Fig. 3
From: A SIRT1-centered circuitry regulates breast cancer stemness and metastasis
PRRX1 underlies CSCs induced by SIRT1 deficiency. a Heat map depicting elevated EMT markers in SIRT1 KO BT549 compared with control cells. b, c Immunoblotting of EMT markers (b) and inducers (c) in Control and SKO BT549. β-Tubulin serves as an internal control. d Immunoblotting of E-Cadherin, Klf4, Aldh1a1, Prrx1 and Sirt1 in Control and Sirt1 KD 4T1 cells. e GSEA showing reduced EMT signatures in SIRT1-depleted and PRRX1-depleted BT549 cells. Normalized enrichment score (NES) and P value are shown in the plot. f Representative photos of cell invasion by Boyden chamber method analysis. Scale bar, 50 µm. Right lower panel, quantification of the percentage of invaded cells. Data represent mean ± SEM (n = 4; ** P<0.01, t-test). g CHIP-qPCR assay using anti FLAG antibody or control IgG in HEK293 cells transfected with FLAG-tagged PRRX1A or PRRX1B plasmid, showing the strong DNA binding of PRRX1B on the KLF4 promoter region (n = 3; * P<0.05, t-test). h Luciferase assays with HEK293T cells co-transfected with empty vector, PRRX1A or PRRX1B constructs together with indicated KLF4 promoter reporter together with control vectors (n = 3; * P<0.05, t-test). i Relative KLF4 mRNA transcripts in wild-type BT549 cells and indicated mutants detected by quantitative RT-PCR. Data represent mean ± SEM (n = 3; * P<0.05, ** P<0.01, t-test). j Representative morphologic photos of indicated cells. Scale bar, 100 µm. k Western blotting analysis of E-Cadherin, N-Cadherin, KLF4, PRRX1, and SIRT1 in indicated cells. Re-introduction of PRRX1 in SKO cells partially restored EMT and reduced KLF4 level. l Mammosphere formation under suspension culture conditions was elevated in indicated cell lines. Data are represented as mean ± SEM (n = 3; ** P<0.01, t-test)
