Fig. 4
Downregulation of RHOB affects the cell cycle status during megakaryocytic and erythroid differentiation. (a-b) GO enrichment analysis of all genes differentially expressed between the siRHOB- and NC-transfected K562 cells with (MK) or without (CON) megakaryocytic induction. The enriched GO terms of the cell cycle-related (a) and cytoskeleton-related (b) pathways after RHOB downregulation are shown. (c-f) The percentages of K562 (c), Meg-01 (d), UT-7 cells (e) and MNCs (f) under megakaryocytic induction (leukaemia cells for 3 days, MNCs for 10 days) in each cell cycle phase were examined by flow cytometry. (g, h) Proliferation of MNCs was determined by a CCK-8 assay. MNCs were transfected with RHOB siRNA or NC and then induced to differentiate into megakaryocytes (g) and erythroid cells (h). (i) The proliferation of K562 cells after RHOB downregulation was assessed by a CCK-8 assay. (j-k) Percentage of cells in each cell cycle phase from RHOB-downregulated K562 cells (j) and MNCs (k) under erythroid induction (K562 cells for 4 days, MNCs for 10 days). Representative flow cytometric plots are shown in the upper panel. (l) The percentage of K562 cells transfected with RHOB siRNA or NC in each cell cycle phase was examined by flow cytometry on Day 3. (m-s) Relative expression of CDK and cyclin genes in K562 (m), Meg-01 (n), and UT-7 cells (o) and MNCs (p) under megakaryocytic induction conditions (leukaemia cells for 3 days, MNCs for 10 days) and in K562 cells (q), TF-1 cells (r) and MNCs (s) under erythroid induction conditions (K562 and TF-1 cells for 4 days, MNCs for 10 days) transfected with RHOB siRNA or NC and analyzed by RT‒qPCR. (t) Protein expression of RHOA, RHOC, CDK4, and cyclin E in K562, Meg-01, and UT-7 cells without induction; K562 cells under erythroid induction; and K562, Meg-01, and UT-7 cells under megakaryocytic induction, all of which were subjected to RHOB downregulation, by western blotting. Representative blot bands are shown in the upper panel. The data are presented as the means ± SDs.
