The immortalized acinar epithelial cell lines from parotid gland (PG C10) and submandibular gland (SMG C10) of sexually mature male Sprague Dawley rats were a kind gift from Dr. David Quissell at the University of Colorado, United States (Quissell et al., 1997; Quissell et al., 1998). Cells were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12 50:50) (Gibco, Thermo Fisher Scientific, Massachusetts, United States), supplemented with 5 mM L-glutamine, 4 mg/mL insulin, 2.5% fetal bovine serum (FBS), 0.8 mg/mL epidermal growth factor (EGF), 0.1 µM retinoic acid, 10 mg/mL hydrocortisone, 5 mg/ml T3 (3,3′,5-triiodo-L-thyronine sodium salt), 1‰ trace element mix (100x) (BioSource International, Camarillo, California, United States) and 50 μg/mL antibiotic (gentamicin) in a humidified atmosphere of 5% CO₂ in air at 37°C. Unless otherwise noted, all chemicals were from Sigma-Aldrich (St. Louis, MO, United States). The experiments were performed on cells from passages 6-9.

H₂O₂ was added from 5, 50, 500 Mm stock solutions in 5 mL of the normal growth medium to achieve final concentrations of 5, 50, 100, 150, and 500 µM. PG and SMG cells were seeded into 6-well plates at a density of 1.5 × 10⁵ cells/well and kept overnight before the growth medium was replaced with a new medium containing the different concentrations of H₂O₂. Cells used for negative control were given new medium without addition of H₂O₂. The cells were then incubated for 24 h before the experiment (Figure 2).

To assess cell viability after H₂O₂ treatment, a Trypan blue dye exclusion test was conducted after 24 h. Initially, 500 μL of 37°C trypsin-EDTA was added to each well, incubating for 10 min to detach cells. After deactivating trypsin with 1.5 mL of culture medium and centrifuging (Heraeus Megafuge 1.0R Refrigerated Centrifuge, Thermo Fisher Scientific, Massachusetts, United States) at 1000 RPM for 5 min, the supernatant was discarded, and the pellet resuspended in 1.5 mL medium. Then, 5 μL Trypan Blue Stain (0.4%) (Invitrogen, Thermo Fisher Scientific, Massachusetts, United States) was mixed with 500 μL cell suspension, and 10 μL of this mixture was placed in Chamber Slides (Invitrogen, Thermo Fisher Scientific, Massachusetts, United States) for analysis with the Countess II Automated Cell Counter (Invitrogen, Thermo Fisher Scientific, Massachusetts, United States), identifying viable/dead cells by size, circularity, and brightness. This procedure was repeated with five biological replicates.

After 24 h treatment with different H₂O₂ concentrations, total RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany), and reverse transcriptions were performed using the Reverse Transcriptase Core Kit (Eurogentec, Seraing, Belgium). qRT-PCR was conducted using AriaMx Real-Time PCR System (Agilent Technologies, Santa Clara, California, United States). Each reaction mixture consisted of 10 μL master mix (Low ROX MasterMix dTTP Blue, Takyon, Eurogentec), 10 μL cDNA, 3 μL nuclease-free water, 2 μL TaqMan® probes: Orai1 (Assay ID: Rn02397170_m1), Stim1 (Assay ID: Rn01506495_m1), Adra1a (Assay ID: Rn00567876_m1), and Chrm3 (Assay ID: Rn00560986_s1). The large ribosomal subunit protein eL27 (RPL27) was used as a control (Applied Biosystems, Thermo Fisher Scientific, California, United States). Comparative quantification of expression was performed using the 2^−ΔΔCT method. RT-qPCR was conducted with five biological and three technical replicates for each probe.

Intracellular oxidative stress was analyzed using the ROS-sensitive probe 5-(and-6-)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA, Invitrogen, Thermo Fisher Scientific, Massachusetts, United States), with three technical replicates per sample. Cells (4 × 10⁴/well) were seeded in a 96-well black plate and incubated overnight. After removing the medium, 1 µM CM-H2DCFDA was added and incubated at 37°C for 30 min in darkness. Next, the CM-H2DCFDA solution was removed, and 100 µL of cell medium containing different H₂O₂ concentrations (0, 5, 50, 100, 150, and 500 µM) was added to each well. During 60 min, DCF (2′, 7′-dichlorofluorescein) fluorescence intensity was measured using a Cytation 3 fluorescence microplate reader (Agilent BioTek, California, United States).

