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Fig S1. Cerulenin induces apoptosis in U266 cells. (A) U266 cells were cultured with Cerulenin (50 &mgr;mol/l) for the indicated times, with or without Z-VAD-FMK (50 &mgr;mol/l) for 1 h pretreatment. Total cell lysates (20 &mgr;g/lane) were subjected to Western blotting using anti-caspase -8, -9, -3, PARP and &alpha-tubulin. FL, CF indicated as full length and cleaved form, respectively. (B, C) U266 cells were treated with indicated dose of Cerulenin for 24 h, with or without Z-VAD-FMK (25 or 50 mmol/l) 1 h pretreatment. Cytotoxicity was determined by MTT assay (B). Values represent mean ± SD of quadruplicate cultures. The percentage apoptotic cells was determined by flow-cytometric analysis for APO2.7 staining (C).

Fig S2. JNK inhibitor blocks Cerulenin-induced cytotoxicity in U266 cells. (A, B) U266 cells were cultured with indicated dose of Cerulenin for 24 h, with or without JNK inhibitor SP600215 (5 or 10 &mgr;mol/l) pretreatment for 1 h. Cytotoxicity was determined by MTT assay (A). Values represent mean ± SD of quadruplicate cultures. JNK inhibitor significantly blocks Cerulenin-induced cytotoxicity (P < 0.05). The percentage apoptotic cells was determined by flow-cytometric analysis for APO2.7 staining (B). (C) Total cell lysates (20 mg/lane) were subjected to Western blotting using anti-caspase -8, -9, -3, PARP and α-tubulin. FL, CF indicated as full length and cleaved form, respectively.

Fig S3. Analysis of inhibition by JNK inhibitor SP600215 and/or ZVAD-FMK in MM cells. (A, B) MM.1S and U266 cells were cultured with indicated dose of Cerulenin for 24 h, with or without JNK inhibitor SP600215 (10 &mgr;mol/l) and/or ZVAD-FMK (50 &mgr;mol/l) pretreatment for 1 h. Cytotoxicity was determined by MTT assay. Values represent mean ± SD of quadruplicate cultures. Although ZVAD-FMK does not affect cytotoxicity by Cerulenin, JNK inhibitor significantly blocks Cerulenin-induced cytotoxicity (P < 0.05).

Fig S4. Cerulenin enhances Bortezomib, Melphalan, and Doxorubicin-induced cytotoxicity in U266 cells. U266 cells were cultured for 24 h with indicated concentrations of Bortezomib (A), Melphalan (B), and Doxorubicin (C) [control media (&squ;), 1.25 nmol/l Bortezomib (&bsl00001;), 2.5 nmol/l Bortezomib (&bsl00001;), 3.75 nmol/l Bortezomib (&bsl00001;); control media (&squ;), 2.5 &mgr;mol/l Melphalan (&bsl00001;), 5 &mgr;mol/l Melphalan (&bsl00001;), 10 &mgr;mol/l Melphalan (&bsl00001;); control media (&squ;), 100 nmol/l Doxorubicin (&bsl00001;), 200 nmol/l Doxorubicin (&bsl00001;), 400 nmol/l Doxorubicin (&bsl00001;)], in the presence or absence of Cerulenin (0–25 &mgr;mol/l). Cell growth was assessed by MTT assays. Data represent mean (±SD) of triplicate cultures. Combination index (CI) of Cerulenin and these agents was analysed using CalcuSyn software.

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BJH_7114_sm_figs1.ppt659KSupporting info item
BJH_7114_sm_figs2.ppt675KSupporting info item
BJH_7114_sm_figs3.ppt54KSupporting info item
BJH_7114_sm_figs4.ppt66KSupporting info item

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