufacturer’s instructions (Ambion). cDNA was synthesized according to the manufacturer’s instructions (M-MLV RT kit, Invitrogen). Gene expression was measured by real-time PCR using the FastStart SYBR Green Master Mix (Applied Biosytems) on a studio 7-instrument (Applied Biosystems). Sequences of qPCR primers are ACTIN Fw: 5′-GGCATGGGTCAGAAGGATT-3′, Rv: 5′-CACACGCAGCTCATTGTAGAAG-3; YAP1 Fw: 5′-CACAGCATGTTCGAGCTCAT-3′, Rv: 5′-GATGCTGAGCTGTGGGTGTA-3′; TAZ Fw: 5′-CCATCACTAATAATAGCTCAGATC-3′, Rv: 5′-GTGATTACAGCCAGGTTAGAAAG-3′; MCM7 Fw: 5′-TCGAGGCATGAAAATCCGGG-3′, Rv: 5’CGCCAGTCGATCAATGTATGACA-3′; ANKRD1 Fw: 5′-AGTAGAGGAACTGGTCACTGG-3′, Rv: 5′-TGGGCTAGAAGTGTCTTCAGAT-3′; CTGF Fw: 5′-GCCACAAGCTGTCCAGTCTAATCG-3′, Rv: 5′-TGCATTCTCCAGCCATCAAGAGAC-3′; p21 Fw: 5′-GGGACAGCAGAGGAAGAC-3′, p21 Rv: 5′-GCGTTTGGAGTGGTAGAAATC-3′.