cell type: Th17 treatment: scramble time point: 72h
Treatment protocol
Isolated CD4+ T cells were activated with plate-bound anti-CD3 (750 ng/24well culture plate and 3.75ug/ 6 well culture plate, Immunotech, Marseille, France) and soluble anti-CD28 (1 μg/ml, Immunotech) in X-vivo 20 serum free medium (Lonza, Basel, Switzerland) supplemented with 2 mM L-Glutamine (#G7513, Sigma, St. Louis, MO, USA), and 50 U/ml Penicillin and cultured at 37°C in 5 % CO2. IL-6 (20 ng/ml, Roche, Basel, Switzerland), IL-1β (10 ng/ml) and TGFβ (10 ng/ml) in the presence of neutralizing anti-IFNγ (1 μg/ml) and anti-IL4 (1 μg/ml) were used to initiate Th17 polarization. Control Th0 cells were cultured in a medium containing only the neutralizing antibodies. All cytokines and neutralizing antibodies were purchased from R&D Systems (Minneapolis, MN, USA) unless otherwise stated. For STAT3 target gene identification, cells were harvested at 2, 12, 24, and 72 h time points. Three biological replicates were prepared, each time including all three different individual STAT3 siRNAs and non-targeting control siRNA.
Growth protocol
Umbilical cord blood was collected from healthy neonates born in Turku University Hospital, Hospital District of Southwest Finland. Mononuclear cells were isolated with Ficoll-Paque (Amersham Biosciences) gradient centrifugation, and CD4+ cell population was purified using magnetic beads (Dynal CD4 Positive Isolation Kit, Invitrogen) according to manufacturers instructions.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using RNeasy Mini Kit from QIAGEN according to the manufacturer's instructions.
Label
Biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol (Expression Analysis Technical Manual, Affymetrix).
Hybridization protocol
Following fragmentation, cRNA were hybridizedAffymetrix GeneChip Human Genome U219 arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station.
Scan protocol
GeneChips were scanned using the Gene TitanTM MC Instrument.
Data processing
Possible batch effects caused by the different hybridizations were corrected for with ComBat tool with default parameters. The data were processed using the robust multi-array average (RMA) algorithm, quantile-normalized (Bioconductor affy package) and log2-transformed. Th17-specific genes were identified between the matched Th17- and Th0-measurements (same time point and culture) in the control-siRNA data using paired and moderated t-statistic (Bioconductor limma package). Genes with false discovery rate (FDR) < 0.1 were defined as changed for each time point. The effect of STAT3 knockdown on the gene expression was assessed using the Th17 measurements from the matched siRNA and scramble samples using the same analysis methods as above.
