GBP-1 restricts epithelial cell proliferation.
SF1: Cell proliferation rates in SKCO15 cells after growth media exchange at the time points indicated. Cells not subjected to media change are indicated as time point 0. Proliferation rates are represented as the fraction of EdU positive cells/Total cells counted (n=3, 5-600 cells per experiment, n.s by ANOVA).
SF2: GBP-1 knockdown by siRNA ameliorates TNF-/IFN-γ induced suppression of cell growth. After 48hrs cytokine treatment, control samples show decreased proportion of EdU positive cells. IECs treated with siRNA against GBP-1 in addition to TNF-/IFN-γ show no significant change in EdU incorporation compared with controls (n.s.). Statistical significance was determined by Fisher’s exact test (, p=0.005, n=2,800 and 2,100).
SF3: Desitometric analysis of westernblot data showing GBP-1 knockdown by siRNA, with or without TNF-/IFN-γ induction (n=4).
SF4: A. GBP-1 regulation of cell proliferation is independent of Erk signaling. SKCO15 cells treated with TNF-/IFN- in addition to control siRNA or siRNA directed against GBP-1. B. As above, SKCO15 cells treated with TNF-/IFN- in addition to control siRNA or siRNA directed against GBP-1. Cyclin D1 levels were assessed by immunoblot.
SF5: GBP-1 does not alter -catenin transcription. -catenin transcript levels were determined by real time PCR. Values shown are normalized to GAPDH under control and GBP-1 overexpression conditions.
SF6: GSK3- inhibition fails to rescue GBP-1 induced suppression of -catenin/TCF transcription. SKCO15 cells stably expressing GBP-1 or GTPase defective GBP-1 were transfected with TCF/lef reporter constructs and treated with a GSK3- inhibitor (AR-A014418, 8hrs 10M).
SF7: TNF-/IFN-γ treatment (48hrs) results in decreased cell proliferation relative to non-treated control SCKO15 cells. Cell proliferation was determined by ELISA-based BrdU incorporation assay after normalization to total protein (n=6, p<0.1).