Supplementary Information

Targeting IκB kinase β/NF-κB signaling in human prostate cancer by a novel IκB kinase β inhibitor CmpdA

Supplementary Figure S1. IKKβ is activated by Akt, mTORC1 and IKKα in prostate cancer PC3 cells.

A.Proposed model indicating IKKβ/NF-κBactivation by Akt, mTORC1 and IKKα. B. Effects of the Akt inihibitor perifosine on Akt, mTORC1, IKKα, IKKβ and NFκB phosphorylation. PC3 cells were treated with DMSO or perifosine (15 or 30 μM) for 2 hours, lysed and Western blots were probed with the indicated antibodies. C. PC3 cells were transfected with IKKα siRNA for 72 hours and analyzed by Western blot. D. PC3 cells were transfected with control or IKKβ siRNA for 72 hours andanalyzed by Western blot. Allresults are representative of three independent experiments.

Supplementary Figure S2. Expressions of EMT- and stemness-related factors in tissue microarrays representative of different stages of prostate cancer. Related to main figure 2.The tissue microarrays represent normal prostate tissue and primary prostate tumors from patients with the following clinical stages: stage II (T2N0M0), stage IV (T3N0M1) and stage IV (T4N1M1).

Supplementary Figure S3. Inhibition of IKKβ/NF-κB by CmpdA in DU145 cells. Related to main figure 3.

A and B. DU145 cells were treated with vehicle control or CmpdA (2 or 5 µ) for 0.5 - 48 hours and phosphorylation status ofIκBα and p65 was examined by Western blot. The results are representative of three independent experiments. C. Cells were treated with different doses of CmpdA for 48 hours and cell morphology was determined. The experiments were repeatedthree times. D. DU145 cells were treated with different doses of CmpdA for 48 hours and cell proliferation was determined by MTT assay (*, P < 0.05). E. Cells were treated with CmpdA at different doses for 48 hours and caspase activity was measured. The experiments were repeated three times(*, P < 0.05; ** P < 0.01; *** P < 0.001). F. Cells were seeded in 6-well plates and treated with CmpdA for 7 days, and colony formation was assessed. G. Cells were treated with CmpdA for 48 hours and the expressions of Survivin, cleaved-caspase-3 and GAPDH were determined. H. Cells were treated with CmpdA at different doses for 48 hours and apoptosiswas examined via flow cytometry.

Supplementary Figure S4. CmpdA inhibits DU145 cell migration. Related to main figure 5.

A. DU145 cells were treated with vehicle control or CmpdA and wound closure was monitored after scratches were made in the monolayers. B. Cells were treated with vehicle control or CmpdA and migration was monitored by the xCELLigence System real-time cell analyzer instrument. C. Cells were treated with different doses of CmpdA for 24 or 48 hours and the expressions of Snail and Slug were determined by Western blot.

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