Expansion of functional TECs in the thymus of Rb family mutant mice. (A) Immunoblot analysis (one of two replicates shown) of protein lysates from pooled CD45⁻ EPCAM⁺ MHCII⁺ TECs from young (2 wk; n = 10), control (Ctrl; 15 wk injected; n = 10), and Mx1-Cre p107-single (15 wk; n = 3 injected, 2 noninjected) thymi against RB, phospho-RB, CCND1, CDK4, and α-Tubulin (loading control). (D) PCR analysis of genomic DNA isolated from CD45⁻ EPCAM⁺ MHCII⁺ TECs for the lox-flanked or Cre-deleted exons of Rb and p130 in 15-wk-old control, non–pI:pC-injected Mx1-Cre p107-Single, and pI:pC-injected Mx1-Cre p107-Single mice (one of four shown). (C) RT-qPCR evaluation of expression of E2F target and S-phase genes in flow-purified TECs from 2-wk-old (n = 2), 3-wk-old (n = 5), and 15-wk-old control mice (n = 3), including E2f1 (p-values for all genes presented as 2–3 wk and 3–15 wk, P = 0.044 and 0.031, respectively), p107 (P = 0.001 and 0.04), PCNA (P = 0.026 and 0.018), bMyb (P = 0.026 and 0.041), Ezh2 (P = 0.0001 and 0.40), and Mcm3 (P = 0.018 and 0.073). (D) RT-qPCR evaluation of expression of E2F target and S-phase genes in flow-purified TECs from control and mutant TECs (15 wk; n = 3 each). Genes analyzed included E2f1 (P = 0.017), p107 (P = 0.004), PCNA (P = 0.033), bMyb (P = 0.026), Ezh2 (P = 0.059), and Mcm3 (P = 0.022). (E) PI analysis of cell cycle activity in TECs from 15-wk-old control and Mx1-Cre p107-Single mice (G1: Ctrl, 98.08 ± 1.18%; Mut, 69.17 ± 9.49% [P = 0.0003]; S: Ctrl, 1.21 ± 0.78%; Mut, 18.86 ± 6.32% [P = 0.0002]; G2: Ctrl, 1.24 ± 0.85%; Mut, 12.13 ± 3.68% [P = 0.0004]; n = 10 control and 4 mutant). (F) BrdU incorporation into mTECs and cTECs in mutant mice (mTEC: 8.95 ± 5.11%; cTEC: 11.7 ± 3.11%; P = 0.54; n = 2). (G) PI analysis of cell cycle activity in mTECs and cTECs from mutant mice (G1: mTEC, 84.17 ± 1.82%; cTEC, 82.4 ± 7.49% [P = 0.70]; S: mTEC, 10.63 ± 0.67%; cTEC, 9.30 ± 3.52% [P = 0.55]; G2: mTEC, 4.84 ± 1.25%; cTEC, 8.11 ± 3.88% [P = 0.02]; n = 2). (H) Expression levels of the mTEC-expressed gene, Aire, assessed by RT-qPCR in control and mutant thymic extracts, normalized for cell number by Eva1 levels (n = 4; P = 0.58). (I) Quantification of maturing cTEC subpopulations as identified by CD40 and CD205 expression (CD40⁻ CD205⁻: Ctrl, 2.01 ± 0.44%; Mut, 1.89 ± 0.08% [P = 0.91]; CD40⁺ CD205⁻: Ctrl, 6.59 ± 4.00%; Mut, 7.23 ± 5.23% [P = 0.62]; CD40⁻ CD205⁺: Ctrl, 9.61 ± 1.54%; Mut, 10.06 ± 0.96% [P = 0.79]; CD40⁺ CD205⁺: Ctrl, 81.77 ± 5.17%; Mut, 81.14 ± 2.11% [P = 0.65]; n = 3). (J) Quantification of maturing mTEC subpopulations as identified by CD80 and UEA1 staining (CD80⁻ UEA1⁺: Ctrl, 27.34 ± 2.67%; Mut, 27.35 ± 6.82% [P = 0.87]; CD80⁺ UEA1⁺: Ctrl, 51.18 ± 2.84%; Mut, 48.24 ± 8.39% [P = 0.86]; n = 3). (K) Expression levels of Il7 and Scf assessed by RT-qPCR in control and mutant thymic extracts, normalized by Eva1 (n = 4; p-values: Il7 = 0.29 and Scf = 0.34). (L) Immunofluorescence staining of control (left) and mutant (right) thymi with antibodies for K5 and K8 (top row); β5t, CD31, and PDGFR-β (second row; white arrows indicate blood vessels); K14, UEA1, and DAPI (third row); or K5 and CD205 (bottom row; one of two shown). Bars, 100 µm. Asterisks indicate P < 0.05; ns indicates no significant difference. All error bars indicate standard error.