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. 2022 Mar 28;60(5):54. doi: 10.3892/ijo.2022.5344

Figure 5.

Figure 5

HDAC6 knockdown (KD) enhances adavosertib-induced cell death in CAL27 cells. (A) Knockdown efficiency of HDAC6 in CAL27 cells with three different HDAC6 siRNAs was assessed by western blotting. (B) CAL27 cells were transfected with siLuc or three different siHDAC6 siRNAs (#2-4) and then treated with or without Adv (1 µM) for 24 h. Expression of DDR-related proteins was detected by western blotting. Expression of HDAC6 and acetylated (ac)-α-tubulin were monitored to assess the KD efficiency. Representative data of three independent experiments are shown. (C and D) CAL27 cells were transfected with siLuc or three different siHDAC6 siRNAs (#2-4) and then treated with various concentrations of Adv up to 24 h. Cells were monitored using IncuCyte live cell imaging system, and dead cell number was assessed using PI staining. Dose-dependent (C) and time-dependent (D) cell death numbers are shown. Representative data of three independent experiments are shown. n=3, bar, mean ± SD. *P<0.05 vs. siLuc. (E) The synergistic effect of cell death in the combination of Adv treatment and HDAC6 KD shown in (C) and (D) was assessed and is summarized. Adv, adavosertib; RCS, ricolinostat; HDAC6, histone deacetylase 6; DDR, DNA damage response.