Bhatnagar et al, 2010 Cell Death and Disease Manuscript # CDDIS T

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1 Bhatnagar et al, Cell Death and Disease Manuscript # CDDIS--98-T Supplemental Materials. Supplemental Figure Legends Supplemental Figure (A) WPE-NA and WPE-NB6 cells were treated with 4 nm of Docetaxel or 4 μg/ml of Cisplatin for 4h and apoptosis was analyzed by annexin V staining as described in Materials and Methods. All experiments have been repeated three times, data shown are mean values + SD (p<., *p<.). (B) Proteins were isolated from the indicated cells, western blots were done using indicated antibodies. Supplemental Figure Various prostate cancer cells were treated with the indicated doses of Docetaxel or Cisplatin for 4 hours and apoptosis was analyzed using the Cell Death Detection Elisa PLUS kit as described in Materials and Methods. All experiments have been repeated three times, data shown are mean values + SD. Supplemental Figure 3 (A) Proteins were isolated from the indicated cell lines, western blots were done using indicated antibodies. (B) WPE-NA cells were transfected with negative control or anti-mir-5, anti-mir-3 inhibitors. 4h after transfection, cells were treated with 4 nm Docetaxel for 4h. Apoptosis was analyzed using the Cell Death Detection Elisa PLUS kit. All experiments have been repeated three times, data shown are mean values + SD (*p<.5). Supplemental Figure 4 WPE-NA and WPE-NB6 cells were treated with 4 nm of Docetaxel or 4 μg/ml of Cisplatin for 4 hours. Total RNA was isolated from the cells and real-time PCR analysis was performed as described in Materials and Methods. (A) WPE-NB6 cells. (B) WPE-NA cells. Data shown are mean values + SD from two independent experiments, (*p<.5, p<.). Supplemental Figure 5 (A) Empty vector transfected or Bcl-w stable expressing WPE-NA cells were treated with 4 nm of Docetaxel or 4 μg/ml of Cisplatin for 4h, and apoptosis was measured by annexin V staining. (B) Empty vector transfected or EF6 stable expressing WPE-NA cells were treated with 4 nm of Docetaxel or 4 μg/ml of Cisplatin for 4h, and apoptosis was measured by annexin V staining. (C) Negative control mirna or mir-5 stable expressing WPE-NB6 cells were treated with 4 nm of Docetaxel or 4 μg/ml of Cisplatin for 4h, and apoptosis was measured

2 by annexin V staining. (D) Negative control mirna or mir-3 stable expressing WPE- NB6 cells were treated with 4 nm of Docetaxel or 4 μg/ml of Cisplatin for 4h, and apoptosis was measured by annexin V staining. The experiments have been repeated three times, data shown are mean values + SD (p<., *p<.). Supplemental Figure 6 (A) Empty vector transfected or three independent clones of Bcl-w stable expressing WPE-NA cells were treated with 4 nm of Docetaxel or 4 μg/ml of Cisplatin for 4h, and apoptosis was analyzed using the Cell Death Detection Elisa PLUS kit. (B) Empty vector transfected or three independent clones of EF6 stable expressing WPE-NA cells were treated with 4 nm of Docetaxel or 4 μg/ml of Cisplatin for 4h, and apoptosis was analyzed using the Cell Death Detection Elisa PLUS kit. (C) Negative control mirna or three independent clones of mir-5 stable expressing WPE-NB6 cells were treated with 4 nm of Docetaxel or 4 μg/ml of Cisplatin for 4h, and apoptosis was analyzed using the Cell Death Detection Elisa PLUS kit. (D) Negative control mirna or three independent clones of mir-3 stable expressing WPE-NB6 cells were treated with 4 nm of Docetaxel or 4 μg/ml of Cisplatin for 4h, and apoptosis was analyzed using the Cell Death Detection Elisa PLUS kit. The experiments have been repeated three times, data shown are mean values + SD (*p<.5, p<., *p<.).

3 Table mirnas that are differentially expressed between WPE-NA and WPE-NB6 cells mirna Ratio (WPE- NB6/WPE- NA) % of Increase (+) or Decrease (-) mir-7a mir-3a-5p mir mir mir mir mir mir-6a mir mir

4 RWPE- WPE- NB NB4 NB6 kd NA NB6 * Bcl-xL 3. Mcl XIAP 5. Bid 5. Bax Tubulin 59. Bhatnagar, et al. Supplemental Figure

5 Absorbance 45 nm RWPE- NB4 NB 4 Cisplatin (μg/ml) C. D. Absorbance 45 nm Rv Du45 PC-3 4 Cisplatin (μg/ml) Absorbance 45 nm RWPE- NB4 NB 4 Absorbance 45 nm Rv Du45 PC-3 4 Docetaxel (nm) Docetaxel (nm) Bhatnagar, et al. Supplemental Figure

6 Bcl-w EF6 Tubulin kd Absorbance 45 nm NC Inhi mir-5 Inhi mir-3 Inhi * * Control Docetaxel Bhatnagar, et al. Supplemental Figure 3

7 Relative mirna levels mir-3 mir-5 * Ctr Cisplatin Docetaxel Relative mirna levels mir-3 mir-5 Ctr Cisplatin Docetaxel Bhatnagar, et al. Supplemental Figure 4

8 5 4 3 Vec Bcl-w * Vec EF6 C. D mir NC mir-5 * mir NC mir-3 Ctr Cisplatin Docetaxel Ctr Cisplatin Docetaxel Bhatnagar, et al. Supplemental Figure 5

9 Absorbance 45 nm Vec Bcl-w. Bcl-w. Bcl-w.3 * * Absorbance 45 nm Vec EF6. EF6. EF6.3 * C. Absorbance 45 nm NC mir-5. mir-5. mir-5.3 D. Absorbance 45 nm NC mir-3. mir-3. mir-3.3 * * Bhatnagar, et al. Supplemental Figure 6

Pro-apoptotic signalling through Toll-like receptor 3 involves TRIF-dependent

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