*4.4. E*ff*ects of Ca2*<sup>+</sup> *on iSGECs mRNA Expression*

Calcium concentrations in media affect SGEC expression of pro-acinar genes, including AQP5, ORAI1, STIM1 and STIM2 [5,28]. Jang et al. demonstrated an increase in the activity of the store-operated Ca2<sup>+</sup> entry (SOCE) system that upregulates AQP5 expression through the nuclear factor of activated T-cells 1 (NFAT1) [28]. To evaluate changes produced by an increase in Ca2+, we cultured iSGECs in 1.2 mM Ca2<sup>+</sup> for three days and then assessed mRNA expression by qRT-PCR. Early iSGEC-nSS2 cultures (p-14) did not exhibit any alterations in expression of acinar markers when cultured in 1.2 mM Ca2<sup>+</sup> (Figure 5A,B). Late iSGEC-nSS2 (p-80) cells supplemented with 1.2 mM Ca2<sup>+</sup> demonstrated an increase in AQP5 and ANO1 mRNA expression and decreases in other acinar markers (AMY1A, ORAI1, STIM1, SLC12A2, TRPC1) (Figure 5A,B). Moreover, a change in ductal characterization marker KRT19 was decreased in p-14 iSGEC-nSS2 cells cultured in 1.2 mM Ca2<sup>+</sup> (Figure 5C,D). Late (p-80) iSGEC-nSS2 responded differently via an increased expression of tight junction component ZO-1 and myoepithelial marker α-SMA. The lack of response to 1.2 mM Ca2<sup>+</sup> in iSGC-nSS2 p14 cells reiterates the possibility of a de-differentiated cell population seen in Figure 4A,B, where p-80 demonstrated a response to increased Ca2<sup>+</sup> that was more similar to other SGEC publications [5,28].

**Figure 5.** *Cont*.

**Figure 5.** Changes in mRNA expression mediated by 1.2 mM Ca2<sup>+</sup>. **Legend.** Changes in acinar (**A**–**C**) and characterization markers (**D**–**F**) gene expression in iSGECs when cultured in 1.2 mM Ca2<sup>+</sup> for 72 h. Changes in gene expression of iSGEC-nSS2 are indicated with arrows: increased (**red**), decreased (**green**), and no change (**yellow**). (**B**) iSGEC-nSS2 p-14 did not display changes in expression whereas late passaged (p-80) exhibited increases in acinar markers AQP5 and ANO1. AMY1A, ORAI1, STIM2, SLC12A2, and TRPC1 expression decreased in 1.2 mM Ca2<sup>+</sup> p-80 cells. (**C**) Overall, characterization markers exhibited little change in 1.2 mM Ca2<sup>+</sup> supplemented medium. (**D**) Ductal cell marker (KRT19) decreased in early passage (p-14) iSGEC-nSS2 to levels similar in late (p-80) cells. Conversely, ZO-1 and α-SMA expression decreased in later passaged (p-80) iSGECs with 1.2 mM Ca2<sup>+</sup> and did not change in early (p-14) cells. Results calculated by Student's *t*-test and *p*-values corrected by the Holm-Šídák multiple comparisons post hoc test (\* *p* < 0.05). Error bars represent mean +/− SEM.
