Fig 2: Effects of NF-?B inhibition on H. pylori-induced apoptosis and PUMA expression (WT mice were treated with H. pylori alone or in combination with 8 mg/kg of the NF- ?B inhibitor Bay 117082 for 3 days). a IHC staining of PUMA in gastric tissues from the treated mice. b TUNEL (brown) staining of gastric tissues from the treated mice. c TUNEL (green) IF staining of gastric tissues from the treated mice. d The apoptotic index was calculated by counting TUNEL signals in 100 randomly selected glands following TUNEL staining, as in c. N = 4/group, the results are presented as the mean ± SEM, **P < 0.01 compared with the control.

Fig 3: P65 directly binds to the PUMA promoter to activate its transcription following H. pylori treatment.a Expression of p-p65 (S536), p65, and PUMA on AGS cells after treatment with H. pylori at the indicated time points was analyzed by western blotting. b AGS cells were treated with 10 mmol/L BAY 11–7082 for 1 h and then with H. pylori or 10 ng/mL TNF-a for 24 h. Left, nuclear fractions were isolated from cells and analyzed for p65 expression by western blotting; right, western blot analysis of PUMA and ß-actin expression in whole-cell lysates after H. pylori or TNF-a treatment. c WT AGS cells were transfected overnight with pCMV or I?BaM and then treated with H. pylori for 24 h. The expression levels of PUMA, p-I?B, I?B, and p-p65 were analyzed by western blotting. d AGS cells were transfected with either a control scrambled siRNA or a p65 siRNA for 24 h and then treated with H. pylori for 60 h. p65 and PUMA expression was probed by western blotting. e Left, schematic representation of the genomic structure of PUMA highlighting the PUMA promoter fragments (Frag) A–E of the PUMA promoter, followed by treatment with H. pylori. Reporter activities were measured 24 h later by a luciferase assay.

Fig 4: AKT inactivation induces Puma expression and neuronal apoptosis via a GSK3ß-dependent mechanism.CGNs maintained for 7 days in high potassium medium were treated with or without the PI3K inhibitor LY294002 (30 µM, LY) in the presence or absence of the GSK3ß inhibitor SB415286 (30 µM, SB). A, RNA was collected 8 hours post treatment and Puma mRNA levels were determined by qRT-PCR (n = 3,* p<0.05). B, Protein extracts were collected 12 hours post-treatment and Puma protein levels were analyzed by western blot. C, The fraction of apoptotic neurons was quantified after 24 hours by Hoechst staining (n = 3, *p<0.05).

Fig 5: PI3K/AKT inhibition induces Puma expression and Puma-dependent apoptosis in CGNs. A, CGNs maintained in high potassium medium (K25) were treated with the PI3K inhibitor LY294002 (30 µM) or vehicle and protein extracts were collected after 12 hours and Phosph-AKT, Phospho-GSK3ß and Puma protein levels were assessed by western blot. B, CGNs were infected with recombinant adenovirus expressing either CA-AKT or GFP at 10 MOI and after 7 days neurons were treated with or without LY294002 (30 µM). RNA was collected at 8 hours and Puma mRNA levels were quantified by qRT-PCR (n = 4, *p<0.05). C, CGNs derived from Puma+/+ and Puma-/- littermates were treated with or without 30 µM LY294002 (LY) and the fraction of apoptotic neurons was quantified after 24 hours by Hoechst staining.