Validated primers

mRNA was extracted using RNeasy® Plus Mini Kit (Qiagen, Valencia, CA) and cDNA prepared using the GoScript™ Reverse Transcription System (Promega, Madison, WI) with a 1:1 mixture of random and Oligo (dT)₁₅ primers according to manufacturer’s instructions. All RT-qPCR reactions were performed using the GoTaq® RT-qPCR Master Mix System (Promega). Validated primers were purchased from Qiagen Inc. (Valencia, CA): human Cyp1b1 – QT00209496, Cyp1a1 – QT00012341, Twist1 – QT00011956, Snai1 – QT00010010, Snai2 – QT00044128, VIM – QT00095795, Twist2 – QT02454004, FN1 – QT00038024, Notch1 – QT01005109, Notch2 – QT00072212, Aldh1a1 – QT00013286, Aldh1a3 – QT00077588, Pou5f1 – QT00210840, Sox – QT00237601, Nanog – QT01844808, Dppa3 – QT01667197, Msi1 – QT00025389, Human Bmi1 – QT00052654, Tgfb1 – QT00000728, Ahr – QT02422938, and Gapdh – QT01192646. RT-qPCR reactions were performed using a 7900HT Fast Real-Time PCR instrument (Applied Biosystems, Carlsbad, CA), with hot-start activation at 95 °C for 2 min, 40 cycles of denaturation (95 °C for 15 sec), and annealing/extension (55 °C for 60 sec). Relative gene expression was determined using the Pfaffl method [103 (link)] and the threshold value for Gapdh mRNA was used for normalization.

The purification of total RNA from fibroblasts was carried out by using Aurum Total RNAMini Kit (Bio-Rad, Hercules, California, USA) according to the manufacturer’s protocol. 0.5 microgram of total RNA was then reverse-transcribed to generate cDNA for PCR by using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, California, USA). Semi-quantitative determination of mRNA levels were performed by real-time qRT-PCR, using SYBR Green (Bio-Rad, Hercules, California, USA). Reactions were performed in duplicate for each sample. Multiple reactions were performed in a volume of 20 μL containing 10 μL of 2× PCR master mix, 1× of validated specific primers (Qiagen, Venlo, The Netherlands), and 2 μL of cDNA template. Amplifications were performed in the Stratagene MX3000P Real-Time PCR Detection System (Stratagene, San Diego, CA, USA), using the following cycle program: denaturation step at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 1 min, and extension at 72 °C for 30 s. The relative mRNA expression levels were calculated by using the comparative CT method (ΔΔCT). Quantitative normalization for each sample was performed by using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Validated primers (Qiagen, Venlo, The Netherlands) for qRT-PCR are provided in Table S1.

The ON-TARGETplus SMARTpool siRNAs targeting RICK, ANKHD1, LDOC1, CHMP5, DOCK7, PPP1R12C, PPP2R3B, SDCCAG3, or TRIM41 and a non-targeting control were purchased from Dharmacon (Lafayette, CO). HEK293T cells were transfected with siRNAs at a final concentration of 50 nM, using DharmaFECT1 (Dharmacon) in 6 wells plates. One day after, HEK293T cells were split at 10⁵ cells/well in 24 well plates. Cells were transfected the following day with a NFκB Luciferase reporter construct (Cignal, SABiosciences) and 0,2 ng of plasmid expressing mycNOD2 or empty vector, using Lipofectamine 2000 (Invitrogen). The following day, cells were stimulated or not with 10μg/ml MDP during 6h and dual reporter measurements were carried out as recommended by manufacturer’s instruction (Promega). Specificity and efficiency of each siRNA was verified by RTQPCR analysis using validated primers (SABiosciences) on RNA made 48h hours after siRNA transfection in parallel transfection experiments. 70 percent or more of target mRNA inhibition was considered as a successful siRNA experiment.

The procedure to perform gene expression analysis was conducted according to Moyano et al. [4 (link)] and results were analyzed according to Livak and Schmittgen [44 (link)]. Validated primers (SA Biosciences) for mRNAs encoding beta-actin (Actb, housekeeping gene; PPM02945B), Psd95 (PPH01848A), Spn (PPM34114A), Syp (PPM03241A), Wnt3a (PPM04720C), Nmdar1 (PPM04235A), β-catenin (PPM03384A), Cyclin D1 (PPM02903F), c-Myc (PPM02924F), Ptp1b (PPM05101F), Hdac2 (PPM04361F), and Vglut2 (PPM35412A) were used with the SA Biosciences PCR master mix (Real-Time SYBR Green, PA-012) to run qPCR in a CFX96. A qPCR in which cDNA was not included was used as a negative control.