As RT-qPCR showed the most significant changes at high H₂O₂ concentrations, Western blot was performed only with 100 and 150 µM H₂O₂. Parotid and submandibular gland acinar cells were exposed 24 h to 100 and 150 µM H₂O₂ concentrations, and untreated cells were used as controls. After removing the medium, the cells were washed with 1 mL PBS, and 500 μL of 37°C trypsin-EDTA was added to each well for 10 min to detach cells. After deactivating trypsin with 1.5 mL of culture medium and centrifuging (Heraeus Megafuge 1.0R Refrigerated Centrifuge, Thermo Fisher Scientific, Massachusetts, United States) at 1,000°RPM for 5 min, the supernatant was discarded. The pellet was resuspended in 100 μL CelLytic^™ MT Cell Lysis Reagent (Sigma-Aldrich). Then, 1 μL EDTA and protease inhibitors (Thermo Fisher Scientific, Rockford, IL, United States) were added and mixed with a homogenizer every 10 min for at least 30 min while kept on ice. The lysed samples were centrifuged for 15 min at 13,000 rpm to remove insoluble material. The BioRad protein assay (Bio-Rad, Hercules, CA, United States) was used to determine total protein concentrations using gamma-globulin as a standard. Total protein (35 μg) was mixed with SDS sample buffer and PBS and heated to 70°C for 5 min. Equal sample volumes were loaded in each well in a Bolt^™ Bis-Tris Plus Mini Protein Gels, 4%–12%, 1.0 mm, WedgeWell^™ format (Invitrogen, Carlsbad, CA, United States). After gel electrophoresis, proteins were transferred to 0.45 μm Immobilon®-P PVDF Membrane (Merk, Darmstadt, Germany). The membranes were blocked for non-specific binding with casein blocking buffer (Sigma Aldrich, Darmstadt, Germany) over night. Next, membranes were incubated overnight with unlabeled rabbit anti-Orai1 (Alomone Labs, Israel), mouse anti-Stim1 (Alomone Labs), rabbit anti-Chrm3 (Bioss, Massachusetts, United States), rabbit anti-alpha1a adrenergic receptor (Invitrogen, Thermo Fisher Scientific, Massachusetts, United States), mouse anti-beta actin (Proteintech, United States) at 4°C. Beta-actin was used as a loading control. The membranes were washed in Tris buffered saline with Tween® 20 (TBST) (Sigma Aldrich, St. Louis, MO, United States) and incubated in secondary antibody for 1 h at room temperature. After additional washing in TBST, the membranes were incubated for 10 min in 1-Step^™ NBT/BCIP Substrate Solution (Thermo Fisher Scientific, Rockford, IL, United States).

Untreated control cells and cells exposed to 100 and 150 μM H₂O₂ were seeded on 13 mm glass coverslips (VWR®, Pennsylvania, United States) at a density of 250,000 cells per well in a 24-well plate. The cells were incubated for 24 h, then fixed in 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, cells were blocked in bovine serum albumin (BSA) 1% overnight. Before staining, cells were permeabilized with 0.1% Triton-X100 in 0.1% sodium citrate (w/v), then blocked with Avidin 10 μg/mL for 30 min. After washing with PBS, cells were blocked with Biotin 1 μg/mL for 30 min. Next, the coverslips were immersed in 5% normal horse serum (ThermoFisher Scientific, Massachusetts, United States) and incubated overnight with 2 μg/mL unlabeled rabbit anti-Orai1 (Alomone Labs, Israel) and 2 μg/mL unlabeled mouse anti-Stim1 (Santa Cruz Biotechnology, Dallas, TX, United States) at 4°C. After washing, cells were incubated with biotinylated horse anti-mouse IgG (Vector Laboratories), donkey anti-rabbit IgG (ThermoFisher Scientific), and Cy3-conjugated streptavidin (ThermoFisher Scientific). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, ThermoFisher Scientific). The coverslips were mounted with a polyvinyl alcohol mounting medium containing DABCO (1,4-diazabicyclo [2.2.2] octane). Photographs were taken using a Nikon E90i microscope equipped with DS-Ri1 camera using NIS-elements software (Nikon Instruments Europe, Amstelveen, Netherlands). The photographs were analyzed using Adobe Photoshop CS6. The fluorescence intensity was measured in nine areas of images with ImageJ.

