Figure 2. Cell aggregation, HF induction ability, and collagen expression of replicative senescent hDPCs. (A) SA-β-gal staining in young (P5) and old (P13) passage cells. Scale bar, 200 μm. Quantification for n > 200 cells per group. p16 (B) and p21 (C) mRNA expression by qRT-PCR. (D) Representative image of spheroids from young (P5) or old (p13) passage cells is shown on (top), and the size in diameter of spheroids (μm) is quantified (right). Scale bar, 200 μm. (E) 3D reconstruction of spheroids. Scale bar, 500 μm. (F) Patch assay. Senescent hDPCs (P13) failed to induce new HFs (n = 3). Scale bar, 500 μm. (G) COL13A1, COL15A1, and COL23A1 mRNA expression by qRT-PCR. (H) Immunofluorescence staining of hDPC spheroids with COL13A1, COL15A1, COL23A1, and DAPI for nuclei. Scale bar, 200 μm. All quantitative data are shown as the mean ± standard deviation (SD) of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.005; #p < 0.05; ##p < 0.01. Asterisk indicates Student's t-test. Sharp indicates one-way ANOVA.