Fig. 2

SIRT1 deficiency induces cancer stemness via upregulation of KLF4. a Representative images showing mammospheres derived from control and SIRT1 KO BT549 cells. Scale bar, 100 µm. b Representative Immunoblots showing loss of SIRT1 in SIRT1 KO BT549. c Quantification of mammosphere-forming efficiency in two passages. Noted that SIRT1 deficiency significantly increased the number of mammospheres. Data represent mean ± SEM from three independent experiments. (*P<0.05, **P<0.01, t-test). d Gene set enrichment analysis (GSEA) of RNAseq data showing differentiated cell identity signature is reduced in SIRT1-depleted BT549 cells. Normalized enrichment score (NES) and P value are shown in the plot. e Stemness-related gene expression determined by real-time RT-PCR. The Y-axis values are relative fold change for gene transcripts normalized to GAPDH. Data represent mean ± SEM (n = 3; *P<0.05, **P<0.01, t-test). f Levels of SIRT1 and four stemness core factors in ALDH⁺ Vs ALDH^- and CD24⁻CD44⁺ Vs non-CD24^-CD44⁺ breast cancer cells, assessed by Affymetrix array HU133 Plus 2.0. (*P<0.05, t-test). g, h Stable knockdown of KLF4 in SKO BT549 results in downregulation of ALDH1 on mRNA and protein levels, analyzed by real-time RT-PCR (n = 3; * P<0.05, ** P<0.01, t-test) and Western blotting. Reconstituting KLF4 into parental cells elevated ALDH1 expression. i, j ALDH1 activities of indicated cells were assessed by ALDEFLUOR assay. DEAB was used to establish baseline fluorescence and to define ALDEFLUOR-positive region. Error bars represent SEM (n = 3; ** P<0.001, t-test). (k, l) Indicated cells were grown in ultra-low attachment surface plates at a density of 500 per well. Assays were conducted after 10 days. k Representative images showed typical mammospheres with scale bars (100 µm). l Quantification of mammosphere-forming efficiency of parental and mutant cells. The symbol ** indicates P<0.01 Vs control groups