FIGURE 6.

Acetylation of GAPDH at K254 promotes tumor cell proliferation. A, verification of GAPDH expressing A549 stable cell lines. A549 cells stably expressed GAPDH^WT and GAPDH^K254Q were transfected with shRNA-resistant WT or K254Q of GAPDH thus the knockdown and put back stable cells were generated. GAPDH knockdown efficiency and re-expression level were determined by Western blot. B, GAPDH^K254Q is compromised to support cell proliferation. GAPDH^WT and GAPDH^K254Q cells were seeded as the same number in each well. Cell numbers were counted every 24 h. Error bars represent cell numbers ±S.D. for experiments performed in triplicate. C, absorbance at 450 nm was measured after adding CCK-8 solution to each well of the plate and incubating for 2 h. Error bars represent cell numbers ±S.D. for experiments performed in triplicate. D, expression of GAPDH^WT and GAPDH^K254Q in xenograft. Whole cell lysate were prepared from either original stable A549/GAPDH^WT and A549/GAPDH^K254Q pools or xenograft tumors, followed by Western blot. E—G, GAPDH^K254Q is defective in supporting tumor growth in vivo. Xenograft was performed using the A549 cell lines characterized in A. 7 weeks after injection, mice were sacrificed and tumors were extracted and photographed (E). Tumor diameters were measured, and the volumes were calculated (F). Tumor weight was measured (G). The p value was calculated by paired t test.