Fig 1: IR transiently downregulates expression of genes involved in saliva secretion. (A-D) Immunofluorescent staining shows colocalization of M3R+ (red) with MIST1+ (green) acinar cells (A) on control. M3R expression does not change at 3 h (B), 24 h (C) or 48 h (D) after IR using the Cs source. (E-H) AQP5 (red) localization at the apical surface of MIST1+ (green) acinar cells in control (E) does not change at 3 h (F), 24 h (G) or 48 h (H) post-IR. Scale bars: 100µm. (I) Expression of Mist1 mRNA was significantly reduced at 3 h post-IR (**P=0.002), but recovered by 24 h post-IR (P>0.1). (J) Relative expression of M3r mRNA did not change post-IR using the Cs irradiator (P>0.1). (K) Aqp5 mRNA expression was decreased at 3 h post-IR (*P=0.023), but recovered by 24 h post-IR (P>0.05). (L) Western blot of MIST1, AQP5, CX32, E-CAD, GAPDH and ß-actin proteins in control and irradiated SMGs using the Cs source (n=3). (M,N) Densitometric analysis of MIST1 (M) and CX32 (N) MIST1: *P=0.041; CX32: **P=0.003. (O) Densitometric analysis of AQP5 protein (*P<0.05). (P) Densitometric analysis of E-CAD protein (one-way ANOVA; F=0.215, P=0.883). (Q) Following IR using SARRP, relative expression of Mist1 mRNA was significantly decreased in both irradiated and CL SMGs at 3 h (***P<0.001, *P<0.05, respectively), at 24 h (***P<0.001, *P<0.05, respectively) and at 48 h (*P<0.05) versus control. Mist1 expression was significantly lower in the IR-treated SMGs compared to the CL SMGs (##P<0.01). (R) IR using SARRP also decreased the relative expression of M3r mRNA in both irradiated and CL SMGs at 3 h (**P<0.001, *P<0.01, respectively) versus non-irradiated control. (S) Following IR using SARRP, relative expression of Aqp5 mRNA was decreased in both irradiated and CL SMGs versus control at 3 h (***P<0.001, **P<0.01, respectively), 24 h (**P<0.01, *P<0.05, respectively) and 48 h (**P<0.01, **P<0.01, respectively). Aqp5 expression was significantly lower in the IR-treated SMG compared to the CL SMG (#P<0.05). Error bars indicate mean±s.e.m. Statistical analysis was performed compared to control (non-irradiated) using one-way ANOVA with Dunnett's post-hoc test, or comparing irradiated and CL SMGs using two-way ANOVA with Bonferroni test (n=3-5 each group).