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-κB activation 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 and analyzed by Western blot. All results 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.

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A and B. DU145 cells were treated with vehicle control or CmpdA (2 or 5 µ) for 0.5 - 48 hours

and phosphorylation status of IκBα 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 repeated three

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 apoptosis was

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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Supplementary Figure S1.

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Supplementary Figure S2.

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Supplementary Figure S3.

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Supplementary Figure S4.

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