Figure 3 |. Lineage tracing of Ck4-expressing Dist-Luminal-C cells.

a, Schematic of the targeting strategy to generate C4T (Ck4^CreERT2/+;Rosa26^td-Tomato/+) mice to label Ck4-expressing cells by tdTomato expression. b, Timeline of Luminal-C cell labeling and prostate regression-regeneration in the C4T mouse prostate. c, Co-immunofluorescence of Ck5, Ck8, Ck14 and Trp63 with endogenous tdTomato in the distal region of the intact C4T mouse 2 weeks after tamoxifen injection. d, Percentage of tdTomato⁺ cells among Ck5⁻ cells, Ck5⁺ cells (n = 1,491), Ck14⁻ cells, Ck14⁺ cells (n = 1,219), Trp63⁻ cells, Trp63⁺ cells (n = 1,685), Ck8⁻ cells and Ck8⁺ cells (n = 1,641) in the 10 week C4T mouse prostate. e, Co-immunofluorescence of Tacstd2 and Ck4 with endogenous tdTomato in the distal region of the intact C4T mouse prostate 2 weeks after tamoxifen injection. f, Immunostaining of Ck5, Ck8 and Trp63 with endogenous tdTomato in the regenerated prostate after one regression-regeneration cycle. g, Percentage of tdTomato-positive cells among Ck8-positive luminal cells in the distal region of the intact and regenerated C4T mouse prostate after one or three regression-regeneration cycles (n = 6 mice/group). h, Immunostaining of Ck5, Ck8 and Trp63 with endogenous tdTomato in the regenerated prostate after three regression-regeneration cycles. i,j, Percentage of tdTomato⁺ cell clones (i) and average colony size (j) in the intact and regenerated prostate after one or three regression-regeneration cycles. k-m, Luminal-C cells drive prostate luminal cell regeneration by a self-renewal mechanism (k). Quantification of tdTomato-labeled cell progeny showed that Tacstd2⁺/tdTomato⁺ Dist-Luminal C cells both self-renew to replenish Tacstd2⁺ Luminal-C cells and differentiate into Tacstd2⁻ daughter luminal cells (n = 6 mice/group) (l). The percentage of Tacstd2⁺/tdTomato⁺ Dist-Luminal C cells was sustained during the regression-regeneration period (n = 6 mice/group) (m). n, Immunostaining of Tacstd2, Nkx3.1 and Hoxb13 with endogenous tdTomato in the regenerated prostate after one regression-regeneration cycle. Six mice were used for each experiment. Data show mean ± standard deviation (g, j, l and m), two-way ANOVA (i) and unpaired two-tailed Student’s t test (g,j,l and m). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001, (d,g,i,j,l,m). Scale bars, 50 μm (c,e,f,h,n).