*2.1. Gene Expression of TRPA1 in Human IVD Tissue*

Gene expression of TRPA1 was tested in human non-degenerated (*n* = 4) and degenerated (*n* = 20) IVD tissue in relation to disc region, disease type, pain score (for degenerated discs only), grade, and age. In the degenerated tissue, TRPA1 was found expressed in 20% of tested donors (four out of 20). Although only expressed in a subset of degenerated donors, TRPA1 was found in both annulus fibrosus (AF) and nucleus pulposus (NP), in an age range of 39–76 years, at pain scores 2 (= intense) and 3 (= disabling) and at Pfirrmann grades 2 and 3 (Table 1). TRPA1 was only expressed in one non-degenerated NP sample (in one out of four donors: male, 17 years old, grade 1, <sup>2</sup>−d*C*<sup>t</sup> = 4.57 × <sup>10</sup>−5). Interestingly, TRPA1 was found expressed in cells isolated from human fetal IVD tissue (*<sup>n</sup>* = 4; 2−d*C*<sup>t</sup> = 0.0463 ± 0.04742). It is known that TRPA1 can associate with TRPV1, thereby regulating its intrinsic properties independently of intracellular calcium. Table 1 summarizes the expression of TRPV1. A manuscript that provides details on the expression of TRPV1 in IVD tissue is currently in revision [26].

**Table 1.** Gene expression of TRPA1 and TRPV1 in human intervertebral disc (IVD) tissue. Some donor tissues were divided into nucleus pulposus (NP) and annulus fibrosus (AF), resulting in more AF and NP samples (*n* = 21 in region, pain score, grade) than a total number of donors (*n* = 20).


### *2.2. Gene Expression of TRPA1 and TRPV1 in Human IVD Cells Treated with Pro-Inflammatory Cytokines*

TRPA1 and TRPV1 can be involved in modulating inflammation in both neuronal and non-neuronal cells [25]. Therefore, changes in the gene expression of TRPA1 and TRPV1 were tested in IVD cells stimulated with pro-inflammatory cytokines IL-1β and TNF-α (both 5 and 10 ng/mL) (*n* = 5) (Table 2). TRPA1 was under the detection limit in most untreated IVD cells, while its gene expression tended to increase with IL-1β treatment (*p* = 0.07 for IL-1 5 ng/mL) and it significantly increased with TNF-α (Figure 1A). TNF-α, but not IL-1β, significantly reduced gene expression of TRPV1 (Figure 1B). The induction of an inflammatory-catabolic shift upon cytokine treatment was demonstrated by an increase of IL-6, IL-8, cyclooxygenase-2 (COX-2), nerve growth factor (NGF), matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 3 (MMP3), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and a reduction in COL2A1. Tissue inhibitor of matrix metalloproteinase 1 (TIMP1), tissue inhibitor of matrix metalloproteinase 2 (TIMP2), Aggrecan and COL1A1 were unchanged (Supplementary Figure S1). In IVD cells seeded in three-dimensional (3D) alginate beads and treated with IL-1β (5 ng/mL, 15 days, *n* = 4–10), gene expression of TRPA1 significantly increased on day 1, 8, and 15 (Figure 1C), while the gene expression of TRPV1 remained unchanged (Figure 1D). Immunostaining confirmed TRPA1 protein induction upon IL-1β treatment (5 ng/mL) (*n* = 3) (Figure 1E). Cell viability in alginate beads was monitored by Calcein/Ethidium homodimer staining and an average of 87% of living cells per treatment and time point was found (Supplementary Figure S2).

**Figure 1.** Gene expression of TRPA1 and TRPV1 in IVD cells treated with 5 and 10 ng/mL interleukin-1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α). Gene expression of (**A**) TRPA1 and (**B**) TRPV1 in two-dimensional (2D) culture (Graph shows 2−dd*C*<sup>t</sup> (mean <sup>±</sup> SD, *<sup>n</sup>* = 5). Gene expression of (**C**) TRPA1 and (**D**) TRPV1 in IVD cells cultured in three-dimensional (3D) alginate beads and treated with IL-1β for 15 days. Graph shows 2−dd*C*<sup>t</sup> (mean <sup>±</sup> SD, *<sup>n</sup>* = 4–10). Asterisks indicate statistical significance (\* *p* < 0.05, \*\* *p* < 0.01, Kruskal–Wallis test and Dunn's multiple comparison test). (**E**) Protein expression of TRPA1 in IVD cells treated with IL-1β and untreated (DAPI = blue, TRPA1 = green). Negative control images show cells without secondary antibody. Scale bar is 50 μm.



*2.3. Gene Expression of Other TRP Channels in Human IVD Cells Treated with Pro-Inflammatory Cytokines*

Changes in the gene expression of other TRP channels, namely TRPC1, TRPC3, TRPC6, TRPV2, TRPV4, TRPV6, TRPM2, TRPM7, and TRPM8, were also tested in IVD cells upon cytokine stimulation (IL-1β and TNF-α, both 5 and 10 ng/mL) (*n* = 5). IL-1β significantly induced gene expression of TRPV4 and reduced TRPC6. TNF-α significantly activated the gene expression of TRPV2. The expression of other TRP channels showed no clear association with a pro-inflammatory treatment in the tested experimental settings (Figure 2A–G). Gene expression of TRPM2 and TRPM8 was under the detection limit in all of the treatment groups.

**Figure 2.** Gene expression of TRP channels in IVD cells treated with 5 and 10 ng/mL IL-1β or TNF-α. Gene expression of (**A**) TRPC1, (**B**) TRPC3, (**C**) TRPC6, (**D**) TRPV2, (**E**) TRPV4, (**F**) TRPV6, and (**G**) TRPM7. Graphs show 2−dd*C*<sup>t</sup> (mean <sup>±</sup> SD, *<sup>n</sup>* = 5). Asterisks indicate statistical significance (\* *<sup>p</sup>* < 0.05, \*\* *p* < 0.01, Kruskal–Wallis test and Dunn's multiple comparison test).
