Fig. 6

CHTM1 involves p38-AIF1 to modulate metabolic stress-induced cell death and is deregulated in human lung cancers. CHTM1-knockdown and scrambled A549 cells were glucose/glutamine starved for 4 h in the presence and absence of p38 kinase inhibitor SB203580. (a, left panel) Representative phase contrast photomicrographs, and (Right panel) crystal violet staining showing glucose/glutamine starvation-induced cell death was abrogated by p38 inhibitor SB203580 in CHTM1-deficient A549 cells. (b) Trypan blue exclusion assay showing metformin-induced cell death was prevented by p38 inhibitor SB203580 in CHTM1-deficient A549 cells. (c) Western blot analyses showing p38 inhibition blocks glucose/glutamine starvation-induced cytosolic accumulation of AIF1 in CHTM1 knockdown A549 cells (compare lanes 8&9 with lanes 11&12). (d) Representative Western blot showing CHTM1 expression in matching tumor (T) and adjacent normal (N) from same lung cancer patients. Same blot was also probed with anti-Sam50, another mitochondrial protein. As is shown, Sam50 does not show expression pattern similar to CHTM1, suggesting that the increase in CHTM1 is not due to generalized increase in mitochondrial contents. Samples were obtained from the Cooperative Human Tissue Network, an NCI supported network. (e) Immunohistochemistry-based detection of CHTM1 (brown color) in representative normal and tumor tissues from lung cancer patients. Samples were also stained with hematoxylin (blue color). Scale bar, 50 μM. Samples were purchased from Biomax (Rockville, MD) as formalin-fixed, paraffin-embedded tissue array slides. (f) Overall results of CHTM1 overexpression in lung cancer samples compared to matched normal tissue samples