(A) Absence of p-mTOR staining in lymphatics of normal trachea. (B) Uniformly strong p-mTOR staining in lymphatics. The epithelium was excluded from the confocal projection of the tracheal whole mount. (C) Absence of p-S6RP staining in lymphatics of normal trachea. (D) Strong p-S6RP staining in lymphatic sprouts (arrows) in CCSP/VEGF-C mice after doxycycline (3 days). Arrowheads mark p-S6RP staining of other cell types. (E) Absence of p-4E-BP1 staining in lymphatics of normal trachea. (F) Strong p-4E-BP1 staining in a subset of lymphatic endothelial cells in CCSP/VEGF-C mice on doxycycline (3 days). (G) The boxed region in F shown at higher magnification. Arrowheads mark p-4E-BP1–positive cells. (H) Phospho-4E-BP1 staining in a lymphatic sprout (arrowhead) in CCSP/VEGF-C mice on doxycycline (2 days, P23). (I) Transient increase in number of p-4E-BP1–stained cells during doxycycline exposure (red boxes, P21–P28) and 14 days after withdrawal of doxycycline (red stripped box, P42). n = 3–10 mice/group. *P < 0.05 vs. vehicle, ANOVA. (J) BrdU labeling of p-4E-BP1–stained cells after doxycycline (3 days). BrdU labeling (green) in lymphatic endothelial cells is more abundant than p-4E-BP1 (red). Prox1 (white in bottom panels) is not restricted to the nuclei of dividing cells (arrowheads, left panels). Some p-4E-BP1–stained cells lack BrdU labeling (arrowheads, right panels). (K) Numerous p-4E-BP1–stained cells were observed after doxycycline alone (2 days, P21-P23) but not when doxycycline was given concurrently with rapamycin. (L) An increased number of p-4E-BP1–stained cells (red box) was observed after treatment with doxycycline (2 days, P21–P23). The increase was prevented when doxycycline was given concurrently with rapamycin (20 mg/kg, green box). *P < 0.05 vs. baseline, ANOVA, n = 5 mice/group. Scale bar: 50 μm (A–D, G, and K); 200 μm (E and F); 10 μm (H). Box and whisker plots show the median, first and third quartiles, and maximum and minimum.