Quercetin (chemical purity ≥ 95%, HPLC), Alcian Blue, Carbamoylcholine chloride (Cch) and fluorescent probe DPBA (2-Aminoethyl diphenylborinate) were purchased from Sigma-Aldrich (USA). Periodic acid-Schiff (PAS) was purchased from Dako-Products, BAPTA-AM, U73122, bisindolylmaleimide (BIM) and PD98059 were purchased from Calbiochem (USA).

LS174T and Caco-2 cells were obtained from the ATCC (American Type Culture Collection, USA) and routinely grown in 75 cm2 flask. LS174T were grown in Advanced modified Eagle medium (A-MEM, GIBCO) containing 4.5 g/l glucose, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin (Sigma-Aldrich). Caco-2 cells were grown in Dulbecco's modified Eagle medium (DMEM, Sigma-Aldrich, USA), containing 4.5 g/l glucose, supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin (Sigma-Aldrich, USA). The cells were kept in a 5% CO₂ and 95% air atmosphere at 37°C.

LS174T and Caco-2 cells were grown to semi-confluence in 60-mm dishes in presence or absence of quercetin (25/50 μM), then were washed twice with PBS and cell lysates were obtained in RIPA buffer, containing 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 2.5 mM Na-pyrophosphate, 1 mM β-glycerophosphate, 1 mM NaVO4, 1 mM NaF, 0.5 mM Phenylmethylsulfonyl fluoride (PMSF) and a cocktail of protease inhibitors (Roche, USA). The cells were kept for 15 min at 4°C and disrupted by repeated aspiration through a 21-gauge needle. Cell lysates were centrifuged for 15 min at 13,000 rpm and the pellets were discarded. The protein concentration was determined by Lowry assay. Fifty micrograms of total proteins were subjected to sodium dodecyl sulfate 7.5% polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Filters were incubated with specific rabbit polyclonal antibody that cross reacts with p-PKCα (Ser657, Upstate) or a specific rabbit polyclonal antibody against p-Erk1-2 (Santa Cruz Biotecnology, INC.), then incubated with a peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (GE-Healthcare, UK). Peroxidase activity was detected with the enhanced chemiluminescence (ECL) system (GE-Healthcare). Protein bands were revealed by ECL and, when specified, quantified by densitometry using Scion Image software. Densitometric values were normalized to α-tubulin.

The supernatants of LS174T were loaded onto the BIO-Dot SF apparatus according to the protocol provided by the manufacturer (BIO-RAD). Filters were incubated with specific mouse polyclonal rabbit antibodies that cross reacts with MUC2 or MUC5AC (Abcam), before being washed three times in Tris-Buffered Saline-Tween 20 0.1% (TBS-T) and then incubated with a peroxidase-conjugated secondary rabbit antibody (GE-Healthcare, UK). After washing with TBS-T peroxidase activity was detected with the enhanced chemiluminescence (ECL) system (GE-Healthcare). Protein bands revealed by ECL were quantified by densitometry, using Scion Image software; densitometric values were normalized for intracellular protein content, that was unchanged in samples treated with quercetin respect to controls. Moreover, total protein content of the cell media from quercetin treated cells were comparable to that of control samples.

Intracellular calcium levels in LS174T cells were measured using the Fluo-4 NW calcium indicator kit (Life Technologies), according to the manufacturer's instructions. Briefly, the LS174T cells were grown in 96 well microplates (20000/w) for 18 h, then were washed with assay buffer and loaded with Fluo-4 probe in the presence of 5 mM Probenecid for 30 min at 37°C and then 30 min at room temperature. Fluorescence was recorded every 6 s using the Flouroskan Ascent–FL (Thermo electronic corporation) with excitation at 485 nm and emission at 538 nm. Fluorescence was recorded for 1 min before loading cells with quercetin or carbachol and was then monitored for an additional 10 min. Blank samples fluorescence was also measured and subtracted from all experimental points.

Ten micrograms of total RNA from LS174T and Caco-2 cells were obtained. Trizol (Invitrogen, no. 15596-026) method has been used for isolation and purification of RNA. RNA was isolated including a DNase digestion step. These standardized RNA isolation procedures guarantee high-quality RNA. Using the Agilent 2100 Bioanalyzer platform (Agilent Technologies) RNA samples were quality-checked documenting the identification of 18-S and 28-S ribosomal RNA (rRNA) peaks. The yields were 9–15 μg, and the RNA Integrity Number (RIN) was between 8.2 and 10.

