Figure 1.
Core TRN elements isolation workflow.
Key TFs were selected based on differential gene expression analysis and text mining. Left flow represents steps of the differential gene expression analysis. Gene expression profiling of primary human monocytes and that of human dermal fibroblasts were archived by using Human WG-6 v3.0 Expression beadschips (n = 3). Right flow represents the text mining. The text mining ranked the co-occurrence of “Monocyte” and “TF Name”. Finally, those two methods were integrated and isolated top 20 TFs as core monocyte TRN elements.
Table 1.
The monocyte core TRN elements.
Figure 2.
Regulatory relationship of monocyte's core TRN elements.
(A) Relative expressions to monocyte are represented in colors. Higher relative expression is depicted as intensifying green color. The value of each relative expression is the average of biological replicates (n = 3). (B) Illustration of hierarchical network of monocyte core TRN elements. Each node (circle) indicates monocyte core TRN elements and green nodes represent the identified TRN inducers. When the TRN inducers or NR4A2 upregulate the gene expression to more than 5% of that of monocyte, an edge was drawn. An edge from an upper node to lower node indicates positive regulation.
Figure 3.
Cell feature assessments in reconstructed fibroblasts.
(A) Morphological changes were visualized by microscopy. The cells were stained with Pallodin-Rhodamin (Yellow), Hoechst33342 (Blue), and Whole Cell Stains (Red). (B–D) Phagocytic latex beads were visualized (B) and the mean intensity of the ingested beads were confirmed by flow cytometry (C–D). (B) Beads-ingested cells are indicated by white arrows. Red color represents DID cell membrane staining, blue color represents nuclei, and green color represents the latex beads. (C) Beads ingested cells was quantified based on the beads fluorescence by flow cytometry in FIB-mock, FIB-SPI1 and DIB-4Fs 2 houes after beads addition.. The cells were cultured with 0.002 v/v%. Vertical and horizontal axes represent cell count and fluorescent intensity, respectively. Beads ingested cells were gated. The analysis was performed in triplicates, showing the similar results. (D) The flow cytometric analysis of the phagocytosis was summarized. Black, blue, and red lines represent FIB-mock, FIB-SPI1, and FIB-4Fs, respectively. The mean fluorescent intensity of ingested beads was measured by flow cytometry. The vertical axis and horizontal axis represent mean beads fluorescent intensity and incubation time, respectively. Error bars represent standard deviation (s.d.) (E) The expression change of TNF, IL6, IL1A, IL1B, IL8, CCL2, CXCL10 and IFNB1 induced by LPS treatment were measured by qRT-PCR in FIB-mock, FIB-SPI1, and FIB-4Fs. The cells were treated with LPS for 24 hours at the final concentration of 10 µg/µl. The bar represents the relative expression of LPS-treated cells as compared to untreated cells and error bar represents s.d. These experiments were repeated three times. * represents P-value≤0.05, ** represents P-value≤0.01 (t-test). The scale bar is 50 µm.
Figure 4.
Four TRN-inducers adopted inflammatory cytokine secretion in response to LPS.
FIB-mock and FIB-4Fs were treated or untreated with 10 µg/ml LPS. The cytokine levels of supernatant medium were assessed using the Proteome Profiler Human Cytokine Array, Pannel A. Array images were collected by LAS-3000 imaging system (A). The intensity of each spot was determined by Multi Gauge softwere (B).
Figure 5.
Four TRN-inducers adopted chemotaxis activity towards CCL2.
Chemotaxis activity was measured by performing a transwell assay. FIB-mock and FIB-4Fs were cultured in transwells and incubated in the lower-chamber containing 5 µM of CCL2. The cells that migrated to the bottom of transwell were stained with Calcein-AM. Relative signal intensities were calculated by comparing fluorescent intensities of CCL2 treated to untreated cells (n = 3). ** represents P-value≤0.01 (t-test).
Figure 6.
Four TRN-inducersfactors activated wider portion of monocyte TRN than SPI1 alone.
(A) Flow cytometric analysis shows expression of CD14 and CD45 and no expression of CD11b in both FIB-SPI1 and FIB-4Fs. All experiments were done in duplicates. (B–C) Bar plots represent the change of up-regulated or down-regulated genes in monocyte specific genes (B) and fibroblast specific genes (C). Total length of the bar represents total number of monocyte specific genes or fibroblast specific genes (100%). The black part is 22-fold grater and P–value<0.05 (t-test) and gray part represents 22-fold less and P-value<0.05 (t-test), respectively, comparing to the FIB-mock. (D) All genes were categorized into either Up-regulated (Up), Down-regulated (Down) or No Change (NC) by applying the same cut-off (B–C). Medians for each dataset are indicated by black centerlines, upper quartiles are indicated by upper edges of the box, and lower quartiles are indicated by lower edges of the box. Maximum and minimum values are marked as end of lines extending from the boxes. ** represents P-value≤0.01 (Wilcoxson rank sum test). (E) Histogram shows distribution of the fold-change of monocyte specific and up-regulated by SPI1 (2-fold or greater and P-value<0.05 (t-test)). Horizontal axis represents the log value of the fold-change as compare to FIB-mock, and vertical axis represents frequency. Upper histogram is for FIB-SPI1, and lower histogram is for FIB-4Fs.
Table 2.
Expression of monocyte markers.
Table 3.
Enriched Gene Ontology.
Figure 7.
Motif activity revealed reconstructed monocyte TRN.
(A) The activity of 10 monocyte-associated motifs were shown as a line chart in FIB-mock, FIB-SPI1, and FIB-4Fs. Vertical axis represents the motif activity. (B) SPI1 motif activity was shown as bar plot. Each bar represent an average of biological replicates and error bar represent s.d. (n = 3).