C jun

Nesfatin-1 (GTX00739, polyclonal), IL-1β (GTX74034, polyclonal), β-actin (GTX109639, polyclonal), p85 (GTX111068, polyclonal), Akt (GTX121937, polyclonal), c-Jun (GTX112974, polyclonal) and p65 (GTX102090, polyclonal) antibodies were purchased from GeneTex (Hsinchu, Taiwan). We bought ON-TARGETplus siRNAs from Dharmacon (Lafayette, CO, USA). qPCR primers and probes, as well as PCR Master Mix, were bought from Applied Biosystems (Foster City, CA, USA). The phosphorylated forms of p85 (17366, polyclonal), Akt (4060, polyclonal), c-Jun (9165, polyclonal) and p65 (3033, polyclonal) antibodies were bought from Cell Signaling (Danvers, MA, USA). AP-1 and NF-kB luciferase plasmids were purchased from Stratagene (La Jolla, CA, USA). TRIzol, a reverse transcription kit and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). Recombinant human nesfatin-1 and the IL-1β ELISA kit (900-K95) were purchased from PeproTech (Rehovot, Israel). Other chemicals not already mentioned were bought from Sigma-Aldrich (St. Louis, MO, USA). Pharmacological inhibitors for PI3K (1 μM LY294002 catalog number: 440202), Akt (1 μM Akti, catalog number: A6730), c-Jun (10 μM, Tanshinone IIA catalog number: T4952), p65 (3 μM TPCK catalog number: T4376; 1 μM PDTC, catalog number: P8765) were supplied by Sigma-Aldrich (St. Louis, MO, USA).

Human siRNAs of c-Src (SASI_Hs01_00112905), PDGFR (SASI_Hs02_00341109), p42 (SASI_Hs01_00124656), JNK1 (SASI_Hs01_00010440), p38 (SASI_Hs01_00018464), c-Jun (SASI_Hs02_00333461), ATF2 (SASI_Hs01_00147372), and scrambled siRNAs were sourced from Sigma (St. Louis, MO, USA). Transient transfection of siRNAs (100 nM) was performed using a Lipofectamine^TM RNAiMAX reagent according to the manufacturer’s instructions.

Human siRNAs of scrambled, S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, and c-Fos were from Sigma (St. Louis, MO). Transient transfection of siRNAs was carried out using Metafectene transfection reagent (Biontex, Germany). siRNA (100 nM) was formulated with Metafectene transfection reagent according to the manufacturer’s instruction.

Human scrambled, MMP-2, MMP-9, p65, p50, c-Jun, c-Fos, p85, p110, Akt, mTOR, and IL-6 siRNAs were from Sigma (St. Louis, MO). Transient transfection of siRNAs was performed using a Lipofectamine 2000 Transfection Reagent according to the manufacturer's instructions.

The mechanism of action was assessed using antibodies against ERK2 from Santa Cruz (Dallas TX), AKT from Millipore (Billerica, MA), c-JUN and BAD from Cell Signaling (Beverly, MA), phosphorylated c-JUN (T91/93) from Abcam (Cambridge, MA), phosphorylated c-JUN (S63/73), the phosphorylated forms of ERK, S6 ribosomal protein (91B2), and BAD, from Cell Signaling, phosphorylated AKT S473 from Millipore, BH3-interacting domain death agonist (Bid) from R&D Systems (Minneapolis, MN), inactive and active forms of caspase 3 from Santa Cruz and R&D Systems, caspase 8 and caspase 10 from MBL (Woburn, MA), and caspase 9 from Cell Signaling, and α-tubulin and β-actin from Sigma (St. Louis, MO). All secondary antibodies were affinity purified with no cross-reactivity with other species. Peroxidase-conjugated secondary antibodies were obtained from Pierce (Rockford, IL), Promega (Madison, WI), and Jackson ImmunoResarch (West Grove, PA). Alexa Fluor 488 and 594-conjugated secondary antibodies (Molecular Probes, Eugene, OR) were used as specified by the manufacturer. Tamoxifen used to obtain a positive control signal for the phosphorylation analysis of c-JUN was from Sigma.

To determine the signaling pathways elicited by PM10, NPDFs were pretreated with the following signaling pathway inhibitors: SB203580 (p38 inhibitor, 10 μmol/L; Sigma-Aldrich), C-Jun (curcumin, 10 μmol/L; Sigma-Aldrich) or BAY117082 (NF-kb inhibitor, 2.5 μmol/L; Sigma-Aldrich). After a 1 h treatment of the inhibitors, the cells were incubated with PM10 (200 μg/mL) for 6 h and then lysed in LIPA buffer. Lysates were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with blocking solution and incubated with antibodies to NF-kb (Cell signaling Technology, Danvers, MA, USA), p38 (Cell signaling Technology), C-Jun (Cell signaling Technology), phospho-NF-kb p65 (Cell signaling Technology), phospho-p38 (Cell signaling Technology), phospho-C-Jun (Cell signaling Technology), and β- actin. Blots were visualized using horseradish peroxide-conjugated secondary antibodies and an Enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL, USA).

ChIP assays were performed as previously described (Gazin et al., 2007 (link)) using the following antibodies: ZNF304 (described above), KAP1 (Bethyl Laboratories), SETDB1 (Millipore, Billerica, MA), DNMT1, DNMT3A, and DNMT3B (all from Imgenex, San Deigo, CA), cJUN (Millipore), H3K27me3 (Cell Signaling Technology), H3K9me3 (Millipore), H3K4me3 (Abcam), EZH2 (Millipore) and BMI1 (Abcam). The CDX1 antibody (Chan et al., 2009 (link)) was kindly provided by Walter Bodmer (University of Oxford, UK). ChIP products were analyzed by qRT-PCR (see Supplementary file 1 for primers). Samples were quantified as percentage of input, and then normalized to an irrelevant region in the genome (∼3.2 kb upstream from the transcription start site of GCLC). Fold enrichment was calculated by setting the IgG control IP sample to a value of 1.