Fig 1: Distinct CDK6 Levels Govern the G0 Exit Kinetics of LT- and ST-HSCs(A) Log2 signal intensity for CDK6 mRNA probe. Shown are individual measures (black circles: LT-HSCs, red squares: ST-HSCs, green triangles: GMPs) and the median and interquantile ranges (horizontal bars); n = 3. All multiple comparisons have been tested.(B) Immunofluorescence for CDK6 protein in LT- and ST-HSCs sorted from CB (left panel) or cultured for 4 days (right panel). Representative pictures and histograms of CDK6 fluorescence density are normalized to the fluorescence density of the IgG control in the same population. Positivity threshold was set over the median + 1 SD of the IgG control distribution and the percetnage of positive cells is indicated. 100–570 cells are analyzed with n = 3 CB samples. Scale bar represents 10 μM.(C) Normalized median CDK6 fluorescence density. Mean ± SEM is shown; n = 3 CB samples. GMPs, granulocyte-monocyte progenitors.(D) Immunofluorescence for CyclinD3 protein in LT-HSCs, ST-HSCs, and GMPs from freshly isolated CB (Day 0, left panel) or after 4 days of culture (Day 4, right panel). Shown are histograms of CyclinD3 fluorescence density normalized to the fluorescence density of the IgG control in the same population. Positivity threshold (dotted line) was set over the median + 1 SD of the IgG control distribution. n = 135–315 cells analyzed for day 0 and n = 49–245 cells for day 4.(E) Normalized median CyclinD3 fluorescence density at the indicated time points. Mean ± SEM is shown; n = 3 CB samples. ∗∗p < 0.05 by paired t test.(F) Time course analysis of CDK6 and CyclinD3 upon stimulation by mitogenic signals. Percentages of CDK6+ (top panel) or CyclinD3+ (bottom panel) cells in each of the indicated populations at the indicated time points after isolation from CB are shown. Mean ± S.E.M shown. n = 3 CB samples, except for day 2, where n = 2 CB samples. ∗∗p < 0.05 by paired t test.See also Figure S3.
Fig 2: Expression of miR16 and its target genes in GBM cell lines SGH44, U87 and U251 and their GSCs. (a) MicroRNA microaary profiling using miRCURY LNA Array. Green block in heatmap were miRNA low expression areas, in which miR16 was found. (b) Identification of target genes of miR16 by luciferase reporter assay in U251 cells. Schematic diagram of Bcl2, CDK6, CCND1, CCNE1 and SOX5 3′-UTR pMIR-REPORT constructs (left). Comparison of sequences between mature miR16 and the wild-type (Wt) or mutant (Mut) putative target sites in the 3′-UTR of Bcl2, CDK6, CCND1, CCNE1 and SOX5. Cells were co-transfected with pMIR-REPORT containing the empty (Ctrl), WT or mutant target site of the Bcl2, CDK6, CCND1, CCNE1 and SOX5 3′-UTR plus miR16 or NC mimic for 48 h. The luciferase activity was normalized to the Renilla activity and presented as relative activity to the corresponding NC (assigned as value ‘1’). Values denote the mean±s.e.m. of three independent assays. *P<0.05.
Fig 3: miR16 inhibitor promoted the tumor growth in vivo. Nude mice received intracerebral transplantation of U251 or U251-GSCs transfected with miR16 mimic, inhibitor or scrambled control. (a) Comparison of tumor size between groups of miR16 inhibitor, miR16 mimic group and scrambled control; (b) Expression level of miR16 and its target genes, including Bcl2, CDK6, CCND1, CCNE1, SOX5 and Ki67 in tumor xenograft tissues were examined by qRT-PCR and immunohistochemistry. Green block were low expression areas.
Fig 4: CDK6 Levels Determine the Duration of Quiescence Exit in the HSC Pool(A–C) Cell division duration of single LT- and ST-HSCs after exposure to mitogenic signal in the presence or absence of PD033299 (50 nM). (A) Percentage of cells from the indicated populations that divided after 100 hr in culture. (B) Cumulative first division kinetics (excluding dead cells). Data from a representative CB example are shown. Curve is least-squares sigmoid fit. R2 > 0.99. (C) Mean time to first division (hours) (tfirstDiv = logEC50).(D–G) Cell division duration of single LT- and ST-HSCs after exposure to mitogenic signal with or without CDK6 EE. (D) Cumulative first division kinetics (excluding dead cells) of indicated populations transduced with indicated lentiviral vectors. Data from a representative CB are shown. Curve is least-squares sigmoid fit. R2 > 0.99. (E) Mean time to first division (hours) (tfirstDiv = logEC50). (F) Time of cell cycle transit of indicated populations in hours.(G) Expansion curves of LT- and ST-HSCs in culture. Shown is the average number of cells per single cell plated at the indicated time points after culture initiation. Data are from one representative experiment out of three. Time 0 represents the time of exposure to mitogenic stimulus.In (A), (C), (E), and (F), individual CB samples are shown; gray lines connect parameters from the same condition. ∗∗p < 0.05 by paired t test. See also Figure S4.
Fig 5: CDK6 EE LT-HSCs Outcompete Wild-Type HSCs without Exhaustion(A–D) NSG mice were injected with sorted Lin− CD34+ CD38− cells transduced with CDK6 EE or control (LUC) lentiviral vectors (GFP+ cells) and untransduced competitive cells (GFP−). Bone marrow was harvested at indicated time points post-transplantation and analyzed by flow cytometry. (A) Percentage of GFP+ cells among engrafted human hematopoietic cells (CD45+). Time 0 corresponds to percentage of GFP+ cells before injection in four independent CB samples. 4 weeks post-transplantation: n = 13 LUC and 14 CDK6 EE mice; 20 weeks post-transplantation: n = 25 LUC and 23 CDK6 EE mice. (B) Lymphoid to myeloid ratio (percentage of CD19+/CD33+) among GFP+ cells at 20 weeks post-transplantation. n = 25 LUC and 23 CDK6 EE mice. In (A) and (B), boxplots represent median, 25th, and 75th percentiles and whiskers represent min and max. Gray boxes, LUC; white boxes, CDK6 EE. (C) Percentage of GFP+ cells among LT-HSCs at 20 weeks post-transplantation. (D) Absolute number of LT-HSCs at 20 weeks post-transplantation. In (C) and (D), n = 6 mice from two CB samples. Individual mice, median, and interquantile range are shown. In (A)–(D), ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01 by Mann-Whitney test.(E and F) CDK6 EE LT-HSCs expand over serial transplantation. LUC, CDK6 EE (GFP+), or untransduced (GFP−) LT-HSCs were sorted from primary transplanted mice (n = 2 pools of three to five mice) and injected at four different doses into secondary NSG mice. (E) Engraftment levels (percentage of CD45+ cells) 12 weeks after secondary transplantation (>0.01% CD45+ GFP+ or CD45+ GFP−) at the two highest doses (200 and 400 cells/mouse). Individual mice, median, and interquantile range are shown. (F) Summary table of number of mice engrafted at each dose tested and estimation of LT-HSC frequencies in each group by the ELDA statistical method. See also Figure S5.
Supplier Page from Abcam for Anti-Cdk6 antibody [8H4]