Fig 1.
Identification of TCN as a novel myeloid differentiation inducer.
(A) Effects of ATRA and components of the kinase inhibitor screening library on NBT positive cells. ATRA was used at a final concentration of 100 nM and kinase inhibitors were used at 10 μM, unless noted otherwise. The NBT assay was performed 72 hours after the addition of these agents. (B) Summary of CD11b gene expressions by real-time PCR. The relative CD11b expression was calculated from the fold expression of CD11b, determined by the copy number of CD11b divided by the copy number of the internal control, GAPDH, compared with control values. The data shown are the mean values of experiments performed in triplicate.
Fig 2.
Effects of ATRA and kinase inhibitors on the expression of CD11b in NB4 cells analyzed by FCM.
(A) The percentages of CD11b positive cells after the addition of the indicated kinase inhibitors are shown as representative histograms of at least three independent experiments. (B) Time course FCM experiment to examine CD11b positive cells after the addition of TCN. Lower panels are controls, to which the solvent dimethylsulfoxide (DMSO) was added. (C) The expression of CD11b in the presence of the indicated doses of TCN and controls (upper panels). The indicated amount of Akt inhibitor SH-5 was added as a comparative experiment (lower panels).
Fig 3.
Morphologic analyses of NB4 and HL-60 cells in the presence or absence of TCN or ATRA.
(A, C) NB4 cells (A) or HL-60 cells (C) were collected and subjected to Wright-Giemsa staining, with and without ATRA (100 nM) or TCN (10 μM) treatment. Magnification = ×400. (B, D) A violin plot of the percentages of the N/C ratio of each cell type, using NB4 cells (B) or HL-60 cells (D). Result of ATRA [16], shown in gray, is presented for comparison (***p<0.001; N.S. not significant).
Fig 4.
Analyses of TCN-induced differentiation by differentiation markers.
The TCN-induced differentiation of NB4 and HL-60 cells and control cells was examined by the real-time PCR analysis of several markers. The expressions of (A, E) CD11b, (B, F) CD11c, (C, G) CD14, and (D, H) MPO, were examined in the presence of the indicated doses of TCN (A-D) or ATRA (E-H). Relative mRNA expression was calculated from the copy number of the target gene adjusted by GAPDH, in each TCN-treated sample (2.5, 5, 10 μM), or ATRA-treated sample (1, 10, 100 nM), which was then divided by the control sample values. The data shown were obtained from three independent PCR amplifications (average ± SD; *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl).
Fig 5.
TCN activates ERK, and the MEK inhibitor U0126 diminishes the induction of CD11b expression by TCN.
(A) Cells were cultured in the presence or absence of TCN or ATRA, for indicated periods, and analyzed for ERK and p38 MAPK activity by western blotting. MEK inhibitor U0126 is also added in the indicated sample. The membranes were probed with an anti-phospho-ERK rabbit polyclonal antibody or anti-phospho-p38 mouse monoclonal antibody. Next, the membranes were stripped and re-probed with an anti-total-ERK mouse monoclonal antibody or anti-total-p38 mouse monoclonal antibody to verify equal protein loading. Indicated numbers are relative density, calculated by Image J 1.54 software, obtained from the amount of density from the band of phospho-ERK, or phosphor-p38, divided by total-ERK, or total-p38. (B) FCM experiment of the effect of TCN on CD11b expression in the presence (0.1, 0.5, 2.5 μM) or absence of the U0126.
Fig 6.
Results of the microarray analysis.
(A) Gene expression profile of NB4 cells comparing TCN treated vs control cells. (B) Result of pathway analysis using DAVID (https://david.ncifcrf.gov/home.jsp) software from 348 genes. The x-axis shows the negative log base 10 p-value. The higher the number, the higher the significance. (C-L) Total RNAs from NB4 cells cultured in the presence or absence of ATRA and/or TCN for 72 h were analyzed by real-time PCR with specific primers for (C) IL-1β, (D) CD3D, (E) IL5RA, (F) ITGA6, (G) CD44, (H) ITGA2B, (I) CD37, (J) CD9, (K) CSF2RA, and (L) IL3RA. Relative mRNA expressions were calculated from the copy number of the target gene adjusted by GAPDH, in each TCN-treated sample (2.5, 5, 10 μM), which were divided by the control sample values. The data presented were obtained from three independent PCR amplifications and reproducibility was confirmed using different batches of cDNA. Statistical significance was determined by one-way ANOVA followed by Dunnett’s test. *p<0.05, **p<0.01, ***p<0.001 vs control.