Figure 4.
mRNASeq of single cells with known signaling dynamics reveals dynamics-specific target genes. (A) DGEA comparisons and their meaning. The comparison identifying genes truly controlled by NfκB dynamics is circled in cyan (n = 231 cells, N = 3 biological replicates, 2 independent sequencing runs). (B) Top: DGEA between TNFα unstimulated vs stimulated (black) and between OSC vs TRA cells (both TNFα-stimulated) (magenta). Genes (n = 281) that are both upregulated by TNFα and differentially expressed between OSC and TRA dynamics (green) are the most promising candidates to be modulated by NfκB dynamics. Bottom: mean expression of all detected genes in OSC vs TRA cells. Most TNFα-responsive and dynamics-correlated genes are higher expressed in OSC cells, which activate a superset of TRA target genes. Bar graphs represent numbers above (left) and below (right) of the parity line. (C) Correlation of extracted features (supplemental Table 1) and the 281 gene candidates from (B). OSC-associated feature scores correlate with many of these genes. This confirms the visual scoring in (B) and suggests many NfκB target genes are more responsive to OSC than to TRA dynamics. (D) Dynamics-dependent expression of selected NfκB pathway genes. (E) Top: TRA vs OSC cells can be identified by CD14 and CD115 mRNA expression detected by scRNAseq (top right quadrant: 66% OSC; other quadrants: 77% TRA). Bottom: CD14 vs 115 gating of unstimulated cells to identify GMPM (67% OSC) vs GMPGM (76% TRA). (F) Gene expression changes between TRA and OSC cells (x-axis) and between unstimulated GMPM and GMPGM (y-axis) allow the identification of dynamics-specific target genes (cyan, significantly different between OSC and TRA but not between GMPM and GMPGM). Enrichment in the top left and bottom right quadrant confirms that GMPM/GMPGM are predominantly OSC/TRA, respectively. (G) GSEA of OSC and TRA cells, corrected for initial cell type differences (for details, see text). (H) Selected target genes that are activated by either TRA or OSC dynamics only (see also supplemental Figure 4C and supplemental Data 5).

mRNASeq of single cells with known signaling dynamics reveals dynamics-specific target genes. (A) DGEA comparisons and their meaning. The comparison identifying genes truly controlled by NfκB dynamics is circled in cyan (n = 231 cells, N = 3 biological replicates, 2 independent sequencing runs). (B) Top: DGEA between TNFα unstimulated vs stimulated (black) and between OSC vs TRA cells (both TNFα-stimulated) (magenta). Genes (n = 281) that are both upregulated by TNFα and differentially expressed between OSC and TRA dynamics (green) are the most promising candidates to be modulated by NfκB dynamics. Bottom: mean expression of all detected genes in OSC vs TRA cells. Most TNFα-responsive and dynamics-correlated genes are higher expressed in OSC cells, which activate a superset of TRA target genes. Bar graphs represent numbers above (left) and below (right) of the parity line. (C) Correlation of extracted features (supplemental Table 1) and the 281 gene candidates from (B). OSC-associated feature scores correlate with many of these genes. This confirms the visual scoring in (B) and suggests many NfκB target genes are more responsive to OSC than to TRA dynamics. (D) Dynamics-dependent expression of selected NfκB pathway genes. (E) Top: TRA vs OSC cells can be identified by CD14 and CD115 mRNA expression detected by scRNAseq (top right quadrant: 66% OSC; other quadrants: 77% TRA). Bottom: CD14 vs 115 gating of unstimulated cells to identify GMPM (67% OSC) vs GMPGM (76% TRA). (F) Gene expression changes between TRA and OSC cells (x-axis) and between unstimulated GMPM and GMPGM (y-axis) allow the identification of dynamics-specific target genes (cyan, significantly different between OSC and TRA but not between GMPM and GMPGM). Enrichment in the top left and bottom right quadrant confirms that GMPM/GMPGM are predominantly OSC/TRA, respectively. (G) GSEA of OSC and TRA cells, corrected for initial cell type differences (for details, see text). (H) Selected target genes that are activated by either TRA or OSC dynamics only (see also supplemental Figure 4C and supplemental Data 5).

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