Genespring 14

The data obtained after extraction were further analyzed using GeneSpring 14.9.1 (Agilent, United States). The analysis aimed to determine differentially expressed genes (DEGs) in the PBMC of the patients treated with agomelatine and primary HN culture treated with MEL.
To identify up- and downregulated gene lists, we used restricted cutoff with fold change >2 and a significant p-value of <0.05 according to previous indications (Dalman et al., 2012 (link)). Significant differences in gene expression were determined via moderated Student's t-test. The differences were considered statistically significant at p < 0.05. According to the Benjamini–Hochberg procedure, the obtained p-values were corrected using the false discovery rate (FDR) as multiple testing correction methods (Cheng and Pounds, 2007 (link)).

Gene expression microarray analysis was performed in PSP-PMCs (n = 3) and BHDS-PMCs (n = 3) using a SurePrint G3 Human GE v3 8 × 60 K Microarray (Agilent Technologies, Inc., Santa Clara, CA, USA). The array contained 26,083 Enterz gene RNA probes (excluding non-coding RNA). The 6 samples were not well-clustered in each group using the total 26,083 genes and 1 PSP-PMCs sample was clustered into BHDS-PMCs group using 1799 genes when the level of differentially expressed gene was set at a fold change > 1.5 (Supplementary Fig. S1 online). We realized that only this PSP patient remained to be in pneumothorax under the chest tube drainage for about 2 weeks before surgery, i.e., the time when we collected pleural lavage fluid for the isolation of MCs. Therefore, we excluded this sample and used the results from 2 PSP-PMCs and 3 BHDS-PMCs samples. 3378 genes were identified as differential expression genes with a fold change > 1.5; 1637 and 1741 genes were up- and down-regulated, respectively, in BHDS-PMCs compared with PSP-PMCs. We evaluated Gene Ontology (GO) terms significantly affected (p < 0.01 and false discovery rate (FDR), Q < 0.001) among these genes. The assessment of GO terms and the generation of heatmaps were conducted using GeneSpring14.9.1 (Agilent Technologies, Santa Clara, CA, USA).

Microarray analysis was performed in samples obtained from Ad5-porIFNα- and PBS-treated animals at 1 day post treatment, prior to challenge with EBOV. Total RNA was extracted from PBMC and BALP, and the concentrations were determined using a NanoDrop (NanoDrop Technologies) and shipped to MOGENE LC (St. Louis, MO, USA) for the microarray assay. Microarray analysis was performed using an Agilent porcine 4x44K microarray (Agilent Technologies) and following the manufacturer’s protocols. Microarray slides were scanned on an Agilent high resolution C scanner and images processed using the Agilent Feature Extraction software. The quality of samples was analyzed using the ArrayQualityMetrics package and the data imported into GeneSpring 14.8 (Agilent) for further analysis. The data was normalized using a shift to the 75th percentile; thereafter, the value was further transformed as log2. All gene IDs from genes that were up- or down-regulated > 2-fold were then imported into the DAVID bioinformatics suite (https://david.ncifcrf.gov/, accessed on 26 July 2019) for detecting overrepresentation of signaling pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) [33 (link)] and gene ontologies (GO) [34 (link)].
FDR-values and p-values were calculated by the DAVID bioinformatics suite using an adapted method for multiple testing with independent statistics [35 (link)].