Figure 3. Reduced histone expression promotes replication stress.

Naive CD4⁺ T cells from young adults were activated with anti-CD3/anti-CD28 beads and transduced with control (shCtrl) or NPAT (shNPAT) shRNA lentivirus for 6 days. (A) Immunoblots for NPAT in lentivirally transduced GFP⁺ cells and summary data of normalized intensities from 4 young adults (mean ± SEM). (B) Expression of indicated histone genes in sorted shRNA⁺ cells. Results, normalized to ACTB, are presented relative to shCtrl⁺ cells from 6 young adults (mean ± SEM). Differences in expression of all histone genes are statistically significant (P < 0.01), except for HIST1H1B (P = 0.03), HIST1H3B (P = 0.06), and HIST1H3D (P = 0.03), after correction for multiple comparisons using Holm’s step-down adjustment. (C) Immunoblots for histone H3 and H4 from shRNA⁺ cells and summary data of normalized intensities from 6 young adults (mean ± SEM). (D) Activated cells were pulsed with BrdU for 1 hour. Representative flow plots of BrdU incorporation and DNA content and summary data of frequencies (n = 5, mean). (E) Activated cells were pulsed with EdU for 2 hours, followed by BrdU for 1 hour. Percentage of BrdU^–EdU⁺ S-exit cells among EdU⁺ cells from 3 young adults (mean). (F) Immunoblotting for p-RPA32 (S8), p-CHK1 (S345), γH2aX (S139), and p21 on sorted shRNA⁺ cells (left) and summary graphs of mean normalized intensities from 6 young adults (right, mean ± SEM). Comparisons by 2-tailed, paired Student’s t test (A–F). *P < 0.05, **P < 0.01.