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. 2020 Aug 6;295(40):13887–13901. doi: 10.1074/jbc.RA120.015288

Figure 7.

Figure 7.

Deregulation of FAAP20 acetylation impairs FANCD2 activation. A, left panel: defective FANCD2 activation upon depletion of p300 or CBP. U2OS cells transfected with the indicated siRNA were treated with 100 ng/ml of MMC for 6 h, and cell lysates were analyzed by Western blot. Where indicated, cells were transfected with pcDNA3–Flag–FAAP20 K83Q/K152Q (versus empty vector) for 40 h before MMC treatment. Right panel: quantification of the FANCD2–Ub:FANCD2 ratios (L:S), via Fiji (ImageJ). Error bar indicates mean ± S.D., n = 3 from three independent experiments, *, p < 0.05, ns, not significant, Student's t test. B, representative FACS plots of EdU cell cycle analysis. EdU+ S phase cells are gated, and % cell populations are represented as an average from two independent experiments. C, the FAAP20 KQ cannot rescue the FANCD2 activation defect caused by co-depletion of p300 and CBP. U2OS cells transfected with the indicated siRNA were treated with 100 ng/ml of MMC for 6 h, and cell lysates were analyzed by Western blot. Where indicated, cells were transfected with pcDNA3–Flag–FAAP20 KQ (versus empty vector) for 40 h before MMC treatment. D, depletion of p300 and CBP accelerates the degradation of endogenous FAAP20. U2OS cells transfected with siRNA control or p300 and CBP together were treated with 100 μg/ml of CHX for the indicated times, and endogenous FAAP20 levels were analyzed by Western blot. siRNA FAAP20-transfected cells were used for control to confirm the specificity of FAAP20 signals. E, left panel: defective FANCD2 activation upon overexpression of HDACs. U2OS cells were co-expressed with Flag–HDAC2 and HDAC3 along with an empty vector or Flag–FAAP20 KQ, and cell lysates were analyzed by Western blot. Right panel: quantification of the FANCD2–Ub:FANCD2 ratios (L:S), via Fiji (ImageJ). Error bar indicates mean ± S.D., n = 2 from two independent experiments, *, p < 0.05, ns, not significant, Student's t test. F, U2OS cells transfected with the indicated plasmids were treated with 1 μm MMC for the indicated times, and chromatin-enriched fractions were subjected to anti-Flag IP, followed by Western blot using acetyl-Lys specific and FAAP20 pS113 CPD antibodies.