*2.2. LSG Biopsies and Culture of Salivary Gland Epithelial Cells (SGECs)*

Atrium Health institutional review board (Charlotte, NC, USA) approval (IRB Protocol #: 08-16-24E) was granted for this study and all patients gave informed consent.

Anti-Ro (SSA) serum-negative (-) xerostomic patients undergoing a LSG biopsy for the assessment of pSS according to the 2016 ACR/EULAR classification criteria were asked to participate in this study [6]. Xerostomic nSS and pSS patient clinical characteristics are listed in Table 1. Remaining LSG biopsy tissue was transported to the research laboratory in complete SGEC explant medium supplemented with 5x antibiotic/antimycotic solution (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).


**Table 1.** Patient demographics and clinical features.

Labial salivary gland biopsies used in this study were collected from one pSS and two sicca patients (nSS). All patients were negative (−) for serum Anti-Ro (SSA) and were not taking disease-modifying anti-rheumatic drugs (DMARDs). \* The patient with the lowest focus score had the lowest salivary flow rates (unstimulated/stimulated). NA: Schirmer's test was not performed due to patient objection or information not listed in electronic medical records.

SGEC cultures were established according to the methods outlined by Jang et al. and are briefly explained as follows [3]. LSG tissue was minced into 0.5–1 mm<sup>2</sup> fragments and placed in T-75 flask with 3.5 mL of complete SGEC explant medium supplemented with 1x antibiotic/antimycotic solution (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and grown (37 ◦C, 5% CO2). Complete SGEC explant medium consisted of 1:3 Ham's F12 and DMEM supplemented with 2.5% FBS (VWR, Radnor, PA, USA), 20 μg/mL EGF (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 200 μg/mL insulin (MP Biomedicals, Santa Ana, CA, USA), 100 ng/mL hydrocortisone (Sigma-Aldrich, Saint Louis, MO, USA), and 1x antibiotic/antimycotic solution (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). After 72 h, 5 mL of SGEC explant media was added. Cells at 80–90% confluency were split 1:3 into T-75 flasks with 0.05% trypsin +0.045 mM EDTA. Trypsin was neutralized by soybean trypsin inhibitor (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at a 1:1 ratio (*v*/*v*). For remaining passages, SGEC sub-culturing medium was used. SGEC sub-culturing medium consisted of Epi-life BasalTM medium (0.06 mM Ca2+) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA, Catalogue# MEPI500CA) supplemented with 1x human keratinocyte growth supplement (HKGS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA, Catalogue# S0015). Fibroblasts were gradually removed from culture using a combination of 0.02% EDTA or 0.01% trypsin.

## *2.3. Transduction of SGECs by Lentiviral SV40Lt Particles*

During passage number 2 (p-2), SGECs were spit into 6-well (VWR, Radnor, PA, USA) tissue culture-treated plates at a confluency of 60–70% and allowed to adhere for 24 h. Cell medium was removed and replaced with 1 mL SV40Lt lentiviral supernatant (ABMgood, Richmond, Canada, Catalogue# G258) diluted with 1 mL of SGEC sub-culturing medium (2 mL total) and polybrene

(Sigma-Aldrich, Saint Louis, MO, USA) at a final concentration of 4 μg/mL. After 48 h, SV40Lt lentiviral cell medium was replaced with SGEC sub-culturing medium and grown for 24 h. Medium was removed and cells were supplied with 1 mL SV40 lentiviral supernatant diluted with 1 mL of SGEC sub-culturing media (2 mL total) and polybrene at a final concentration of 4 μg/mL, for 48 h.

SV40Lt lentiviral cell medium was removed and replaced with SGEC sub-culturing medium and cells were grown to 80–90% confluency before being split into 6-well tissue culture-treated plates at a ratio of 1:6. For cell passages after SV40Lt lentiviral transfection, cells were split 1:6 up to passage 10. The remaining passages were split at 1:3 using T-75 (Corning Life Sciences, Tewksbury, MA, USA) tissue culture-treated flasks. To determine increased expression of pro-acinar markers in high Ca2<sup>+</sup> supplemented medium, all iSGECs at early passage 14 (p-14) and only iSGEC-nSS2 at passage 80 (p-80) were subjected to 1.2 mM Ca2<sup>+</sup> in SGEC medium for 72 h prior to experimentation [3].
