Supplementary Figure 1. Relative T-type Ca2+ channel gene expression in (A) A2780 and (B) A2780Cis cells 72 hours after transfection with specific siRNAs normalized to control (siSCRMBL). Graph represents data from ≥ three independent experiments.

Supplementary Figure 2. Down regulation of T-type Ca2+ channel gene expression with specific siRNAs decreases survivin protein level. A2780Cis cells were transfected with selected siRNA for 72 hours as described in Materials and Methods. Whole cell extracts were isolated and analyzed by immunoblot using specific antibodies to the indicated proteins.

Supplementary Figure 3. Effects of Mibefradil on AKT activity and its role in survivin stabilization. (A) Cells were treated with 10 µmol/L Mib for 1 hour and then stimulated with insulin (10 µg/mL) for the indicated times. AKT activity was confirmed by detecting phosphorylation of its direct substrates of AKT kinase, GSK-3β serine 9 (GSK- 3β pS9) and PRAS40 threonine 246 (PRAS40 pT246). (B) Increased AKT phosphorylation, FoxM1 expression and survivin expression in cells co-treated with 10 µmol/L Mib for 1 hour in the presence of Calyculin A (CalA, 10 nmol/L).

Supplementary Figure 4. Effects of carboplatin and T-type Ca2+ channel inhibition on survivin expression. (A) A2780 and A2780Cis cells were treated with the indicated concentrations of Mib, carboPt or the combination for 24 hours prior to immunoblotting for survivin, PARP or p38 (loading control). (B) A2780 cells were transfected with selected siRNA and 48h post-transfection treated with the indicated concentrations of carboPt for 24h prior to immunoblotting for survivin or GAPDH (loading control). (C) A2780Cis and A2780 cells were treated with either 6 μmol/L Mib, 30 μg/ml carboPt or the combination for 24 hours and blotted with antibodies to the indicated proteins.

Supplementary Figure 4.

Supplementary Table 1. Sequence of siRNA targeted against T-type channel subunit CACNA1 (Life Technologies).

Name / Sequence / Accession Number
siCACNA1G-ORF (1) / 5'-UUGUAGAGGACUUUGUUCC / NC_000017.11
5'-GGAACAAAGUCCUCUACAA
siCACNA1G-ORF (2) / 5'-AUUUCCUCCAGCGUGAUGC / NC_000017.11
5'-GCAUCACGCUGGAGGAAAU
siCACNA1H-ORF (1) / 5'-UGCAGAGCCUGGUGAGAAA / NC_000016.10
5'-UUUCUCACCAGGCUCUGCA
siCACNA1H-ORF (2) / 5'-GCAACAUCCUGGAGGCCUU / NC_000016.10
5'-AAGGCCUCCAGGAUGUUGC
siSCRMBL / 5'-GACGAAAGACCACUCAAUU / -
(non-targeted) / 5'-AAUUGAGUGGUCUUUCGUC

Supplementary Table 2. Sequence of primers designed for RT-qPCR amplification for gene expression.

Name / Sequence / Accession
Number / PCR Product (bp)
CACNA1G_F / AAAGAGGCTGGTGAAGACGA / NM_018896.3 / 101
CACNA1G_R / TCCTGGTCAACACACTCAGC
CACNA1H_F / TCGAGGAGGACTTCCACAAG / NM_001005407.1 / 176
CACNA1H_R / TGCATCCAGGAATGGTGAG
CACNA1I_F / AGGATGAGCTATGACCAGCG / NM_001003406 / 151
CACNA1I_R / CAGAGAGCAGGGACTCATGC
BIRC5_F / TCCGCAGTTTCCTCAAATTC / NM_001012271.1 / 139
BIRC5_R / GTTGCGCTTTCCTTTCTGTC
GAPDH_F / TTGAGGTCAATGAAGGGGTC / NM_002046.5 / 117
GAPDH_R / GAAGGTGAAGGTCGGAGTCA
b-actin_F / GTTGTCGACGACGAGCG / NM_001101.3 / 93
b-actin_R
b-actin_F / GTTGTCGACGACGAGCG / NM_001101.3 / 93
b-actin_R / GCACAGAGCCTCGCCTT
/ GCACAGAGCCTCGCCTT
GUS_F / CCGACTTCTCTGACAACCGACG / NM_001293104.1 / 147
GUS_R / AGCCGACAAAATGCCGCAGACG

Supplementary Table 3. Sequence of primers designed for amplification of genomic DNA at the BIRC5 promoter containing and in close proximity to FoxO binding sequence (BS, Fig 3D).

Name / Sequence / Accession Number / PCR Product (bp)
BIRC5_M1(3)F / CATGGTGAAACCCCATCTCT / NG_029069.1 / 242
BIRC5_M1(3)R / CGCCCCCATTCTTTAATACA

Supplementary Table 4. Combination indexes calculated for sequential treatment with the indicated concentrations of Mibefradil and carboPt in selected ovarian cancer cell lines (Fig 4A).

Combination Index (CI) ± SEM
Mib 1 µmol/L / Mib 3 µmol/L / Mib 6 µmol/L
Cell line / carboPt (1 µg/mL)
A2780 / 1.0 ± 0.22 / 0.89 ± 0.05 / 1.6 ± 0.21
A2780Cis / 0.34 ± 0.01 / 0.58 ± 0.026 / 0.6 ± 0.1
IGROV-1 / 0.25 ± 0.07 / 0.17 ± 0.08 / 0.8 ± 0.15
SKOV3.ip1 / 1.1 ± 0.26 / 1.0 ± 0.22 / 1.2 ± 0.26
OVCAR-3 / 0.4 ± 0.09 / 0.33 ± 0.01 / ND
ES2 / 0.8 ± 0.64 / 0.6 ± 0.41 / ND

Data was analyzed using Chou-Talalay method (as described in Material & Methods) and represent averaged values from ≥ two independent experiments ± SEM, ND- no data. CI<1 indicates drug synergy, CI=1 indicates additive effect, CI>1 indicates drug antagonism. CIs were derived from the equation: , where fa- fraction affected, fu- fraction unaffected, D-dose, Dm- the dose that produces 50% growth inhibition, m- shape of the curve (slope).