Research Paper Advance Articles

Figure 1. An inverse correlation between DNMT1 and SPINT2 emerges at the early transition (E-to-M) to senescence in RS of primary HDF. (A, B) Heatmap (A) and correlation plot (B) depicting DNMT1 and SPINT2 expression dynamics throughout RS process. Transcriptomic data were obtained from our previously established time-series RS model of primary HDF (GSE41714). The different stages of RS—early (E), middle (M), advanced (A), and very advanced (VA)—are indicated. (C–E) RS of primary HDFs was induced as described in the 'Experimental Procedures' section. (C) qRT-PCR analysis of DNMT1 and SPINT2 mRNA expression. ^**p < 0.01 vs. DT2 by Student’s t-test. (D) Western blot analysis of DNMT1, SPINT2, and p21 protein expression. (E) Cell size (upper) and granularity (lower) were assessed by side scatter (SSC) and forward scatter (FSC), respectively, using flow cytometry. ^**p < 0.01 vs. DT2 by Student’s t-test. Bar graphs represent mean ± standard deviation (SD) from three independent cultures. Representative SSC and FSC patterns of DT2 and DT11 cells are shown in the right panel. (F) SA-β-gal assay was performed at each stage. Representative images are shown and quantifications of SA-β-gal-positive cells are presented below each image. ^**p < 0.01 vs. DT2 by Student’s t-test.