*2.2. Subset of Endodontic Tissue Molecules*

Morio et al. reported 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 [6]. We used these CCBMs as (i) wet lab experimental data to validate claims from the bioinformatics data, and (ii) to assess the ability of UV irradiation to activate or inhibit cellular pathways related to immune functions. For this, levels of CCBM expression were calculated as the expression log2 ratios of the mean of each treatment after UV irradiation, divided by the mean of the respective control for the same treatment (Figure 2).


**Figure 2.** Expression Log2 ratios of 13 chemokine, cytokine, and biomarker (CCBMs) concentrations reported in tissue culture media of HEPM cells and gingival fibroblasts at 0, 24, and 48 h after treatment with 255 nm, 405 nm, or 255/405 nm UV irradiation. Expression was calculated as the log2 ratio of the mean of each treatment after UV irradiation over the mean of the untreated control for that same cell type, UV irradiation wavelength, and time period. Groups are shown as a heatmap, where blue represents inhibition (negative values), white represents midpoint, and orange represents activation (positive values).

#### *2.3. Analysis*

We used ingenuity pathway analysis (IPA, Qiagen, Germantown, MD, USA) to assess whether the molecules in this study were related to the activation of innate and immune mechanisms. Two types of analysis were performed.

IPA core analysis was run on the list of 32 molecules from the literature dataset in Table 1 and used to assess whether the IPA canonical pathway and IPA diseases and function annotations were predicted to be associated with relevant diseases, immune pathways, and immune functions. Statistical significance was calculated using Fisher's exact test and significant *p* values (*p* < 0.05) were reported.

IPA comparison analysis was run on the expression log2 ratios of the 13 molecules and used to assess whether the IPA canonical pathway and IPA diseases and function annotations were predicted to be activated or inhibited after UV irradiation. Statistical differences were determined using activation z-scores calculated from the mean of each treatment expression log2 ratio. The activation z-score was determined by IPA as reported by Kramer et al. [11] and makes predictions based on the direction of gene activation or inhibition.

IPA comparison analysis was also used to assess whether the IPA canonical pathway predicted any effects of the activated or inhibited 13 molecules on downstream regulation of gene expression for other innate or adaptive immune functions.