To confirm the expression patterns of genes MUC2 and MUC5AC, we performed a quantitative RT-PCR using the comparative Ct method. Transcript levels of the target genes were normalized to G6PD (the internal control) after correcting for differences in amplification efficiencies. RT-PCR reactions (n = 3) were performed for each gene of interest using a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). All genes investigated have previously been identified and sequences were available in GenBank. Primers for qRT-PCR analysis were designed using the Primer3 program (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi).

The final PCR reactions contained: 0.4 μM of each primer; 0.25 × SYBR Green (Invitrogen); 4 mM MgCl₂ and as template 5 μl of cDNA reverse transcribed from a standardized amount of total RNA (0.3 μg). qRT-PCR was performed using Hotstart Taq polymerase (Qiagen) in a final volume of 20 μl. All quantitative reactions were subjected to: 95°C for 15 min followed by 45 cycles at 94°C for 15 s, 59°C 15 s and 72°C 15 s. Melting curve analysis was applied to all reactions to ensure homogeneity of the reaction product.

Potential contamination was assessed by including non-reverse transcribed total RNA (genomic DNA contamination) and controls without template, observing no products in these reactions.

Primers used in these studies are the following: Human MUC2: (F), CGA CTA CTA CAA CCC TCC GC (R), GGG AGGAGT TGG TAC ACA CG; Human MUC5AC: (F), GAC TGT CAT CCT CTG TGC G (R), CAC CTCGTA GTT GAG GCA CA; Human G6PD: (F), ACA GAG TGA GCCC TTC TTC AA (R), ATA GGAGTT GCG GGC AAA G.

Cells were grown to semiconfluency in 60-mm culture dishes. Following the method of Grootaert et al. (2016), after detachment by trypsin, cells were suspended in 1 mL of phosphate buffered saline (PBS) and fixed overnight with 1% formaldehyde at room temperature. Next, cells were incubated with the fluorescent probe DPBA, (0.2%) in DMSO (0.3%), for 1 h at room temperature, whereas control cells were incubated whit DMSO (0.3%) in water without stain. After two washes in PBS, the samples were resuspended in 500 μl of PBS and analyzed by flow cytometry using FACSCAN (BD, Heidelberg, Germany) and data were analyzed using Flowing 2.5.1 software.

LS174T cells were grown for 24 h on glass coverslip. Then, the medium was removed and cells immediately fixed in 3.7% Paraformaldehyde. Next, the cells were oxidized with 1% periodic acid (Sigma-Aldrich, USA) for 10 min. After washing with water, cells were treated with Schiff's reagent (Dako-Products) for 20 min and then rinsed with water. The cells were counterstained with hematoxylin (Sigma-Aldrich, USA) and then the coverslips were briefly washed with distilled water, and finally mounted on glass slides for microscopy examination. Cells were analyzed with an optical microscope (20x).

LS174T cells were grown for 24 h on glass coverslip. The day after the medium was removed and cells fixed in 3.7% Paraformaldehyde. Then the cells were incubated in Acetic Acid Solution (3%) for 3 min and next with Alcian Blue (pH 2.5) solution for 30 min at room temperature. Following the treatment, the cells were washed in Acetic acid solution to remove Alcian Blue excess and then in water. The coverslips were mounted on glass slides for microscopy examination. Cells were analyzed with an optical microscope (20x).

The supernatants of LS174Tcells, grown in absence and in presence of quercetin (50 μM), were loaded onto the BIO-Dot SF as previously described. Next the membranes were placed in 5% non-fat milk in tris buffered saline, 0.1% Tween 20 (TBST, Bio-Rad Laboratories) at room temperature overnight to block the non-specific binding sites. Filters were incubated in 1% periodic acid for 15 min, rinsed with distilled water and stained with Schiff's reagent for up to 60 min at room temperature. Glycoprotein bands were quantified by densitometry using ImageJ software.

Slot-Blot membranes of LS174T supernatants cells, obtained and treated as previously described, were incubated in Acetic Acid Solution (3%) for 3 min. Then membranes were washed with distilled water and stained with Alcian Blue (pH 2.5) solution for 30 min at room temperature. Glycosaminoglycan bands were quantified by densitometry using ImageJ software.

Statistical differences were evaluated using a Student's t-test for unpaired samples.

