Supplementary Figure 4: Proximity ligation assay shows distinct proximity between histones (old versus new) and lagging strand-enriched DNA replication machinery components in GSCs.

(a) A representative GSC showing PLA signals between the lagging strand-enriched component PCNA and new H3-GFP and a representative GSC showing PLA signals between the lagging strand-enriched component PCNA and old H3-mKO. (b) Quantification of the number of PLA puncta per nucleus between PCNA and histones (old versus new) in GSCs. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. PLA puncta between PCNA and new H3-GFP: 11.4; n=28; between PCNA and old H3-mKO: 8.5; (n=31), *: P< 0.05, based on Mann-Whitney U test. Shapiro-Wilk normality test P = 0.0013, data not normally distributed. PCNA + H3-mKO (old H3) GSC Shapiro-Wilk normality test P = 0.2467; data normally distributed. Mann-Whitney U two-tailed test: Mann-Whitney U = 297.0. P = 0.0366. For PCNA + H3-GFP (new H3) GSC, Shapiro-Wilk normality test P = 0.0013, data not normally distributed. For PCNA + H3-mKO (old H3) GSC, Shapiro-Wilk normality test P = 0.2467; data normally distributed. Mann-Whitney U two-tailed test: Mann-Whitney U = 297.0. P = 0.0366 (c) Quantification of PLA signals in two negative control experiments: first, PLA experiments were performed between histones and a cytoplasmic protein Vasa¹³; second, PLA signals were counted in non-replicating somatic hub cells. Both showed very low signals. Vasa PLA mean = 1.3, n = 52; Hub PLA mean = 0.2; n = 44. Scale bars: 5μm.