Analysis of Signaling Pathways using Antibody Microarrays and RNA Interference

The study of complex cellular signaling pathways requires powerful and specific tools for the manipulation and quantification of protein levels and activity. We have developed a new set of tools to streamline the experimental approach for pathway analysis. We present application data showing the integration of these tools for the analysis of cellular signaling pathways. The human lung carcinoma A549 cell line was used to study the signaling pathways involved in cytokine release in response to TNFa stimulation. Cell supernatants from TNFa-stimulated A549 cells were analyzed on four different ExcelArrayTM antibody microarrays (Inflammation I, Inflammation II, Angiogenesis, and Chemotaxis), enabling the quantitation of 41 unique analytes. TNFa stimulation resulted in increased expression of multiple cytokines, including IL-8. The cytokine expression profile was then analyzed following inhibition of several key signaling pathways using RNA interference. Knockdown of NFkB protein in A549 cells using a new NFkB SuperSignal® siRNA/Western Blot kit resulted in significant inhibition of cytokine production. In a different application, ExcelArrayTM antibody microarrays were used to study whether genetic differences in Toll-like receptors (TLRs) account for differences in the inflammatory response in T.gondii infection. Antibody microarray data showed unique cytokine responses for TLR-4 and TLR-9 agonists in human monocytes. These results highlight the utility of multiplexed antibody microarrays in cell signaling studies. Introduction Antibody microarrays enable the simultaneous quantitation of multiple analytes (1-7). We have constructed five human 12-plex cytokine/chemokine sandwich microarrays featuring many improvements over currently available alternatives (Table 1). These microarrays are printed on PATHplusTM microarray slides. The PATHplusTM surface allows for increased dynamic range, high signal-to-noise and excellent reproducibility compared with conventional derivatized glass and nitrocellulose pad slides. Thoroughly characterized antibody pairs are used as capture and detection reagents. Streptavidin-conjugated DyLightTM 649 is utilized as the detector molecule, providing superior photostability and fluorescence intensity relative to other fluorescent dyes. The general microarray assay layout is illustrated in Figure 1. We present work demonstrating the use of antibody microarrays in studying signal transduction pathways, including the TNFa pathway and Toll-like Receptor (TLR) signaling. Materials and Methods Cell Culture A549 human non-small cell lung carcinoma cells from ATCC (CCL-185) and human monocytes were cultured in ATCC media according to product recommendations. Cytokine release was stimulated with TNFa (25 ng/ml), PHA (5 mg/mL), ConA (5 mg/mL), or PMA(5 mg/mL). Appropriate vehicle controls were performed for each cell treatment. Cell culture supernatants were collected at indicated times and either assayed immediately or stored at -20°C. siRNA Transfection and Western Blot Analysis siRNA transfections were performed according to recommendations provided by Thermo Scientific with minor modifications. The siRNA and antibodies used were from Thermo Scientific SuperSignal siRNA/ Western kits (www.piercenet.com). The day before transfection, A549 cells were plated at a density of 7x104 per well in a 12 well plate. The cells were transfected with 100 nM either Non-Targeting Pool (negative control), NF-kB siRNA, c-jun siRNA, AKT-1 siRNA or a combination of 50 nM each of c-jun and NF-kB siRNA. Forty-eight hours post-transfection, the media was replaced with fresh media (nonstimulated) or media containing 25 ng/ml TNFa (stimulated) and incubated for an additional 24 hrs. The cell culture supernatants were collected and assayed for cytokine/chemokine release. After harvesting the supernatants, the A549 cells were washed once with cold PBS and lysed directly in wells using 1X SDS PAGE sample buffer. Equivalent volumes of cell lysates were separated on a 4-12% gradient polyacrylamide gel and transferred to nitrocellulose membrane. The membranes were processed using Thermo Scientific SuperSignal® siRNA Chemiluminescent Detection Module (cat. # 82200). Antibody Microarray Analysis Cell culture supernatants were assayed for cytokine/chemokine concentration using Thermo Scientific ExcelArrayTM Inflammation I (#81002), Inflammation II (#81003), Angiogenesis (#81005), Chemotaxis (#81006), or MMP/TIMP (#82012) microarrays. Assays were performed according to product instructions. Briefly, 100 uL standard (1000-12.3 pg/ml) or sample was applied to each well and incubated at room temperature for 2 hours. After sample incubation, the microarrays were washed with wash buffer. Pre-titered biotinylated detector antibody was applied to the microarray and incubated at room temperature for 1 hour. Again, the microarray was washed and streptavidin-linked DyLightTM 649 was applied to the microarray and incubated for 45 minutes. The microarray was washed and dipped in a final rinse solution. Microarrays were imaged using an Alpha Innotech AlphascanTM microarray imager. Spot densitometry was performed using ArrayvisionTM software. Conclusions ExcelArrayTM antibody arrays were used to demonstrate TNF • a stimulation of 12 cytokines and chemokines in A549 cells. NF • kB, c-Jun and Akt gene silencing using SuperSignal® siRNA/Antibody kits results in a modified cytokine response to TNFa stimulation. Knock-down of TIMP-1 and TIMP-2 proteins was demonstrated on a MMP/TIMP • ExcelArrayTM antibody array. Antibody microarray analysis of Toll-like receptor (TLR) signaling demonstrated unique • cytokine profiles for TLR-4 and TLR-9 agonists. References Schematic of TNFa Signaling Pathway Figure 4. Western Blot analysis of siRNA Treated A549 lysates using SuperSignal® siRNA/Antibody Kits Figure 5: Total cell lysate was loaded on polyacrylamide gels, separated by SDS PAGE and transferred to blotting membranes for subsequent immunoblotting. NFkB, c-Jun and Akt antibodies were used to probe the membranes for expression levels of these specific targets. Lane numbers correspond to cell lysates obtained from the following cell treatments: (1) mock transfection, (2) ON-TARGETplus siCONTROL Non-Targeting Pool (OTPNTP), (3) ONTARGETplus NF-kB siRNA, (4) ON-TARGETplus c-Jun siRNA, (5) ON-TARGETplus NF-kB + c-Jun siRNA and (6) ON-TARGETplus Akt siRNA. NFB