Fig. 6

Increased anoikis resistance in ovarian cancer cells is involved intrinsic apoptotic pathway. a The Affymetrix GeneChip 3' Expression Array revealed a panel of apoptosis-related genes (Fold change >2) induced in KLF12 knockdown OVCA433 cells which showed increased anoikis resistance. b Proteome Profiler (Human Apoptosis Array Kit) showed that 8 out of 35 Intrinsic Apoptosis-related factors were commonly upregulated in miR-141 enforced expression or KLF12 knockdown OVCA433 cells. c A schematic diagram illustrates that there are several Sp1 binding sites located along the promoter of survivin, confirming Sp1 is able to transcriptionally upregulate survivin. KLF12 is able to interfere the interaction of Sp1 with the promoter of survivin and the transcriptional activity. d ChIP assay showed the competitive occupancy of Sp1 by KLF12 on the promoter of survivin. (Upper) Position weight matrix (PWM) for SP1-binding sequence motif derived from Jaspar database. ChIP assays showed the occupancy of Sp1 on 3 SBEs (Red color) of the Survivin promoter in A2780cp cells were inhibited by KLF12. (Lower) Schematic diagram shows the positions of seven higher binding affinity SBEs on the survivin promoter. e Luciferase reporter assay using Survivin promoter luciferase reporter construct (luc-Survivin) demonstrated that Sp1 could significantly elevate the luciferase reporter signals from ~3-5.5 folds in HEK293 and ~48 to 70% in A2780cp by Sp1 dose dependently (200 to 1000 ng/well) (Upper). Using luc-survivin and pCMV6-AC-GFP-Sp1 (600 ng/well) and co-transfected with varied amount of pCMV6-KLF12 (200–800 ng/well), results showed that the Sp1-upregulated survivin signals were remarkably reduced from 5 to 2 folds in HEK293, and from 158% to 45% in A2780cp by KLF12 in a dose-dependent manner (200 – 1000 ng/well). f Western blot analysis showed that transient transfection of Sp1 could upregulate, while KLF12 could inhibit, survivin and XIAP expressions in A2780cp and OVCA433 cells. g Western blot analysis confirmed that knockdown of Sp1 had no effect on KLF12 expression but significantly reduced the expression of survivin and XIAP in two Sp1-enriched ovarian cancer cell lines, OVCA433 and SKOV3