Since LS174T cells produce large amount of secretory mucins released in the extracellular space (Van Klinken et al., 1996) we used these cells to study the effects of quercetin on mucin secretion. These cells are similar to intestinal goblet cells since they contain intracellular mucous granules as shown by Alcian blue and Shiff-PAS staining for glycoproteins and glycosaminoglycans, respectively (Figures 1A,B). By Slot-Blot of cell media and staining of the membranes with Alcian blue and Shiff-PAS, we also observed that in LS174T cells the treatment with 50 uM quercetin for 24 h increases glycoproteins and glycosaminoglycans secretion (Figures 1C,D). To evaluate the effects of quercetin on MUC2 and MUC5AC secretion, LS174T cells were incubated for 2, 4, or 24 h with two different concentrations of quercetin, 25 and 50 μM, and then mucin secretion was measured by Slot Blot analysis. Secretion levels of MUC2 protein after 4 and 24 h incubation with both doses of quercetin were significantly higher than that of untreated control cells (Figure 2A). MUC5AC secretion levels were significantly increased in cells incubated with 25 and 50 μM of quercetin for 24 h (Figure 2B). compared to control cells. In these experimental conditions quercetin did not affect cell viability as shown by trypan blue staining of cells (Supplementary Figure 1A), indicating that the increased levels of secreted mucins cannot be ascribed to cell lysis.

We also evaluated the effect of quercetin on MUC2 and MUC5AC gene expression (Figures 2C,D). The mRNA levels of MUC2 and MUC5AC measured by RT PCR were significantly increased by the treatment with 25 and 50 μM quercetin respect to untreated control samples and the increase was time-dependent. Further studies were carried out in intestinal epithelial Caco-2 cells. Similar induction of MUC2 and MUC5AC mRNA levels were observed in Caco-2 cells treated with quercetin (Figures 2E,F). Even in these cells, quercetin did not affect cell viability (Supplementary Figure 1B).

To evaluate whether quercetin permeates LS174T and Caco-2 cell membranes flow cytometry experiments using the fluorescent probe DPBA for the staining of flavonoids (Grootaert et al., 2016) were performed. The Figures 3A,B shows a significant increase of DPBA fluorescence in LS174T and Caco2 cell lines incubated with quercetin.

The release of secretory mucins in the extracellular space is a Ca²⁺-dependent event (Yang et al., 2013). For this reason, we evaluated the effects of quercetin on intracellular Ca²⁺ levels by fluorimetry using a FLUO4 fluorescent probe in the presence of Probenecid (5 mM) which has the function of holding the probe inside the cells. The Figure 4A shows intracellular calcium transients kinetics after quercetin stimulation of LS174T cells. In these experiments, 1 mM Cch was used as a positive control to induce a significant and rapid increase of intracellular calcium concentrations. The incubation with 50 μM quercetin induces intracellular Ca²⁺ levels in LS174T cells over time compared to untreated control cells and cells treated with 25 μM quercetin. To demonstrate that quercetin-induced mucin secretion is dependent on the increase of intracellular Ca²⁺ levels, LS174T cells were incubated with 50 μM quercetin in the presence and absence of an intracellular calcium chelator, 1,2-Bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), and the respective culture media were analyzed using Slot-Blot analysis (Figures 4B,C). In the presence of BAPTA-AM, quercetin failed to induce the secretion of MUC2 and MUC5AC.

The increase of intracellular calcium by quercetin suggests the involvement of PLC in the signaling pathway mediating the effects of this flavonoid on MUC2 and MUC5AC secretion and expression. PLC enzyme catalyzes phosphatidylinositol 4,5-bisphosphate (PIP2) conversion to inositol-1,4,5-triphosphate's (IP3) and diacylglycerol (DAG) and IP3 is major contributor to intracellular Ca²⁺ release (Tran et al., 2016). Therefore, to evaluate whether PLC signaling mediates quercetin effects we used the PLC inhibitor U73122. Slot Blot and RT-PCR analysis showed that pretreatment of LS174T cells with U73122 completely reversed the increase of secretion (Figures 5A,B) and mRNA (Figures 5C,D) levels of MUC2 and MUC5AC by quercetin.

We further investigated the molecular mechanisms involved in quercetin-dependent increase of mucin secretion in LS174T and mucin gene expression in LS174T and in Caco-2 cells by evaluating the involvement of ERK1-2 and PKCα signaling pathways. Quercetin significantly induced P-PKCα and P-ERK1-2 levels compared to untreated LS174T (Figures 6A,B) and Caco-2 cells (Figures 6C,D). Furthermore, pretreatment of cells with the PKCα-β-γ inhibitor bisindolylmaleimide (BIM) and the mitogen activated protein kinase kinase (MEK) inhibitor PD98059, prevented the effects of quercetin on the secretion of MUC2 and MUC5AC (Figures 7A,B) in LS174T and mRNA mucin expression (Figures 7A,D), in both cell lines demonstrating that PCKα/ERK1-2 pathway mediates quercetin effects.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.