*3.2. Subset of 13 Endodontic Tissue Molecules*

IPA analysis also predicted that different wavelengths of UV irradiation might selectively regulate gene expression for innate or adaptive immune functions. IPA comparison analysis of the log2 ratios of the concentrations of 13 CCBMs in tissue culture media of HEPM cells and gingival fibroblasts at 0, 24, and 48 h after treatment with 255, 405, or 255/405 UV irradiation identified differences in expression across 18 observations, representing 2 cell lines, 3 UV treatments, and 3 time periods (Figure 3). The expression log2 ratios of the 13 CCBMs varied from −2.6245 to 1.6114. Relevant IPA canonical pathways in Figure 3 were related to cellular stress and injury (wound healing signaling pathway, the CLEAR signaling pathway, HIF1α signaling pathway, and autophagy), cytokine signaling (IL17 signaling, IL6 signaling, and NF-κB signaling), growth factor signaling (regulation of the epithelial mesenchymal transition by growth factors pathway), and organismal growth and development (ID1 signaling pathway). IPA comparison analysis predicted that UV irradiation activated or inhibited pathways depending upon the cell type, wavelength of treatment, and time after treatment. Numerous IPA canonical pathway annotations were inhibited shortly after irradiation (0 h) but activated at 24 and 48 h. Fibroblasts and HEPM cells both were strongly activated by 405 nm and 255/405 nm UV irradiation treatments (Figure 3).

Within the wound healing signaling pathway, for example, after 0 h of 255, 405, and 255/405 nm irradiation, fibroblasts and HEPM cells (observations 1–3, 10–12) had negative z-scores, predicting inhibition of pathway signaling, whereas after 24 and 48 h after 255, 405, and 255/405 nm irradiation, fibroblasts (observations 4–9) and after 48 h of 255, 405, and 255/405 nm irradiation, HEPM cells (observations 16–18) had positive z-scores, predicting activation of pathway signaling. There were also conditions predicted to activate some pathways yet inhibit others (observations 3, 9, 12, 15, and 18). Here, treatment with cells with 255/405 nm irradiation contained both inhibited and activated pathway signaling. After 48 h of 255/405 nm irradiation, HEPM cells (observation 18) had a positive z-score but contained both inhibited and activated pathway signaling (Figure 4A–C). Signaling was predicted to occur through binding of molecules to the TNF receptor, EGFR, and TGFBR and signaling through JNK and ERK1/2 to transcription factors NF-κB, CEBPB, and AP-1. This was predicted to activate additional CCBMs, leading to proinflammatory responses, disruption of desmosomes, chemoattraction of leukocytes, migration and proliferation of fibroblasts and cells, collagen matrix remodeling, and wound healing pathways.

**Figure 3.** IPA comparison analysis of 18 observations from fibroblasts (observations 1–9) and HEPM cells (observations 10–18) at 0 h (observations 1–3, 10–12), 24 h (observations 4–6, 13–15), and 48 h (observations 7–9, 16–18) after treatment with 255 nm (observations 1, 4, 7, 10, 13, and 16), 405 nm (observations 2, 5, 8, 11, 14, 17), or 255/405 nm (observations 3, 6, 9, 12, 15, and 18) irradiation. Groups are shown as a heatmap, where blue represents inhibition (negative values), white represents midpoint, and orange represents activation (positive values). Numerous IPA canonical pathways were inhibited shortly after irradiation (0 h) but activated at 24 and 48 h. Fibroblasts and HEPM cells both were strongly activated by 405 nm and 255/405 nm UV irradiation treatments.

**Figure 4.** *Cont*.

**Figure 4.** Schematic diagrams of the wound healing signaling pathway, prepared using Ingenuity Pathway Analysis software (IPA, Qiagen, Germantown, MD), showing both inhibited and activated pathway signaling in HEPM cells 48 h after (**A**) 255 nm irradiation, (**B**) 405 nm irradiation, and (**C**) 255/405 nm irradiation. Signaling starts via TNF binding to the TNF receptor; EGF and TGFA binding to the EGFR; and TGFB binding to the TGFBR. These pathways signal through TRADD and TRAF2 to JNK and through RAS, RAF, and MEK to ERK1/2. Signaling continues to NF-κB, CEBPB, and AP-1 to activate additional CCBMs, leading to proinflammatory responses, disruption of desmosomes, chemoattraction of leukocytes, migration and proliferation of fibroblasts and cells, collagen matrix remodeling, and wound healing pathways. Pathway molecules in red indicate activation and molecules in green indicated inhibition. Signaling connections in orange indicate pathway activation and signaling connections in blue indicate pathway inhibition.
