Supplemental Figure S1.

A: PKA inhibition does not affect CS growth. To determine whether PKA inhibition altered CS growth, CS bacteria were cultured in media with and without PKA inhibitor KT5720. The experiment was repeated in biological triplicate. No significant difference was identified between the groups. B: PKA inhibition does not affect CS binding. A binding assay was performed to measure whether the ability of the CS bacteria to bind to epithelial cells (IEC-6) was altered in the presence of KT 5720. The addition of PKA inhibitor KT5720 to the cell culture media was assessed at different concentrations, and no significant differences were identified (P = 0.14). This experiment was performed at three different concentrations, each in biological triplicate. C: cAMP levels in the CS bacteria do not account for the increased cAMP found in our model of NEC. cAMP levels were assessed in control IEC-6 and FHs 74 Int cell lines. These levels were then compared with cAMP levels measured in media containing CS without any epithelial cells. No significant difference was found compared with the IEC-6 cell line (P = 0.15). Although there was a significant difference compared with the FHs cell line controls (P = 0.01), the increase seen was very small compared with the increases seen in our models for NEC. Each experiment was performed in biological triplicate. D: Human intestine with active NEC demonstrate sloughing, but cAMP is not increased in whole intestine segments from patients with surgical NEC. Human intestinal tissue was collected from infants undergoing surgery for active NEC perforation. All surgical human NEC samples were obtained from patients with Bell's stage III disease, corresponding with a severity score equivalent of 3. Hematoxylin and eosin staining of paraffin sections of human intestine demonstrates increased inflammatory infiltrate and epithelial sloughing in the NEC group. cAMP was significantly lower than in whole small intestine obtained from age-matched infant control subjects (control versus NEC: P = 0.008). E: Knockdown of CREB by siRNA results in a fourfold decrease in expression. Rat intestinal epithelial cells (IEC-6) were grown to confluence and transfected with control siRNA or siRNA for CREB. RT-qPCR was performed on different samples to confirm knockdown, and a fourfold difference was identified. Data are expressed as means ± SEM. n = 7 infants undergoing surgery for active NEC perforation (D); n = 8 age-matched infant control subjects (D); n = 3 samples (E). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗∗P < 0.0001. cAMP, cyclic adenosine monophosphate; Cont, control; CREB, CREB, cAMP response element binding protein; CS, Cronobacter sakazakii; HFs 74 Int, human small intestinal cell line; IEC-6, rat intestinal epithelial cell line; NEC, necrotizing enterocolitis; PKA, protein kinase A; RT-qPCR, quantitative RT-PCR.