Figure 6.
MCM4 mediates YTHDC1 functions in AML cells. (A) Comparison of the expression level of MCM4 in patients with primary AML bearing various chromosomal translocations vs those in BM HSCs collected from healthy donors. The expression values (detected by using Affymetrix exon arrays) were log2-transformed. The P value was detected by 1-way analysis of variance followed by multiple comparisons vs the HSC group. Normal: AML with normal karyotype; Complex: AML with complex karyotype. (B) Growth curve of MOLM-13 cells expressed Scramble shRNA (Scr), MCM4 shRNA1# (M4sh1#), and MCM4 shRNA2# (M4sh2#); cells were counted every 2 days. The P value was detected by 1-way analysis of variance followed by multiple comparisons vs the Scr group at day 7. (C) Flow cytometric analysis of apoptosis frequency of MOLM-13 cells expressed Scramble shRNA (Scr), MCM4 shRNA1# (M4sh1#), and MCM4 shRNA2# (M4sh2#). Annexin V+ indicated the apoptotic cells. (D) Flow cytometric analysis of differentiated cell frequency of MOLM-13 expressed Scramble shRNA (Scr), MCM4 shRNA1# (M4sh1#), and MCM4 shRNA2# (M4sh2#). (E) Colony-forming units of MOLM-13 cells expressed Scramble shRNA (Scr), MCM4 shRNA1# (M4sh1#), and MCM4 shRNA2# (M4sh2#). Colony number was counted after 7 days of plating. (F) qRT-PCR analysis of MCM4 expression in MOLM-13 cells expressed empty vector control, YTHDC1 WT (YTHDC1 wt), or YTHDC1 W377A W428A mutant (YTHDC1 mut). (G) The mRNA half-life of MCM4 in MOLM-13 cells expressed empty vector control, YTHDC1 WT (YTHDC1 wt), or YTHDC1 W377A W428A mutant (YTHDC1 mut). The P value was detected by using the Student t test between vector and YTHDC1 wt or YTHDC1 wt and mut 8 hours after actinomycin D treatment. (H) Cell growth curve of MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. The P value was detected by using the Student t test between Scr+vector and Ysh2#+vector or Ysh2#+vector and Ysh2#+MCM4 at day 7. (I) Flow cytometric analysis of apoptosis frequency of MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. (J) Flow cytometric analysis of differentiated cell frequency of MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. (K) Colony-forming units of MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. (L-M) Flow cytometric analysis of cell cycle in MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. Gating strategy is shown in panel L. Cells were stained with 5-bromo-2′-deoxyuridine (BrdU) (to determine the S phase) and 4′,6-diamidino-2-phenylindole (DAPI) (to determine the DNA content). (N) Flow cytometric analysis of the percentage of p-γH2AX+ cells in MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. (O) Western blot analysis of p-γH2AX expression in MOLM-13 cells expressed Scramble shRNA (Scr), YTHDC1 shRNA1# (Ysh1#), or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. ACTIN was served as an inner control. (P-Q) Flow cytometric analysis of the percentage of PCNA+ cells in MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. Gating strategy is shown in panel P. Data are presented as mean ± standard deviation; Student t test. **P < .01; ***P < .001. (R) DNA fiber assay. Distributions of the iodo-deoxyuridine (IdU) track length were determined on IdU-labeled DNA fibers from MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. (S) The IdU track lengths of 3 different groups were compared. (****P < .0001, Mann-Whitney U test). Mean lines in the boxes indicate the median value of at least 150 tracks per experimental condition. Scale bar, 5 μm. (T) DNA fiber assay. Distributions of the IdU track length were determined on IdU-labeled DNA fibers from KASUMI-1 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. (U) The IdU track length of 3 different groups were compared. ****P < .0001, Mann-Whitney U test. Mean lines in the boxes indicate the median value of at least 80 tracks per experimental condition. Scale bar, 5 μm.

