Reply to Chen: Methodological and analytical considerations in the study of H3-Dendra2 distribution

A recent comment by Chen (1) questions some conclusions of our study regarding histone segregation in Drosophila stem cells (2).Our detailed point-by-point response, which includes all experimental controls and data re-analyses, is publicly available (3).Herein, we address the central critiques regarding statistical interpretation, methodology, and technical rigor.Chen reanalyzed our data obtained using the Gal4/ Upstream Activation Sequence (UAS) Flp-out system, correctly noting a statistically significant deviation (P < 0.05) from a perfect 1:1 ratio for H3.1 in Intestinal Stem Cell-Enteroblast (ISC-EB) pairs.However, this metric alone cannot support her model of consistent "old" histone (H3.1-GreenFluorescent Protein (GFP)) enrichment in the stem cell (4).A significant P value merely rejects the null hypothesis of perfect symmetry; it does not specify the direction of bias.In fact, the mean H3.1-GFP fluorescence ratio (ISC:EB, log 2 scale) in ISC-EB pairs is less than 0 (SI Appendix , figure S8B in ref.2), which directly contradicts the specific prediction of increased signal in the ISC.Furthermore, our imaging data contradict the predicted unidirectional pattern.We observe