Statistical analyses were performed using GraphPad Prism for Windows (https://www.graphpad.com/; version 9.5.1) and Excel. Shapiro-Wilk test was used to analyze the normality of data. ANOVA and Student’s t-tests were used to compare groups. All tests performed were two-sided with an alpha level of 0.05. A p-value of <0.05 was considered statistically significant. All data are presented from either three or five independent experiments as indicated, with mean ± standard error (SEM).

To assess the impact of H₂O₂ on cell viability, we counted the number of live cells after 24 h treatment and compared them to control (Figure 3). Increased H₂O₂ concentration led to a significant reduction in cell viability for both SMG and PG cells (Figures 3A, B, respectively). There was a clear dose-response relationship, with higher H₂O₂ concentrations consistently associated with lower cell viability. To evaluate whether this reduction in viability corresponded with a general increase in oxidative stress, we also measured the level of intracellular ROS.

We used the CM-H2DCFDA fluorescence assay to examine the impact of various H₂O₂ concentrations on ROS levels in PG and SMG cells. The ROS concentration increased steadily in a dose-dependent manner during the 60 m of the experiment, in both SMG (Figure 3C) and PG cells (Figure 3D).

We examined how H₂O₂ influenced the expression of certain genes in the intracellular Ca²⁺-signaling pathway that regulates primary saliva production in acinar cells. We observed a substantial and significant downregulation of the cholinergic receptor muscarinic 3 subtype (Chrm3) mRNA expression in both PG and SMG cells when exposed to elevated H₂O₂ concentrations (100, 150 µM) in comparison to the control group (Figure 4A). For the alpha-1A adrenergic receptor (Adra1a), there was a slight reduction in both SMG and PG cells (Figure 4B). However, this reduction was only statistically significant in PG cells exposed to 150 µM H₂O₂. Interestingly, we found that the expression of both receptors was significantly higher in SMG compared to PG cells.

Next, we examined the mRNA expression of Stim1 and Orai1, which are components of the SOCE mechanism crucial for saliva production. Figure 4C shows a significant downregulation of Orai1 mRNA expression at 5 and 50 µM H₂O₂ in PG cells, followed by a slight, but not significant increase at higher concentrations. In contrast, in SMG cells, mRNA expression of Orai1 showed no significant changes. Furthermore, there was a gradual decline in Stim1 mRNA expression in both PG and SMG cells with increasing H₂O₂ concentrations, but this reduction was significant only at 150 µM for both cell lines (Figure 4D).

We conducted Western blotting to assess the protein expression levels of Orai1, STIM1, Chrm3, and Adra1a in PG and SMG cells exposed to 100 and 150 μM H₂O₂. Additionally, we used immunofluorescence microscopy to evaluate the expression of Orai1 and STIM1 in PG and SMG cells exposed to the same H₂O₂ concentrations.

Protein expression of Chrm3 was downregulated by H₂O₂ in both SMG and PG cells (Figure 5A). For Adra1a, protein expression decreased in PG cells with increasing H₂O₂ concentrations, whereas in SMG cells, H₂O₂ exposure did not produce notable changes in protein expression compared to the control. Additionally, Orai1 and STIM1 expression were reduced in PG cells following exposure to H₂O₂. In SMG cells, Orai1 expression was comparable to the control when H₂O₂ levels were increased, while STIM1 expression decreased, with a more pronounced reduction at 100 μM H₂O₂ than at 150 μM H₂O₂.

The immunofluorescence microscopy showed somewhat dissimilar effects of H₂O₂ on protein staining in the SMG cells compared to western blot. Both Orai1 and STIM1 staining intensity was increased at 100 µM. At 150 µM H₂O_2, Orai1 was reduced compared to control cells while STIM1 was similar to control levels (Figures 5B, C). For the PG cells, immunofluorescence and western blot results were consistent, the protein staining of both Orai1 and STIM1 showed a reduction in intensity in PG cells when exposed to both H₂O₂ doses (Figures 5D, E).