Confluent fibroblast monolayers were untreated (“media alone”) or stimulated with TGF-β1 (and media) (1 ng/ml; porcine origin; R&D Systems, USA) for 24 hours with or without AZD8055 (1 μM; synthesized and provided by GSK) or rapamycin (100 nM; Merck Chemicals) in standard DMEM (n = 4 biological replicates per condition). Total RNA was extracted with miRCURY RNA Isolation Kit (Exiqon). Polyadenylate-tailed RNA enrichment and library preparation were performed using the KAPA Stranded mRNA-Seq Kit with KAPA mRNA Capture Beads (KAPA Biosystems, Wilmington, MA, USA). Paired-end RNA-seq was performed with the NextSeq sequencing platform (Illumina, San Diego, CA, USA). Read preprocessing and alignment to the genome (Homo sapiens UCSC hg19) were performed using STAR aligner. Counts were normalized using the R package DESeq2, then log2-transformed with an offset of 1, and filtered to remove genes with an average count <1 across all samples. This left 15,959 genes that were then interrogated using the WGCNA package in R. Briefly, gene coexpression similarity was estimated by generating an absolute Pearson product moment correlation matrix. Using a soft threshold of 20, an adjacency matrix was calculated and used to estimate the topological overlap and assign genes to modules. Highly correlated modules were merged using a cut height of 0.06 on the topological matrix dendrogram. Sixty-five modules were returned and assigned colors as names. MetaCore (Thomson Reuters) was used to run pathway enrichment analyses and interaction network analyses on each of the 65 modules. OCN analysis was performed using the MetaBase R packages licensed from Clarivate Analytics and identifies one-step away direct regulators of the dataset that are statistically overconnected with the objects from the dataset. The P value of overconnectivity was calculated using hypergeometric distribution.

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