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. 2019 May 23;9(5):937–951. doi: 10.1016/j.apsb.2019.05.001

Figure 5.

Figure 5

HDACi synergizes with ARS to induce ALAS1 expression. (A)–(B) Western blot validation of heme biosynthetic enzyme ALAS1 in Huh-7 cell and Hep3B cell in the absence and presence of ARS, HDACi [SAHA (A) or LBH589 (B)], or both treatment for 24 h. β-Actin served as loading control. (C) Time courses of ALAS1 of Hep3B cell received ARS, LBH589, or both treatments. The values under each blot are the ALAS1 expression relative to controls at the same time taken as 1 after normalization by β-actin. (D) Real-time PCR was performed for ALAS1 of Huh-7 cell and Hep3B cell in the absence (normalized as 1) and presence of ARS, HDACi (SAHA or LBH589), or both treatment for 24 h. (E) Hep3B cells were transiently transfected with three ALAS1 siRNA or control siRNA (si NC) or remained untreated (control). Knock down efficiency of ALAS1 was examined by Real-time PCR analysis (upper) and Western blot assay (lower). GAPDH served as loading control. (F) Hep3B cells were treated with siRNA as described for (E) 24 h prior to administrate with ARS, SAHA or the combination as indicated for 48 h. Cell viability was measured by MTT assay. (G)–(H) Huh-7 cells were treated as described for (E) and (F). ALAS1 knockdown significantly blocked synergistic antitumor effect of ARS and SAHA. *P < 0.05, **P < 0.01, ***P < 0.001 (compared as indicated), #P < 0.05, ##P < 0.01 (compared with si NC group treated with ARS), †P < 0.05, ††P < 0.01, †††P < 0.001 (compared with si NC group treated with ARS + SAHA) (Student's t-test).