Journal: Clinical and Translational Medicine

Article Title: DNTTIP1 drives leukaemogenesis through MiDAC‐mediated epigenetic silencing of BMF

doi: 10.1002/ctm2.70603

Figure Lengend Snippet: Histone deacetylase 1/2 (HDAC1/2) are critical for deoxynucleotidyl transferase terminal‐interacting protein 1 (DNTTIP1)‐mediated leukaemogenesis. (A and B) Co‐immunoprecipitation (Co‐IP) analysis in RS4;11 (A) and MV4;11 (B) cells stably expressing HA‐tagged DNTTIP1. Cell lysates were immunoprecipitated with anti‐HA (top) or anti‐HDAC1 (bottom) antibodies, followed by immunoblotting with the indicated antibodies. Input lanes represent 5% of total lysate ( n = 3). (C) Proximity ligation assay (PLA, red) detecting endogenous DNTTIP1‒HDAC1 interaction in RS4;11 (top) and MV4‐11 (bottom) cells. Scale bar = 20 µM ( n = 3). (D and E) GST/His‐tagged pull‐down assay showing the interaction between DNTTIP1 and HDAC1. (F) Immunoblot (left) and RT‐qPCR (middle) analyses demonstrating HDAC1 knockdown using two independent short hairpin RNAs (shRNAs) (shHDAC1_1 and shHDAC1_2). Cell counting of RS4;11 cells expressing shHDAC1_1 and shHDAC1_2 compared with control (pLKO) (right) ( n = 3). (G) Flow cytometric analysis of Annexin V/propidium iodide (PI) staining in RS4;11 cells with HDAC1 knockdown (shHDAC1_1 and shHDAC1_2) versus control (pLKO) ( n = 3). (H) Flow cytometric analysis was performed to assess cell cycle distribution in RS4;11 cells with HDAC1 knockdown (shHDAC1_1 and shHDAC1_2) versus control (pLKO) ( n = 3). (I) Immunoblot analyses demonstrating the rescue effect of DNTTIP1 overexpression (DNTTIP1_5) upon HDAC1 knockdown (shHDAC1_2) cells (left). Cell counting analysis demonstrating that overexpression of DNTTIP1 (DNTTIP1_5) partially rescues the growth inhibition induced by HDAC1 knockdown (shHDAC1_2) in RS4;11 cells (middle). Quantification of early (Annexin V + PI − ) and late (Annexin V + PI + ) apoptotic populations in RS4;11 cells with DNTTIP1 overexpression on HDAC1 knockdown ( n = 3). shHDAC1_2 + DNTTIP1_5 versus shHDAC1_2 + EV is denoted by symbol (#). All groups versus pLKO + EV is denoted by symbol (*). (K) Reciprocal Co‐IP analysis in RS4;11 cells demonstrated endogenous interaction between DNTTIP1 and HDAC2 ( n = 3). (L) Immunoblot (left) and RT‐qPCR (middle) analyses demonstrating HDAC2 knockdown using two independent shRNAs (shHDAC2_1 and shHDAC2_3). Cell counting of RS4;11 cells expressing shHDAC2_1 and shHDAC2_3 compared with control (Scr) (right) ( n = 3). (M) Flow cytometric analysis of Annexin V/PI staining in RS4;11 cells with HDAC2 knockdown (shHDAC2_1 and shHDAC2_3) versus control (Scr) ( n = 3). (N) Flow cytometric analysis was performed to assess cell cycle distribution in RS4;11 cells with HDAC2 knockdown (shHDAC2_1 and shHDAC2_3) versus control (Scr) ( n = 3). (O) Immunoblot analysis demonstrating the efficiency of HDAC1/2 double knockdown (left). Cell counting analysis showing that HDAC1/2 double knockdown inhibited proliferation more potently than individual knockdown of either HDAC1 or HDAC2 (middle). Quantification of early (Annexin V + PI − ) and late (Annexin V + PI + ) apoptotic populations in RS4;11 cells with HDAC1/2 double knockdown ( n = 3). All groups versus pLKO + Scr is denoted by symbol (*). (P) Schematic diagram of DNTTIP1 domains (top). HEK‐293T cells were transiently transfected with HA‐tagged DNTTIP1 deletion constructs and cultured for 24 h. Western blot analysis was performed on total cell lysates and immunoprecipitated fractions using HA‐specific antibodies (bottom) ( n = 3). (Q) Cell counting of RS4;11 cells transfected with HA‐tagged DNTTIP1 deletion constructs ( n = 3).

Article Snippet: Controls included GST protein (MCE, HY‐ P70270 ) substituting GST‐HDAC1 (GST pull‐down) and empty NTA‐Ni beads (His pull‐down).

Techniques: Histone Deacetylase Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Western Blot, Proximity Ligation Assay, Pull Down Assay, Quantitative RT-PCR, Knockdown, Cell Counting, Control, Staining, Over Expression, Inhibition, Transfection, Construct, Cell Culture