Fig 2: Identification of asymmetric gene and protein expression in paired daughter cells (PDCs). (A) Freshly isolated HSCs were tracked during culture until first division (35 h) using a time-lapse microscope. A microwell array platform was used for gene expression analysis and a micro-groove platform for protein expression analysis (Figure S2); (B) The gene score values show a wide variation from one gene to another. A stringent threshold (average 10-fold difference in gene expression between PDCs) was put to identify the “asymmetric gene set”; (C) Table showing 12 candidate genes that were seen to cross the 10-fold threshold (fold change values in table); (D) IFNa-treated PDCs show a higher asymmetry index as compared with control PDCs; (E) Representative immunofluorescent images showing asymmetric expression of Glut1, JAM3, and HK2 in 15 different PDCs; (F,G) Analysis of PDCs asymmetry for Glut1 (n = 59 control and n = 72 IFNa), JAM3 (n = 64 control and n = 69 IFNa treatment), and HK2 (n = 32 control and n = 54 IFNa) with IFNa-treated cells or control; (F) PDCs asymmetry in frequency with threshold defining “asymmetric protein expression” used in (H) Glut1 = 1, JAM3 = 1, HK2 = 0.3; (G) PDCs asymmetry. IFNa condition shows significantly higher levels of asymmetric index for Glut1 and HK2 as compared with the control. Boxplot’s interquartile range from the 25th to the 75th percentile. A line and red square show, respectively, median and mean, and the error bar is SD (outliers lower or upper inner fence is equal to 1.5 the interquartile range); (H) The protein expression pattern in PDCs (all PDCs considered) for Glut1, JAM3 and HK2. Asymmetry with one marker (first column) are shown in total percentage (without considering other markers). A double asymmetry (two asymmetric proteins) analysis is shown in the second column. An asymmetry comprising two proteins (for example “P1” and “P2”) on two daughter cells (defined by ca and cb) is either “coinciding”, a cell having high expressions of both proteins “P1” “P2” (phonetically defined by ca P2P1=++, cb P2P1=--), or “diametrical”, a cell having more than one protein “P1” but less than of “P2” ca P2P1=-+ and, conversely, his sister more than one protein “P2” and less than “P1” cb P2P1=+-. Number of pairs analyzed for co-staining as follows: n = 69 IFNa and n = 63 for control Glut1/JAM3, n = 30 IFNa and n = 26 for control Glut1/HK2, and n = 30 for IFNaa and n = 26 for control JAM3/HK2. ** p < 0.01, and * p < 0.05.