Proteomic analysis shows that the overall cellular histone content does not decrease during TED. (A) The number of quantified proteins in the different cellular models; the number of proteins with a LFQ value in at least 1 differentiation stage is indicated. (B) Evolution of the histone/DNA ratio in MEDEP cells during TED. Arbitrary histone quantification values were obtained by summing the LFQ values for all histone proteins. DNA values were obtained from optical density 260 (OD₂₆₀) of deoxynucleotides after DNase digestion of DNA, as previously described²³  and as set forth in the supplemental Methods. At each time, the ratio (arbitrary histone LFQ quantification/DNA OD₂₆₀) was calculated and ratios at each differentiation time were expressed relative to this ratio at T0. (C) Protein quantification using either histones or total cellular protein content as the reference. Total cellular protein content was calculated using the BCA colorimetric assay from 1 million cells (BCA curves) or using the MS LFQ values and a standard histone value of 5.5 pg per cell as reference (MS curves). The histone curves were calculated by using the MS LFQ values and the total cellular protein content determined using the BCA colorimetric assays as the reference. The standard deviation labels of histones curves are smaller than the symbols at each point. (D) Protein quantification at the end of TED using MS LFQ values and either total cellular protein amounts determined by BCA assay (panel C; BCA curves), histones or globins as the reference. A standard histone value of 5.5 pg per cell was used for protein quantification with histones as the reference. Cellular globin contents for MEDEP and primary cells (PCs) were calculated using the heme quantification values presented in supplemental Figure 2G. Red lines display theoretical perfect correlation. Error bars represent the standard deviation of 3 (MEL, G1ER, or MEDEP cells) or 4 independent experiments (PCs, panel D; and MEDEP cells, panel B).