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. 2020 Jun 24;11:3193. doi: 10.1038/s41467-020-16890-6

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Representative gating strategies for CD24^medCD29^hiCD49f^hi mammmary stem cells (MaSC) with contour plots shown for LD (black) and JL (red) tumours (SSC: side scatter). b Frequency of CD24^medCD29^hiCD49f^hi mammary stem cells (MaSC) in LD and JL tumours (n = 13). Data are shown as a scatter dot plot with lines indicating the median with interquartile range (error bars). P-value obtained from an unpaired two-sided t-test. c Mammosphere-formation efficiency (MFE%) of LD and JL tumour cells (n = 3). Data are shown as a scatter dot plot with lines indicating the median with interquartile range (error bars). P-value obtained from an unpaired two-sided t-test. d Circadian clock genes mRNA expressions (FPKM: Fragments Per Kilobase Million) in cancer cells from LD (n = 5) and JL (n = 4) primary tumours. Data presented as box-and-whisker plots. Variability is shown using medians (line in the box), 25th and 75th percentiles (box), and min to max (whiskers). P-values were calculated using DESeq2 and the Wald test based on the negative binomial distribution. Respective log2FoldChange (log2FC) are listed in Supplementary Data 3. e Mammosphere-formation efficiency (MFE%) of MCF12A normal human mammary cells in different circadian phases (n = 3). Blue squares and grey diamonds represent peaks of BMAL1^HIGH/PER2^LOW and BMAL1^LOW/PER2^HIGH expression, respectively. Data are presented as a dot plot with dots indicating the median with interquartile range (error bars). P-value was calculated from a one-way ANOVA/Tukey’s multiple comparisons test. f Tumour-initiation study based on orthotopic injection of primary tumour cells from LD (n = 6) and JL (n = 6) mice in the mammary fat pad (MFP) of host mice (n = 4 per each donor). Tumour-initiation potential was calculated as the percentage of host mice that formed tumours. We set a threshold based on MFP weight at 0.25 g for positivity. P-value was obtained from a binomial two-sided test. The picture illustrates the observed differences between MFP between JL and LD donors. Indicated (n) represent number of independent experiments as biological replicates.