Ly294002

The following inhibitors or reagents were used in this study: LY294002 (Enzo Life Sciences, USA), MK-2206 (Invitrogen, USA), C3 exoenzyme (Cytoskeleton, USA), and pertussis toxin (PTX; Invitrogen, USA). YAP and phospho-YAP (Ser127) were purchased from Cell Signaling (Cell Signaling technology, USA). CTGF, ANG-2, and GAPDH antibodies were from Santa Cruz Biotechnology (Santa Cruz, USA). Alexafluor secondary antibodies were from Life Technologies, USA.

Prostate cancer PC3 cells obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and docetaxel‐resistant PC3/Doc cells, as previously described 11, lung adenocarcinoma H460 and paclitaxel‐resistant H460/RT cells, oral epithelium carcinoma KB cells and the vincristine‐resistant KB/VCR cells, murine PCa RM‐1 cells (The Cell Bank of Chinese Academy of Sciences) and RM‐1/Doc cells (docetaxel‐resistant cell line derived from RM‐1) were cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) supplemented with 10% foetal bovine serum (GIBCO, Grand Island, NY, USA), 100 U/ml penicillin and 100 g/ml streptomycin.
Trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), the PI3K inhibitor LY294002, cycloheximide (CHX), actinomycin (Act D) and sodium tauroursodeoxycholate (TUDCA) were purchased from Sigma‐Aldrich (St‐Louis, MO, USA). The pan‐caspase inhibitor Z‐VAD‐fmk was obtained from Enzo Life Sciences (Plymouth Meeting, PA, USA).
In some experiments, the cells were exposed to Z‐VAD‐fmk, CHX, LY294002 or Act D for 2 hrs before TSA treatment. DMSO was used as the control vehicle.

MDA-MB 231 cells were plated into 24-well plates (10⁵ cells/well) in complete medium. Confluent monolayers were nutrient starved by growing them for 24 h in medium containing 0.5% FBS and then scratched using a 200 μl pipette tip. After washing, cells were treated or not with 10 ng/ml of refp17 or vp17s in complete medium (10% FBS). When reported, starved MDA-MB 231 cells were pretreated with 2.5 μg/ml of mAb to CXCR1 (mAb 330; R&D, Minneapolis, MN, USA) or to CXCR2 (mAb 331; R&D), or with an isotype-matched mAb (2.5 μg/ml; R&D) for 1 h at 37 °C before proteins stimulation. In some experiments, MDA-MB 231 cells were serum starved for 24 h in the presence or absence of inhibitors of PI3K/Akt (LY294002) (20 μM) (ENZO Life Sciences, Farmingdale, NY, USA), Jak/STAT (AG-490) (20 μM) (Sigma-Aldrich, St. Louis, MO, USA) or MEK/ERK1/2 (PD98059) (10 μM) (Calbiochem, Billerica, MA, USA) signaling pathways. Cell migration was evaluated at different time points using an inverted microscope (DM-IRB microscope system, Leica, Buffalo Grove, IL, USA). Cells were photographed using a CCD camera (Hitachi Inc., Krefeld, Germany). Wound closure was monitored over 12 h. In some experiments, in order to count the cells migrating into the wound area or protruding from the border of the wound, cells were fixed before wound closure and stained with Comassie brilliant blue.

All chemicals were purchased from Sigma-Aldrich (Schnelldorf, Germany), if not indicated otherwise. Rapamycin came from Cell Signaling Technology (Frankfurt, Germany), LY294002 and SB203580 from Enzo Life Science (Lörrach, Germany). Antibodies were obtained from the following sources: anti-Akt and anti-phospho-S6 from Cell Signaling Technology (Frankfurt, Germany), anti-HA from Covance (Munich, Germany), anti-nucleolin from Santa Cruz Biotechnology (Heidelberg, Germany), and IRDyes 680LT and 800CW secondary antibodies from Li-COR Biosciences GmbH (Bad Homburg, Germany).

For Western blotting analysis, antibodies against AKT (GTX121937), and GAPDH (GTX100118) were purchased from GeneTex (Hsinchu, Taiwan); anti-pAKT^S473 (CST4060), anti-GSK-3β (CST9315), and anti-pGSK-3β^S9 (CST9323) were purchased from Cell Signaling (Danvers, MA, USA). The PI3K inhibitor LY294002 and Akt inhibitor SH-6 were obtained from Enzo Life Sciences (Farmingdale, NY, USA). CA strains SC5314 and SC5314-GFP were gifts from Dr. Lo Hsiu-Jung (National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Taiwan). A plasmid containing cDNA encoding a GSK-3β mutant GSK-3β^S9A was a gift from Scott Friedman lab (Addgene plasmid # 49492; http://n2t.net/addgene:49492; RRID:Addgene_49492). The cDNAs was subcloned into pLentiviral vector to generate lentivirus carrying GSK-3β^S9A cDNA. Lentiviral pGIPZ carrying either shRNA vectors targeting human LDOC1 (shLDOC1) or GFP-tagged LDOC1 ORF were purchased from GE Dharmacon (Lafayette, CO, USA).

Gentisyl alcohol was purified from a culture of Arthrinium spp., KUC21332, as described previously [10 (link)]. Antibodies against phosphorylated ERK1/2 (Thr²⁰²/Tyr²⁰⁴, catalog number: 9101), JNK (Thr¹⁸³/Tyr¹⁸⁵, catalog number: 4668), P38 (Thr¹⁸⁰/Tyr¹⁸², catalog number: 4511), P90RSK (Thr⁵⁷³, catalog number: 9346), AKT (Ser⁴⁷³, catalog number: 4060), P70S6K (Thr⁴²¹/Ser⁴²⁴, catalog number: 9204), S6 (Ser^235/236, catalog number: 2211), and GSK3β (Ser⁹, catalog number: 9336). Antibodies against total ERK1/2 (catalog number: 4695), JNK (catalog number: 9252), P38 (catalog number: 9212), P90RSK (catalog number: 9335), AKT (catalog number: 9272), P70S6K (catalog number: 9202), S6 (catalog number: 2217), GSK3β (catalog number: 9315) were purchased from Cell Signaling Technology (Beverly, MA, USA). The inhibitors for ERK1/2 (U0126), PI3K/AKT (LY294002), and JNK (SP600125) were obtained from Enzo Life Sciences, Inc. (Farmingdale, NY, USA).

Lapatinib, OSI-906, jnj38877605, MK2206, and U0126 were purchased from Selleck Chemicals. LY294002 and triciribine were purchased from Enzo Life Sciences. GSK2334470 was purchased from MedChem Express. The following antibodies were purchased from Cell Signaling Technology: phospho-EGFR (#3777), phospho-InsR/IGF1R (#3024), phospho-Met (#3077), phospho-Akt (#4060), phospho-Erk1/2 (#9101), phospho-CRAF (#9427), phospho-IRS1 (#3070) and IRS1 (#2382). phospho-IRS1 (pY632) antibody (ab109543) and vinculin antibody (ab73412) were purchased from Abcam. GAPDH antibody was purchased from Shanghai Kangchen. α-tubulin antibody was purchased from Santa Cruz. And β-actin antibody was purchased from Abmart.