*4.2. Characterization of iSGECs by qRT-PCR*

mRNA expression of epithelial and acinar markers was determined by qRT-PCR in iSGECs, SGCLs, and Hela cells. Expression of epithelial and acinar cell markers showed slight deviation among all three iSGECs when grown as a monolayer culture (Figure 3A,B). Compared to SGCLs and Hela, A253 and HMC-3A showed the greatest similarity to iSGEC cultures. Mucoepidermoid carcinomas have been shown to exhibit increased expression of AQP5 and among all cell lines analyzed, HMC-3A expressed AQP5 highest (Figure 3A) [24].

The two consistently over-expressed pro-acinar genes by iSGEC-nSS2 were ANO1 and SLC12A2, compared to both iSGEC-pSS1 and -nSS1 (Figure 3A). We did not observe a difference in the pro-acinar expression of AMY1A, AQP5, STIM1, and STIM2 when comparing iSGEC-nSS2 to both iSGEC-pSS1 and -nSS1.

iSGEC-nSS1 exhibited the highest expression of both VIM and CST3, and the lowest expression of KRT19 (a ductal/epithelial cell maker of salivary glands) among iSGECs, indicating a heterologous mesenchymal phenotype (Figure 3B) [5,25]. KRT5 is a marker for progenitor and basal ductal cells of the salivary gland [26,27]. In agreement with the work by Jang et al. on primary SGECs, KRT5 was the gene with highest expression in iSGECs by qRT-PCR among the characterization markers, which did not differ significantly among all three iSGEC lines [5]. HSG, HSY, and HeLa cells expressed most transcripts consistently at the same level and exhibited a mesenchymal expression pattern reflected by iSGEC-nSS1 with low CDH1 and high VIM expression [25].

(**B**) Characterization markers

**Figure 3.** mRNA expression of acinar and characterization markers in monolayer cultured iSGECs, SGCLs, and HeLa cells by qRT-PCR. **Legend.** (**A**) Pro-acinar markers were highly expressed in all iSGECs. ANO1 and SLC12A2 were expressed highest in iSGEC-nSS2 compared to other iSGECs. (**B**) Characterization markers for epithelial, ductal, progenitor, and myoepithelial expression in all cell lines examined. The epithelial marker ZO-1 is an integral component of tight junctions and was overexpressed, whereas Vimentin (VIM) was under-expressed in iSGEC-nSS2. KRT-19, a ductal cell marker, was differentially expressed in iSGECs. \* Indicates significant difference in expression determined by Student's *t*-test (alpha = 0.05) and corrected using the Holm–Šidák method. Error bars represent mean +/− SEM.
