Article Title: Interleukin-6 trans-signaling is a candidate mechanism to drive progression of human DCCs during periods of clinical latency
Figure Lengend Snippet: IL6 trans-signaling converts non-stem cells into stem-like cells. a MCF 10A spheres cultured without or with IL6, IL6 plus sgp130-Fc or with HIL6. b CFSE-labeled MCF 10A cells were cultured as spheres with or without activators (IL6, HIL6) and inhibitors of classical (an anti-IL6 antibody) and trans-signaling (sgp130-Fc). CFSE-dilution in CD44 high CD24 low , CD44 high CD24 high and CD44 low CD24 high/intermediate cells was determined by flow cytometry at day 4. The CFSE-fluorescence intensity of all cells at day one is included as reference. Data are representative for three 3 independently performed experiments. c The absolute number of CD44 high CD24 low , CD44 high CD24 high , CD44 low CD24 high/intermediate cells (upper panel) and LRCs (CFSE high , lower panel) was determined as cell/bead ratio at day 4 by flow cytometry (N=4-5 per group). d Fold-change correlation analysis comparing gene expression changes induced by IL6 plus sgp130 (classical signaling) and HIL6 (trans signaling) in MCF 10 A cells at 12 and 24 hrs with the gene expression signatures of luminal progenitor (LumProg), mature luminal (MatLum) and mammary stem cell enriched cells (MaSC) according to the study of Lim et al. 35 Nc cor: non-centered correlation between fold-changes, Num: number of common differentially expressed genes. e nLRCs from primary, PKH26-labelled control mammosphere-cultures were sorted by flow cytometry as PKH - cells. f Primary HMECs were cultured as spheres for two consecutive rounds in the absence (N=26) or presence of HIL6 and IL6 (HIL6+HIL6, N=18; IL6+HIL6, N=15; HIL6+IL6, N=14; IL6+IL6, N=17). P values in panel c: one-way ANOVA with Dunett’s multiple comparisons test (post-hoc); panel d: P values according to Student’s t-distribution for Nc cor and hypergeometric testing for Num. panel f: one-way ANOVA with Tukey’s multiple comparisons test (post-hoc); comparisons between groups labeled in red are depicted in the bar graph. Asterisks indicate significance between groups (* P
Article Snippet: For some analyses mammosphere media was supplemented additionally with 10 ng/ml IL6 (Sigma-Aldrich, Germany), 1.5 µg/ml anti-IL6 antibody (Sigma-Aldrich, Germany), 20 ng/ml Hyper-IL6, 0.1 or 10 ng/ml recombinant human sgp130-Fc (R & D Systems, Germany).
Techniques: Cell Culture, Labeling, Flow Cytometry, Fluorescence, Expressing