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. 2015 Oct 25;7(7):7640–7656. doi: 10.18632/oncotarget.6156

Figure 5. MNT knockdown inhibits myeloid differentiation and rescues G0-G1 arrest.

Figure 5

a. HL60 cells were transiently transfected with shMNT (1.0μg) and WCEs was prepared; cells were treated with 1μM ATRA post-36h transfection as indicated. Lysates were resolved on 8% SDS-PAGE followed by immunoblotting with MNT, E6AP and β-actin antibodies. b. HL60 cells were stably transduced with shMNT lentiviral vector and WCEs were prepared post-2, 3 and 4 days of transduction. Lysates were resolved on 8% SDS-PAGE followed by immunoblotting with anti-MNT antibody. c. Graphical representation of FACS analysis of cd11b in HL60 cells transiently transfected with shMNT (1.0μg) and co-treated with 1μM ATRA post-36h transfection. d. Graphical representation of FACS analysis of cd114 in HL60 cells transiently transfected with shMNT (1.0μg) and co-treated with 1μM ATRA post-36h transfection. e. MNT knockdown in HL60 cells via lentiviral transduction of shMNT inhibits granulocytic differentiation. Cells were stained with May-Grunwald and Wright's Giemsa staining. Arrows indicate granulocytes/neutrophils. Scale represent 100μm. f. Graphical representation of G0-/G1 cells post-24 and 48h shMNT (1.0μg) transfection and co-treatment with 1μM ATRA post-36h transfection. g. Effects of MNT knockdown on the protein levels of G1/S transition regulators in HL60 cells. WCEs were harvested post-24 and 48h shMNT (1.0μg) transfection and co-treated with 1μM ATRA post-36h transfection as indicated. Lysates were subjected to immunoblotting with antibodies against indicated G1/S transition regulatory proteins. Data are representative of minimum three independent experiments. Results are given as standard error of mean (+S.E.M.); *P<0.05, **P<0.001, ***P<0.0001. One-way ANOVA with Bonferroni's Multiple Comparison Test was performed using GraphPad Prism Version 5.00.