Fig 2: Inverse expression correlation of miR-7 vs YY1/KLF4 in non-Hodgkin lymphoma (NHL) tissues. (A–D) Correlation of the expression of YY1 vs miR-7, or KLF4 vs miR-7 in DLBCL and follicular lymphoma was analyzed, respectively. Expression correlation analysis of miR-7 vs KLF4/YY1 was evaluated by Pearson correlation in DLBCL (A) (r=-0.4454 p=0.0106) and (B) (r=-0.3415 p=.0301) or follicular lymphoma (C) (r=-0.5229 p=0.0180) and (D) (r=-0.4248 p=0.0385).

Fig 3: Expression of miR-7, KLF4, and YY1 in NHL tissues. (A) Representative micrographs of KLF4 and YY1 expression analyzed by immunohistochemistry, and miR-7 expression evaluated by in situ hybridization in a tissue microarray of patients with NHL are shown. (Top) different markers in biopsies of DLBCL. (Below) representative micrograph of different markers in follicular lymphoma biopsies (original magnification: 4X and 40X in the frame. (B–E) Density expression of miR-7, YY1, and KLF4 were reported and total expressions for each subtype were compared (***p < 0.001).

Fig 4: KLF4 is involved in Polyphyllin III-induced protective upregulation of xCT. (A). Western blot analysis of KLF4 and xCT expression in MDA-MB-231 cells after Polyphyllin III (0–7.5 µM) treatment for 24 h. (B). qRT-PCR analysis of relative KLF4 mRNA expression after Polyphyllin III (0–5 µM) treatment for 24 h. (C,D). Western blot analysis of KLF4 and xCT expression after 5 µM Polyphyllin III treatment for 24 h when transfected (C) with negative control siRNA (siNC) or KLF4 siRNA (siKLF4) and (D) with the control plasmid (Control) or KLF4 plasmid (ovKLF4). (E–G). MDA-MB-231 cells were transfected with negative control siRNA (siNC) or KLF4 siRNA (siKLF4) and then treated with 5 µM Polyphyllin III for 24 h. The error bars show the means ± SD, n = 3 independent repeats, and p values were calculated using a two-tailed paired Student’s t-test in (E) and a two-tailed unpaired Student’s t-test in (F) and (G) (E) The cell viability was assayed (F) The relative levels of GSH were assayed (G) The relative levels of lipid peroxidation were assayed by C11-BODIPY fluorescence. (H–K). KLF4 was overexpressed using plasmid transfection in MDA-MB-231 cells, and then the cells were treated with 5 µM Polyphyllin III for 24 h. The error bars show the means ± SD, n = 3 independent repeats, p values were calculated using a two-tailed paired Student’s t-test in (H) and (I) and a two-tailed unpaired Student’s t-test in (J)–(K) (H) The cell viability was assayed (I) The cells were treated with 5 µM Polyphyllin III in the absence or presence of SAS for 24 h. The cell viability was assayed (J) The relative levels of GSH were assayed (K) The relative levels of lipid peroxidation were assayed by C11-BODIPY fluorescence. (L) Schematic diagram of the mechanism of Polyphyllin III in MDA-MB-231 cells. *p < 0.05; **p < 0.01; ***p < 0.001; n. s., not significant (p > 0.05).

Fig 5: HIFs are required for hypoxia-induced expression of pluripotency factorsA-C. Breast cancer cell lines were exposed to 20% or 1% O2 for 24 h and NANOG (A), KLF4 (B), and SOX2 (C) mRNA levels were determined by RT-qPCR, relative to 18S rRNA, and normalized to the mean value for MDA-MB-231 cells (MDA231) at 20% O2 (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. same cell line at 20% O2 by Student's t test. D and E. HCC-1954 (D) and MCF-7 (E) subclones, which were stably transfected with an expression vector encoding a non-targeting control (NTC) shRNA, or vector encoding shRNA targeting HIF-1a (sh1a) or HIF-2a (sh2a), or vectors encoding shRNAs targeting both HIF-1a and HIF-2a (DKD), were exposed to 20% or 1% O2 for 24 h and RT-qPCR was performed to determine NANOG (D) or KLF4 (E) mRNA levels relative to 18S rRNA. The results were normalized to NTC at 20% O2 (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. NTC at 20% O2; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. NTC at 1% O2 by ANOVA. F. ZR75.1 cells treated with vehicle or digoxin (200 nM) were exposed to 20% or 1% O2 for 24 h and SOX2 mRNA was measured (mean ± SEM; n = 3). *P < 0.05, **P < 0.01 vs. NTC at 20% O2; ###P < 0.001 vs. NTC at 1% O2 by ANOVA. G and H. NTC and DKD subclones of HCC-1954 (G) and MCF-7 (H) were exposed to 20% or 1% O2 for 48 h, whole cell lysates were prepared, and immunoblot assays were performed to analyze HIF-1a, HIF-2a, NANOG and KLF4 protein expression. Actin was also analyzed as a loading control. I. ZR75.1 cells were treated with vehicle or digoxin (200 nM), exposed to 20% or 1% O2 for 48 h, and HIF-1a, NANOG and SOX2 immunoblot assays were performed.