Figure 4
Figure 4. Molecular changes induced by MLN8237 in cell cycle–regulatory and antitumor pathways. Molecular changes induced by MLN8237 in cell cycle–regulatory and antitumor pathways were analyzed in MM cell lines. MM cell lines were cultured in the absence or presence of DMSO or MLN8237 (0.5-1μM) for various times. Protein level changes were determined by either Western blotting or flow cytometry. (A) Relative expression of negative cell cycle–regulatory molecules PP1α and PP2αA in MM cell lines treated with MLN8237 (0.5μM) for the indicated times. Pixel density of each protein band was measured using ImageJ software (1.37v; National Institutes of Health, http://rsb.info.nih.gov/ij/) and normalized with GAPDH expression. Fold expression per control for each band is shown. *Statistical significance (t test, one-tailed distribution, P < .05; n = 3 independent experiments). (B) Intracytoplasmic expression of phospho(Thr183/Tyr185) SAPK/JNK and phospho(Thr180/Tyr182) MAPK in MM1.S cells after 1-hour exposure to DMSO or MLN8237 (0.5μM). Dotted line indicates isotypic control; blue line, DMSO; and red line, MLN8237 (0.5μM for 1 hour). (C) Molecular changes induced by MLN8237 in p53 pathway were determined by Western blotting in MM cell lines. MLN8237 induced expression of p53, p27, and p21 in MM cell lines. MM cell line OPM1 does not express p21. GAPDH was used as a control to determine total protein expression. (D) Intracytoplasmic expression of cell-cycle checkpoint molecules phospho(Tyr15) Cdc2, phospho(Ser345) Chk1, and phospho(Thr68) Chk2 were analyzed in MM1.S cells after 1-hour exposure to DMSO or MLN8237 (0.5μM). Dotted line indicates isotypic control; blue line, DMSO; and red line, MLN8237 (0.5μM for 1 hour). (E) Molecular changes induced by MLN8237 in α-tubulin activation and formation were analyzed by Western blotting in MM cell lines. MLN8237 induced expression of α-tubulin in MM cell lines at early exposure time, whereas there was decreased expression of α-tubulin after 24 hours of MLN8237 treatment. GAPDH was used as a control to determine total protein expression.

Molecular changes induced by MLN8237 in cell cycle–regulatory and antitumor pathways. Molecular changes induced by MLN8237 in cell cycle–regulatory and antitumor pathways were analyzed in MM cell lines. MM cell lines were cultured in the absence or presence of DMSO or MLN8237 (0.5-1μM) for various times. Protein level changes were determined by either Western blotting or flow cytometry. (A) Relative expression of negative cell cycle–regulatory molecules PP1α and PP2αA in MM cell lines treated with MLN8237 (0.5μM) for the indicated times. Pixel density of each protein band was measured using ImageJ software (1.37v; National Institutes of Health, http://rsb.info.nih.gov/ij/) and normalized with GAPDH expression. Fold expression per control for each band is shown. *Statistical significance (t test, one-tailed distribution, P < .05; n = 3 independent experiments). (B) Intracytoplasmic expression of phospho(Thr183/Tyr185) SAPK/JNK and phospho(Thr180/Tyr182) MAPK in MM1.S cells after 1-hour exposure to DMSO or MLN8237 (0.5μM). Dotted line indicates isotypic control; blue line, DMSO; and red line, MLN8237 (0.5μM for 1 hour). (C) Molecular changes induced by MLN8237 in p53 pathway were determined by Western blotting in MM cell lines. MLN8237 induced expression of p53, p27, and p21 in MM cell lines. MM cell line OPM1 does not express p21. GAPDH was used as a control to determine total protein expression. (D) Intracytoplasmic expression of cell-cycle checkpoint molecules phospho(Tyr15) Cdc2, phospho(Ser345) Chk1, and phospho(Thr68) Chk2 were analyzed in MM1.S cells after 1-hour exposure to DMSO or MLN8237 (0.5μM). Dotted line indicates isotypic control; blue line, DMSO; and red line, MLN8237 (0.5μM for 1 hour). (E) Molecular changes induced by MLN8237 in α-tubulin activation and formation were analyzed by Western blotting in MM cell lines. MLN8237 induced expression of α-tubulin in MM cell lines at early exposure time, whereas there was decreased expression of α-tubulin after 24 hours of MLN8237 treatment. GAPDH was used as a control to determine total protein expression.

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