Fig 1: Acinar cell apoptosis is delayed in post-ligated SMGs of ATG5-deficient mice. (a) ATG5 status impinges upon duct ligation-triggered acinar apoptosis. Apoptosis by ApopTag Peroxidase (brown nuclear staining) was visualized using an In situ Apoptosis Detection Kit (left panel). Peak apoptosis was detected in L1 and L3 SMGs of Atg5WT mice, whereas the strongest ApopTag signals were detected in L3 and L7 SMGs of Atg5KO mice. Magnification: × 100, and enlarged view (inset): × 400. Bar: 100 µm. Percent ApopTag-positive cells quantified by dividing the total ApopTag-positive cells by total number of cells examined from 10 randomly chosen fields are shown (right panel). Student's t-test was employed to determine statistically significant differences in percentage of ApopTag-positive cells between Atg5WT and Atg5KO groups. Results are shown as mean±S.D.; *P<0.05. (b) ATG5 deficiency delays caspase-3 activation. Equal amounts of whole SMG lysates from Ctrl, L1, L3 and L7 SMGs of Atg5WT and Atg5KOmice were analyzed on western blots using indicated antibodies. The cleavage of caspase-3 represents caspase-3 activation. (c) Gene expression analyses of Prol1/Muc-10 and Klk1, markers for acinar and duct cells, respectively. SMG duct ligation was performed as described in Figure 2. Expression of the indicated messages in ligated and control SMGs was analyzed by quantitative RT-PCR analyses. Relative gene expression was calculated where respective mean value for control SMG set to 1 for each amplification (N=4). Non-parametric Mann–Whitney test was performed to compare expression levels between respective ligated and control SMGs (* below bars), and between same-day ligated SMGs and control SMGs from Atg5WT and Atg5KO (* above bars). Results are shown as mean±S.D.; *P<0.05; **P<0.01. (d) Autophagy inhibition correlates with resistance to H2O2-induced cell death. Cell viability was measured in m5-7, Atg7-KO MEF, WT MEF and salivary Pa-4 cells treated with indicated concentrations of H2O2 in combination with chloroquine (CQ; 20 µM) or baflomycin A1 (BafA1; 10 nM) for 48 h and compared with that of the control cells. Data were analyzed with ANOVA followed by Bonferroni t-test to determine the statistical differences between treatment and control group (left panels). Representative western blots (right panels), in which total lysates from cells treated for 6 h with vehicle, CQ (20 µM) or BafA1 (10 nM) were stained with indicated antibodies, are shown. Results are shown as mean±S.E.M.; N=3; *P<0.05; **P<0.01; ***P<0.001