Expression of the INK4b-ARF-INKa locus during cellular ageing and differentiation. (A) Organization of the human INK4b-ARF-INK4a locus (not drawn to scale), encoding three distinct proteins, p15INK4b, p14ARFand p16INK4a. The untranslated regions (yellow boxes), the coding sequences of p15INK4b(green), p14ARF(blue) and p16INK4a(red) are indicated. (B) INK4b and INK4a, but not ARF, are upregulated in ageing human diploid fibroblasts (HDFs). RT-qPCR analysis of INK4b-ARF-INK4a expression in neonatal (yellow bar) versus adult (blue bar) HDFs. Bar graphs represent the mean of three independent biological replicate experiments, each analyzed in triplicate by RT-qPCR. mRNA levels are expressed relative to Gapdh. Error bars represent standard error of the mean. (C) INK4b and INK4a are selectively upregulated in ageing human embryonic fibroblasts (HEFs). Comparison of INK4b-ARF-INK4a expression in HEF cells (TIG3) at a low passage doubling (PDL 26, yellow) and high PDL (PDL 64, blue). (D) Flow cytometrical analysis of umbilical cord blood cells showing forward scatter (FSC) on the x-axes and CD34 staining on the y-axes. The immature CD34+ cells (blue) and mature CD34- cells (red) were sorted (left hand panel). Isolated CD34+ cells were reanalysed (middle panel; red dots represent 65% of the population). Following 6 weeks of culture these cells stained negative for CD34 (less than ~15%; right hand panel). (E) INK4b-ARF-INK4a expression in CD34+ (yellow) cells, CD34- (red) cells, and the progeny of seeded CD34+ cells after 6 weeks of culture (blue). (F) Erythroblasts (EB) kept under proliferation conditions for 2 days following induction of differentiation towards erythrocytes. Cells were cytocentrifuged onto glass slides and stained for haemoglobin (brown colour) and with standard cytologic dyes. (G) Expression of INK4b-ARF-INK4a in proliferating (yellow) erythroblst cells and cells differentiating towards erythrocytes (blue).