Histolochemical staining, immunofluorescence and confocal microscopy The histochemical and immunofluorescence assay was per- formed as follows. SSGs were excised and fixed in 4% (v/v) paraformaldehyde in PBS overnight at 4uC. The tissue was embedded in paraffin, and 5-mm sections were cut. The sections were de-paraffinized and rehydrated in a graded ethanol series. Cell morphology was visualized by routine histologic techniques using hematoxylin-eosin (H&E) staining. For immunofluorescence the sections were treated for antigen retrieval by boiling in citrate buffer (10 mM, pH 6.0, 0.05% v/v Tween 20) for 10 min at 100uC and cooled to room temperature for 30 min. Nonspecific antibody binding was blocked by 60 min incubation with 5% (v/v) goat serum and 0.1% (v/v) Triton X-100 in PBS at room temperature. The antibodies for amylase (2 mg/ml, mouse monoclonal IgG; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and c-KIT (CD117, 2 mg/ml, Santa Cruz Biotechnology) were diluted 1:50 and 1:25, respectively, with 1% goat serum and 0.1% Triton X-100 in PBS, and incubated overnight at 4uC. Then the slides were incubated for 60 min at room temperature with secondary antibody Alexa Fluor 555-conjugated donkey anti- mouse IgG (Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 647- conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) at a dilution of 1:500. For nuclear staining, 4,6-diamidino-2- phenylindole (DAPI; 1 mg/ml; KPL, Gaithersburg, MD, USA) was added to samples and incubated for 5 to 7 min at room temperature. Slides were then rinsed and cover-slipped with fluorescent mounting medium (DAKO, Carpinteria, CA, USA). The stained preparations were evaluated using a confocal laser scanning microscope (Model 710, Zeiss). SGIE isolation and colony forming efficiency SSG integrin a6b1-positive cells (SGIE) were immuno-magnetic isolated as we previously described [23,24]. To test their colony forming efficiency, 5000 cells were seeded on irradiated NIH 3T3 fibroblasts for 10 days on a 35-mm Petri dish. Then colonies were fixed with 4% formaldehyde (Sigma) for 10 min at room temperature. After counter-staining with 1% Rhodamine B solution (Sigma), colonies were manually counted under light microscopy Statistical analysis Statistical analysis of MS data was performed as described in above. Statistical analysis of microarray data was performed as described in section above. Saliva flow rate, real-time PCR validation and assessment of colony forming efficiency were performed by One-way ANOVA, followed by Tukey test. Results are presented as mean values 6 SD. P,0.05 was considered statistically significant. Results Effect of IR on saliva secretion and proteome The most fundamental function of the SGs is to produce and secrete saliva. We therefore studied the effect of 15-Gy head and neck IR on saliva output. Fig. 1A demonstrates the effect of IR on saliva secretion before IR and for 12 weeks post-IR. Four weeks post-IR, saliva decreased to 56% of the output in control animals, continued to decrease to 50% at 8 weeks, and then remained at that level until at least 12 weeks post-IR. Fig. 1B shows H&E staining of submandibular salivary glands before and 4, 8 and 12 weeks after IR. Four weeks after IR structural changes were found in parts of the gland, including aberration in acini organization and reduction in the Proteome and Transcriptome in Irradiated Glands numbers of ducts. Eight and 12 weeks after IR gland structure appeared normal without signs of fibrosis. Saliva protein composition was determined by proteomic analysis before and 4, 8 and 12 weeks after IR (Fig. 1C), followed by MS identification (Table S1). Among the protein spots with altered intensities, as determined by 2-DE after IR, three protein groups were detected: those showing increased expression (Fig. 1C, blue ellipses), those showing reduced expression (Fig. 1C, red ellipses), and those with erratic expression (Fig. 1C, black rectangles). MS identification of the up-regulated proteins showed that most of them were derived from the serum, including serum albumin precursor, serotransferrin, immunoglobulins and complement components (Table S1, spots 9, 14–18, and 26, Fig. 1D). MS identification of the down-regulated proteins (Table S1, spots 1–3, 5, 7 and 11, Fig. 1D) showed that most of them originated from the SG, including a-amylase, parotid secretory protein, prolactin- induced protein and von Ebner gland protein 1 and 2. The third group, containing proteins with erratic expression, included proteins derived from both the SG (parotid secretory protein, a-amylase, prolactin-induced protein, von Ebner gland protein 1 and 2, and cysteine-rich secretory protein 1 precursor) and serum (serum albumin precursor, serotransferrin, apolipopro- tein E precursor, immunoglobulins and complement components) (Table S1, Fig. 1D). While the serum-derived proteins tended to return to normal levels 12 weeks post-IR, most of the SG-derived proteins, except for spot 12, tended to be at reduced levels 12 weeks post-IR (Table S1). Effect of IR on SSG transcriptome and SGIE colony forming efficiency We identified 95 genes that were significantly differentially expressed as a result of IR using microarray (Table S2). We used these differentially expressed genes for further functional annota- tion of pathways and functional categories using the David online database (http://david.abcc.ncifcrf.gov/). A significant over-rep- resentation of genes in the following functional categories was observed: endoplasmic reticulum, signal, emp24/gp25L/p24, enzyme inhibitor activity, protein transport, cell redox homeosta- sis, nucleotide binding, golgi apparatus, proteolysis, protein folding and secretory granules. Following IR, two major expression patterns were identified among the differentially expressed genes (Fig. 2A). The first included 81 genes that were down-regulated at least twofold 4 weeks post-IR, and recovered to near control levels 8 and 12 weeks post-IR (Fig. 2B). The second pattern included 14 genes which were up-regulated following IR and then continued to be over- expressed during the 12-week period following IR (Fig. 2C). Within the first pattern, down-regulated (compared to controls) SG-expressed genes included those encoding mucin 19 (Muc19), cadherin 22 (Cdh22), parotid secretory protein (Psp) and proline- rich protein of BstNI subfamily 1 (Prb1), as validated by Taqman real-time PCR (Fig. 3). Muc19 and Cdh22 genes showed ca. twofold down-regulation 12 weeks post-IR. Psp gene showed the same level of down-regulation at week 4 post-IR, and was then up- regulated ca. twofold (compared to control levels) at 8 and 12 weeks post-IR (Fig. 3). Contrary to the microarray results, Prb1 gene was found by real-time PCR to be up-regulated following IR and therefore actually belonged to the second pattern. The second expression pattern demonstrated an at least twofold up-regulation in gene expression following IR (Fig. 2C), and included the remaining 14 genes: cell-cycle regulating genes, and those encoding Mgmt (O-6-methylguanine-DNA methyltransfer- ase), CysS and a-amylase. The latter two genes encode major PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40636