Figure 1
Figure 1. Transfection of THP-1 and NB4 cells with miRNA mimics or antisense inhibitors for miR-29a and miR-142-3p affects myeloid differentiation. (A) The expression levels of miR-29a and miR-142-3p were detected by RT-PCR (left) and real-time PCR (right) in THP-1 and NB4 cells that were transiently transfected with miRNA mimics or mimic scramble control. U6 snRNA was used as an internal control. The values of each group were expressed as mean ± SD for 3 real-time PCR assays. (B) Expression of CD11b in THP-1 and NB4 cells was analyzed by flow cytometry. Numbers in the graphs represent the percentages of positively stained cells at 72 hours of PMA or 96 hours of ATRA treatment (black histograms) compared with untreated cells (red histograms). A representative experiment of 3 is presented. The differences are statistically significant (see supplemental Figure 2B). (C) Representative May-Grünwald-Giemsa staining of THP-1 and NB4 cells transfected with microRNA mimics or scramble, and treated with PMA or ATRA for 72 hours. Images were captured at room temperature using 20× objective with numeric aperture 0.5 and acquired through CCD DP72 camera and Cellsens Standard Version 1.2.1 software (Olympus). The mild differentiated cells were annotated by gray arrows. Mature macrophages and segmented neutrophils after 72 hours of differentiation were annotated by black arrows, and their percentages were marked. More mature monocytes show bluish-gray cytoplasm and a saddle-shaped nucleus. More mature granulocytic cells show polylobular nuclei, a decreased ratio of nuclear area to cytoplasmic area, and decreased cytoplasm staining, corresponding to band cells and metamyelocytes. (D) Expression levels of CD14 or CD11b mRNA were analyzed by real-time PCR. Comparative real-time PCR was performed in triplicate and normalized to GAPDH mRNA. Error bars represent SD. The expression of the mRNA in untreated scramble-transfected cell was normalized as 1. (E) Nitroblue tetrazolium assay in NB4 and HL-60 cells transfected with miR-29a mimic, miR-142-3p mimic, or mimic control. (F) Real-time PCR analysis of the expression levels of miR-29a and miR-142-3p in THP-1 and NB4 cells transfected with miR-29a inhibitor (anti-29a), miR-142-3p inhibitor (anti–142-3p), or inhibitor control (anticontrol). U6 snRNA was used as an internal control. (G) Expression levels of CD14 in THP-1 cells and CD11b in HL-60 cells were analyzed by FACS. A representative experiment of 3 is presented. The differences are statistically significant (see supplemental Figure 2F). *P < .05 (Student t test). **P < .01 (Student t test).

Transfection of THP-1 and NB4 cells with miRNA mimics or antisense inhibitors for miR-29a and miR-142-3p affects myeloid differentiation. (A) The expression levels of miR-29a and miR-142-3p were detected by RT-PCR (left) and real-time PCR (right) in THP-1 and NB4 cells that were transiently transfected with miRNA mimics or mimic scramble control. U6 snRNA was used as an internal control. The values of each group were expressed as mean ± SD for 3 real-time PCR assays. (B) Expression of CD11b in THP-1 and NB4 cells was analyzed by flow cytometry. Numbers in the graphs represent the percentages of positively stained cells at 72 hours of PMA or 96 hours of ATRA treatment (black histograms) compared with untreated cells (red histograms). A representative experiment of 3 is presented. The differences are statistically significant (see supplemental Figure 2B). (C) Representative May-Grünwald-Giemsa staining of THP-1 and NB4 cells transfected with microRNA mimics or scramble, and treated with PMA or ATRA for 72 hours. Images were captured at room temperature using 20× objective with numeric aperture 0.5 and acquired through CCD DP72 camera and Cellsens Standard Version 1.2.1 software (Olympus). The mild differentiated cells were annotated by gray arrows. Mature macrophages and segmented neutrophils after 72 hours of differentiation were annotated by black arrows, and their percentages were marked. More mature monocytes show bluish-gray cytoplasm and a saddle-shaped nucleus. More mature granulocytic cells show polylobular nuclei, a decreased ratio of nuclear area to cytoplasmic area, and decreased cytoplasm staining, corresponding to band cells and metamyelocytes. (D) Expression levels of CD14 or CD11b mRNA were analyzed by real-time PCR. Comparative real-time PCR was performed in triplicate and normalized to GAPDH mRNA. Error bars represent SD. The expression of the mRNA in untreated scramble-transfected cell was normalized as 1. (E) Nitroblue tetrazolium assay in NB4 and HL-60 cells transfected with miR-29a mimic, miR-142-3p mimic, or mimic control. (F) Real-time PCR analysis of the expression levels of miR-29a and miR-142-3p in THP-1 and NB4 cells transfected with miR-29a inhibitor (anti-29a), miR-142-3p inhibitor (anti–142-3p), or inhibitor control (anticontrol). U6 snRNA was used as an internal control. (G) Expression levels of CD14 in THP-1 cells and CD11b in HL-60 cells were analyzed by FACS. A representative experiment of 3 is presented. The differences are statistically significant (see supplemental Figure 2F). *P < .05 (Student t test). **P < .01 (Student t test).

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