Fig. 5

Knockdown of HDAC5 or HDAC6 induced cell cycle arrest. a and b, Cells were passaged in 12-well plates for 24 h. Serum was removed from the medium, and cells were starved with serum-free medium for 24 h. Then, the cells were cultured with fresh medium with serum at four time points: 0, 4, 8 and 16 h. After harvesting and washing with PBS, all of the A375 cells were fixed in ice-cold 70 % EtOH for more than 2 h at 4 °C, centrifuged at 2000 rpm for 5 min, then washed twice in PBS. The cells were incubated at 37 °C Rnase solution (1 mg/ml) for less than 30 min and then stained by propidium iodide (50 µg/ml) for cell cycle analysis with Flow Cytometers Galios (Beckman, USA) using a 488 nm excitation wavelength, gating doublets and clumps using pulse processing and collecting fluorescence above 620 nm. Data were extracted with Modfit software.*p < 0.05 was considered as significant difference