Supplemental Figure S7.

Representative images and graphs of Ki-67, uroplakin 3a (UPK3), and KRT5 staining in keratinocyte growth factor (KGF)–pretreated bladders 1, 3, and 10 days after cyclophosphamide injection. A–F: Triple-label immunofluorescence (IF) for Ki-67 (red), UPK3 (white), and KRT5 (green) in PBS-pretreated (A–C) and KGF-pretreated (D–F) bladder sections 1 day after cyclophosphamide. A and D: Ki-67 staining shows that compared with 6 hours after injury, both the PBS (A) and the KGF (D) groups appear to have more Ki-67⁺ proliferating nuclei 1 day after injury, although the KGF group still appears to have more Ki-67⁺ cells than the PBS group. Dotted lines indicate demarcation between urothelial layer and underlying lamina propria. B and E: Ki-67 and UPK3 merged staining shows that both PBS-pretreated mice (B) and KGF-pretreated mice (E) have Ki-67⁺ proliferating cells that are mostly UPK3⁻ (arrowheads), although a few Ki-67⁺ cells are UPK3⁺ (arrow). C and F: Ki-67 and KRT5 merged staining shows that both PBS-pretreated (C) and KGF-pretreated (F) mice have Ki-67⁺ cells that are mostly KRT5⁺ (arrowheads), including some UPK3⁺ cells, as seen in B (arrow). A and D: Blue indicates DAPI. G: Graph showing that the percentages of UPK3⁺, KRT5⁺, and UPK3⁺/KRT5⁺ proliferating (Ki-67⁺) urothelial cells are not statistically different, although the percentages of KRT5⁺ cells may be trending higher in the KGF group versus the PBS group. H–M: Triple-label IF for Ki-67 (red), UPK3 (white), and KRT5 (green) in PBS-pretreated (H–J) and KGF-pretreated (K–M) bladder sections 3 days after cyclophosphamide administration. H and K: Ki-67 staining shows that compared with 6 hours and 1 day after injury, both the PBS group (H) and the KGF group (K) appear to have more Ki-67⁺ proliferating nuclei 3 days after injury, although the PBS-pretreated group has more Ki-67⁺ cells than the KGF group, including three to four rows of proliferating cells in the former versus one to two in the latter. Dotted lines indicate demarcation between urothelial layer and underlying lamina propria. I and L: Ki-67 and UPK3 merged staining shows that both PBS-pretreated mice (I) and KGF-pretreated mice (L) have Ki-67⁺ proliferating cells that are mostly UPK3⁻ (arrowheads), although rare Ki-67⁺ cells are UPK3⁺ (arrow). J and M: Ki-67 and KRT5 merged staining shows that both PBS-pretreated (J) and KGF-pretreated (M) mice have Ki-67⁺ cells that are mostly KRT5⁺ (arrowheads), except for the UPK3⁺ cell seen in I (arrow). H and K: Blue indicates DAPI. N: Graph showing that the percentages of KRT5⁺ proliferating (Ki-67⁺) cells in the PBS group are statistically higher than the KGF group, whereas the percentages of UPK3⁺ and UPK3⁺/KRT5⁺ proliferating urothelial cells are not statistically different. O–T: Triple-label IF for Ki-67 (red), UPK3 (white), and KRT5 (green) in PBS-pretreated (O–Q) and KGF-pretreated (R–T) bladder sections 10 days after cyclophosphamide administration. O and R: Ki-67 staining shows that the PBS group (O) continues to have robust numbers of proliferating (Ki-67⁺) urothelial cells, whereas the KGF group (R) has few proliferating cells. Dotted lines indicate demarcation between urothelial layer and underlying lamina propria. P, Q, S, and T: Most of the Ki-67⁺ cells in both the PBS-pretreated bladders (P and Q) and the KGF-pretreated bladders (S and T) are UPK3⁻ and KRT5⁺ (arrowheads). U: Graph showing that the percentages of KRT5⁺ proliferating (Ki-67⁺) cells in the PBS group are statistically higher than in the KGF group (as is true for the small number of proliferating UPK3⁺/KRT5⁺ cells), whereas the percentages of UPK3⁺ proliferating urothelial cells are not statistically different. O and R: Blue indicates DAPI. Data are expressed as means ± SEM (G, N, and U). N = 9 (3 planes per bladder and 3 bladders per group; G); N = 6 (2 planes per bladder and 3 bladders per group; N and U). **P < 0.01, ***P < 0.001. Scale bars: 50 μm (A–F, H–M, and O–T). L, lumen.