Fig 3: Proliferation analysis of slow-cycling and frequently dividing LSCs.

Adult 2–3- month- old Krt15-GFP mice were used for all experiments. The limbal region markers in wholemount corneas were defined by Gpha2/K15-GFP, nuclei were detected by DAPI counter staining (A, D, G, I) while Ki67⁺ cells were stained (A) and quantified (B). (C-D). Corneal epithelial debridement was followed by EdU injection and 6 hours later, cells in S-phase (EdU⁺) were identified in wounded or uninjured controls. A schematic illustration is shown in (C) and a typical tile scan confocal image of wholemount immunostaining is shown in (D). (E-G) Water-based EdU administration for 15-days (pulse) was followed by a 30-day chase. Schematic illustration (E), quantification of EdU accumulation in basal cells over time (F), and typical tile scan confocal image of wholemount staining with regional markers (G) is shown. (H-J) Double nucleotide (IdU/EdU) injection with an interval of 1.5 hours was followed (0.5 an hour later) by tissue harvesting and quantitative analysis (see methods) on wholemount staining. Schematic illustration (H), representative image (I), and an estimated cell cycle for each zone (J) is shown (n=15 areas from 3 corneas of 3 individuals). (K) Violin plot diagram of unique molecular identifiers (UMI) RNA reads per cell in each cluster. (L) Violin plot showing the expression of the indicated genes involved in SC quiescence or activation. Y-axis in L represents expression level. Statistical significance was calculated using the One-way ANOVA test followed by the Bonferroni test (*, p-value<0.05). Data represented as mean ± standard deviation, Abbreviations: Peri., periphery; CjB, conjunctival basal; CjS, conjunctival suprabasal; OLB, outer limbal basal; ILB, inner limbal basal; LS, limbal superficial; CB1, corneal basal 1; CB2, corneal basal 2; CW, corneal wing; CS, corneal superficial; M1/M2, mitosis. Scale bars, 50 μm.