Proteome and Transcriptome in Irradiated Glands Figure 4. Gene expression, SGIE colony forming efficiency and immunofluorescence staining during 12 weeks post-irradiation. A. In irradiated glands, c-KIT expression decreases progressively (4W, 8W, 12W) compared to control glands. Immunofluorescence staining shows a similar pattern (bar = 50 mm). B. SGIE colony forming efficiency in control and 4, 8 and 12-week post-IR. The number of rats used in each experiment is indicated inside the bars. *Significantly different at P,0.02. C. a-amylase gene expression is increased (4W, 8W, 12W) compared to control glands. Immunofluorescence staining shows a similar pattern (bar = 50 mm). Data are expressed as means of n = 3. *P,0.05, ***P,0.001. doi:10.1371/journal.pone.0040636.g004 To gain a better understanding of IR damage to SG functionality, results from several of the SG-derived genes (encoding Muc19, Cdh22, Psp, a-amylase and CysS), a DNA repair gene (encoding Mgmt) and proliferating genes (encoding c-KIT) were validated using Taqman real-time PCR (Fig. 3). The main functions of mucus include lubrication and protection of epithelia from environmental insults. The gel-like properties of mucus are generally attributed to the physical properties and structural features of the gel-forming mucins, such as Muc19 [35]. Muc19 was down-regulated twofold 12 weeks post-IR, which might cause the saliva to lose its lubricating and protective properties, and contribute to the xerostomia [3] seen in irradiated patients. Cdh22 belongs to the cadherins, a superfamily of transmembrane glycoproteins which typically mediate calcium- dependent homophilic intercellular adhesion [36]. Cdh22 was found to be downregulated twofold 12 weeks post-IR, indicating damage in signaling and adhesion. Psp, a proacinar cell marker, is associated with rat SSG regeneration [37]: in the adult regener- ating gland, mRNA levels of Psp have been found to be up- regulated compared to ligated glands [37]. The increased expression of Psp 12 weeks post-IR suggests that the salivary glands are attempting to regenerate (see also our discussion on cell- cycle genes below). Saliva contains a prominent family of proline-rich proteins (PRPs) which includes acidic, basic and glycosylated PRPs [38]. Interestingly, ,60-fold up-regulation of Prb1 was observed following IR (Fig. 3). The salivary PRPs have several important activities: they bind hydroxyapatite and calcium, mediate adher- ence of microorganisms, and modify the lubricative properties of saliva. The Prb1 gene encodes a precursor of basic proline-rich salivary proteins and is synthesized mainly in the acinar cells [39]. CysS belongs to the cystatins, natural inhibitors of cysteine proteinases through their tight binding to the proteinases’ active part [40]. CysS is expressed in the acinar cells of the SG [40] and has been reported to play a role in the response to injury [40]. The up-regulation in CysS mRNA levels following IR suggests an attempt to deal with the damage caused to the salivary glands. The results for submandibular expression of a-amylase, i.e. induced PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40636