Serum starvation increased chromatin dynamics. (A) Experimental scheme. Proliferating cells were starved by removing serum from the culture medium. Most of the cells entered the quiescent G0 phase. The starved cells were then stimulated with serum re-addition to re-enter them into the proliferating state. (B) MSD plots (±SD among cells) of nucleosomes in RPE-1 cells (black) and with the serum starvation for 3 d (light blue) or 7 d (dark blue). For each condition, n = 20 cells. Note that the chromatin dynamics increased depending on the serum starvation period. ***, P < 0.0001 by the Kolmogorov–Smirnov test for no starvation versus 3-d starvation (P = 1.1 × 10−8) and for no starvation versus 7-d starvation (P = 1.5 × 10−11). (C) Top: Verification of RNAPII activity of RPE-1 cells without (0 d) or with the serum starvation for 3 d and 7 d by immunostaining of Ser5P of the RPB1 CTD in RNAPII. Bottom: Quantifications of RNAPII Ser5P signal intensity are shown as box plots. The median intensities of Ser5P are 45.5 (n = 38) at 0 d starvation, 24.4 (n = 47) at 3 d, and 17.3 (n = 42) at 7 d. RNAPII activity decreased after serum starvation. ***, P < 0.0001 by the Wilcoxon rank sum test for 0 d versus 3 d (P < 2.2 × 10−16) and for 0 d versus 7 d (P < 2.2 × 10−16). (D) Top: RNAPII activity observed by immunostaining in the RPE-1 cells without (untreated control), with serum starvation for 3 d, or with the re-addition of serum. Bottom: Quantification of RNAPII Ser5P signal intensity is shown as box plots. The median intensities of Ser5P are 54.5 (n = 101) in untreated control, 38.0 (n = 103) in 3-d starvation, and 56.3 (n = 79) in re-addition. Note that RNAPII activity decreased in the G0 phase and was restored with serum re-addition. The Wilcoxon rank sum test shows N.S. (P = 0.20), and ***, P < 0.0001 for untreated versus starvation (P < 2.2 × 10−16) and for starvation versus serum re-addition (P < 2.2 × 10−16). (E) MSD plots (±SD among cells) of nucleosomes in RPE-1 cells without (black) or with serum starvation for 3 d (light blue), and 1 d after serum re-addition (orange). For each condition, n = 39–40 cells. The up-regulated chromatin dynamics were restored to the untreated level upon serum re-addition. The Kolmogorov–Smirnov test shows N.S. (P = 0.93), and ***, P < 0.0001 for untreated versus 3-d starvation (P = 3.2 × 10−7) and for 3-d starvation versus serum re-addition (P = 4.8 × 10−6).
