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. 2017 May 24;9(7):933–949. doi: 10.15252/emmm.201607180

Figure EV3. Epigenetically down‐regulated THAP10 can be reactivated by DNMT inhibitors and HDAC inhibitors.

Figure EV3

  1. Upper panel: Relative quantification of THAP10 levels in U937 (Mock and A/E‐HA) cells. The results represent the average of three independent evaluations ± SD (*P = 0.038, # P = 0.008, two‐sided Student's t‐test was used for the comparisons). Middle panels: RNA levels of THAP10 analysed by agarose electrophoresis. GAPDH was used as a loading control. Lower panels: Protein levels of THAP10 and AML1‐ETO monitored by immunoblot analysis. β‐actin was used as a loading control. Treatment for 16 h with 100 μM Zn2+ was employed to induce the expression of AML1‐ETO.
  2. Human 293T cells were transiently co‐transfected for 48 h with luciferase reporter containing wild‐type sequences of the THAP10 promoter regions, together with increasing amounts (10, 50 and 100 ng) of pcDNA3.0 with AML1‐ETO cDNA or without (293T‐Mock). Data represent the mean of three independent evaluations ± SD for luciferase activity relative to the empty vector (pGL3‐LUC) control. pRL‐TK was used as an internal control.
  3. Chromatin immunoprecipitation (ChIP) using the indicated antibodies or IgG in AML blasts, after which qRT–PCR was performed to evaluate the specificity of protein binding. Triplicate values are defined as mean ± SD.
  4. SKNO‐1 and Kasumi‐1 cells were treated with the DNMT inhibitor 5‐azacytidine (5‐Aza, 2.5 μM) +/− the HDAC inhibitor TSA (3.0 μM) for 40 h, after which RT–PCR was performed to detect the mRNA levels of THAP10. Data represent the normalized values (mean ± SD) of relative THAP10 expression from three independent experiments. Normalized THAP10 level in SKNO‐1 cells was arbitrarily set to 1.
  5. AML‐ETO+ patient blasts were treated with the DNMT inhibitor decitabine (Dac, 2.5 μM) +/− the HDAC inhibitor chidamide (Chi, 3.0 μM) for 40 h, after which RT–PCR was performed to detect the mRNA levels of THAP10. Data represent the normalized values (mean ± SD) of relative THAP10 expression from three independent experiments. Normalized THAP10 level in SKNO‐1 cells was arbitrarily set to 1.
  6. In primary blasts from clinical trial (NCT02886559) patients, qRT–PCR detected the THAP10 levels right after completion of the first therapeutic program. Normalized THAP10 level in patient blasts was arbitrarily set to 1. Triplicate values are defined as mean ± SD.