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. Author manuscript; available in PMC: 2013 Dec 15.

Published in final edited form as: Int J Cancer. 2012 May 14;131(12):2820–2831. doi: 10.1002/ijc.27598

BMAL1 knockdown induces dramatic morphological alterations in ACC-MESO-1 cells. (a) H–E stain showing the nuclear morphological changes identified in ACC-MESO-1 cells after ablation of BMAL1. (b) IF of α-tubulin and DAPI stains. The upper panels represent ACC-MESO-1 cells treated with siRNA control. The lower panels represent the most frequent morphological changes (arrow indicates micronucleation and arrow head indicates multiple nuclei) in single ACC-MESO-1 cell after BMAL1 depletion. The middle panels represent ACC-MESO-1 cells treated with BMAL1 siRNA. (c) Quantification of the binuclear, multinuclear and micronuclear phenotypes in ACC-MESO-1 cells after BMAL1 knockdown. (d) Cell cycle profiling of ACC-MESO-1 cells showing marked decrease of BMAL1-induced cell death and polyploidy formation following mitosis block. ACC-MESO-1 cells transfected with BMAL1 siRNA or control oligos with double-thymidine (black arrows) and without thymidine treatment (arrow heads) were harvested for analyses of DNA content by flow cytometry. Synchronized cells at late G1/early S by double-thymidine escaped from BMAL1 knockdown-induced cell death with marked reduction in subG1 and polyploidy formation. (e) Time lapse microscopic examination showing the aberrant mitosis in ACC-MESO-1 cells transfected with BMAL1 siRNA (white arrow head) and intact mitosis in cells transfected with control oligos (white arrow). (f) Proposed molecular mechanism of BMAL1 knockdown-induced mitotic catastrophe in ACC-MESO-1 cells. Decreasing cyclin B below a critical level results in mitosis skipping and enhances polyploidy formation. Downregulation of p21WAF1/CIP1 results in accumulation of cyclin E which could lead to increased number of polyploidy cells. Wee1 downregulation could contribute to eventual escape from G2/M arrest, which in turns participates in impairment of mitotic integrity. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

BMAL1 knockdown induces dramatic morphological alterations in ACC-MESO-1 cells. (a) H–E stain showing the nuclear morphological changes identified in ACC-MESO-1 cells after ablation of BMAL1. (b) IF of α-tubulin and DAPI stains. The upper panels represent ACC-MESO-1 cells treated with siRNA control. The lower panels represent the most frequent morphological changes (arrow indicates micronucleation and arrow head indicates multiple nuclei) in single ACC-MESO-1 cell after BMAL1 depletion. The middle panels represent ACC-MESO-1 cells treated with BMAL1 siRNA. (c) Quantification of the binuclear, multinuclear and micronuclear phenotypes in ACC-MESO-1 cells after BMAL1 knockdown. (d) Cell cycle profiling of ACC-MESO-1 cells showing marked decrease of BMAL1-induced cell death and polyploidy formation following mitosis block. ACC-MESO-1 cells transfected with BMAL1 siRNA or control oligos with double-thymidine (black arrows) and without thymidine treatment (arrow heads) were harvested for analyses of DNA content by flow cytometry. Synchronized cells at late G1/early S by double-thymidine escaped from BMAL1 knockdown-induced cell death with marked reduction in subG1 and polyploidy formation. (e) Time lapse microscopic examination showing the aberrant mitosis in ACC-MESO-1 cells transfected with BMAL1 siRNA (white arrow head) and intact mitosis in cells transfected with control oligos (white arrow). (f) Proposed molecular mechanism of BMAL1 knockdown-induced mitotic catastrophe in ACC-MESO-1 cells. Decreasing cyclin B below a critical level results in mitosis skipping and enhances polyploidy formation. Downregulation of p21WAF1/CIP1 results in accumulation of cyclin E which could lead to increased number of polyploidy cells. Wee1 downregulation could contribute to eventual escape from G2/M arrest, which in turns participates in impairment of mitotic integrity. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]