MCM4 mediates YTHDC1 functions in AML cells. (A) Comparison of the expression level of MCM4 in patients with primary AML bearing various chromosomal translocations vs those in BM HSCs collected from healthy donors. The expression values (detected by using Affymetrix exon arrays) were log2-transformed. The P value was detected by 1-way analysis of variance followed by multiple comparisons vs the HSC group. Normal: AML with normal karyotype; Complex: AML with complex karyotype. (B) Growth curve of MOLM-13 cells expressed Scramble shRNA (Scr), MCM4 shRNA1# (M4sh1#), and MCM4 shRNA2# (M4sh2#); cells were counted every 2 days. The P value was detected by 1-way analysis of variance followed by multiple comparisons vs the Scr group at day 7. (C) Flow cytometric analysis of apoptosis frequency of MOLM-13 cells expressed Scramble shRNA (Scr), MCM4 shRNA1# (M4sh1#), and MCM4 shRNA2# (M4sh2#). Annexin V+ indicated the apoptotic cells. (D) Flow cytometric analysis of differentiated cell frequency of MOLM-13 expressed Scramble shRNA (Scr), MCM4 shRNA1# (M4sh1#), and MCM4 shRNA2# (M4sh2#). (E) Colony-forming units of MOLM-13 cells expressed Scramble shRNA (Scr), MCM4 shRNA1# (M4sh1#), and MCM4 shRNA2# (M4sh2#). Colony number was counted after 7 days of plating. (F) qRT-PCR analysis of MCM4 expression in MOLM-13 cells expressed empty vector control, YTHDC1 WT (YTHDC1 wt), or YTHDC1 W377A W428A mutant (YTHDC1 mut). (G) The mRNA half-life of MCM4 in MOLM-13 cells expressed empty vector control, YTHDC1 WT (YTHDC1 wt), or YTHDC1 W377A W428A mutant (YTHDC1 mut). The P value was detected by using the Student t test between vector and YTHDC1 wt or YTHDC1 wt and mut 8 hours after actinomycin D treatment. (H) Cell growth curve of MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. The P value was detected by using the Student t test between Scr+vector and Ysh2#+vector or Ysh2#+vector and Ysh2#+MCM4 at day 7. (I) Flow cytometric analysis of apoptosis frequency of MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. (J) Flow cytometric analysis of differentiated cell frequency of MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. (K) Colony-forming units of MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. (L-M) Flow cytometric analysis of cell cycle in MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. Gating strategy is shown in panel L. Cells were stained with 5-bromo-2′-deoxyuridine (BrdU) (to determine the S phase) and 4′,6-diamidino-2-phenylindole (DAPI) (to determine the DNA content). (N) Flow cytometric analysis of the percentage of p-γH2AX+ cells in MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. (O) Western blot analysis of p-γH2AX expression in MOLM-13 cells expressed Scramble shRNA (Scr), YTHDC1 shRNA1# (Ysh1#), or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. ACTIN was served as an inner control. (P-Q) Flow cytometric analysis of the percentage of PCNA+ cells in MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. Gating strategy is shown in panel P. Data are presented as mean ± standard deviation; Student t test. **P < .01; ***P < .001. (R) DNA fiber assay. Distributions of the iodo-deoxyuridine (IdU) track length were determined on IdU-labeled DNA fibers from MOLM-13 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. (S) The IdU track lengths of 3 different groups were compared. (****P < .0001, Mann-Whitney U test). Mean lines in the boxes indicate the median value of at least 150 tracks per experimental condition. Scale bar, 5 μm. (T) DNA fiber assay. Distributions of the IdU track length were determined on IdU-labeled DNA fibers from KASUMI-1 cells expressed Scramble shRNA (Scr) or YTHDC1 shRNA2# (Ysh2#) with or without MCM4 expression. (U) The IdU track length of 3 different groups were compared. ****P < .0001, Mann-Whitney U test. Mean lines in the boxes indicate the median value of at least 80 tracks per experimental condition. Scale bar, 5 μm.

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