Fig 2: ASIV decreases HIF-1α and p-ERK1/2 protein levels, and increases p27 and p21 protein levels in HPASMCs. (A and B) Western blot analysis of HIF-1α, p-ERK1/2, p27 and p21 protein levels in HPASMCs under normoxic or hypoxic conditions. (C-E) HIF-1α, p-ERK1/2, p27 and p21 protein levels, respectively (relative to normoxia group). Values are means ± SEM of 3 independent experiments. *P<0.01 compared with the normoxia group; ##P<0.01, compared with the hypoxia group. ASIV, astragaloside IV; HPASMCs, human pulmonary artery smooth muscle cells; N, normoxia; H, hypoxia.

Fig 3: RUNX3 promotes the tumorigenic phenotypes of KGN cell in vitro. (A) Ectopic expression of RUNX3 in KGN cells was examined by immunoblotting. β-actin was used as the loading control. (B) Cell growth was determined by the neutral red uptake assay and expressed as the fold change relative to day 1. (C) Anchorage-independent growth was examined by the soft agar assay and the number of colonies formed by KGN/Vector and KGN/RUNX3 cells were counted. (D) Cell motility was determined by the scratch assay. Images were captured under the phase contrast microscope at 100× magnification. (E) The mRNA level of CCND2 (cyclin D) and CDK1B (p27) was measured by quantitative reverse transcription-PCR (qRT-PCR) and expressed as the fold change relative to the vector-only control cells. (F) Cyclin D2 and p27Kip1 protein levels were examined by immunoblotting. β-actin was used as the loading control. Data in (B,C,E) are shown as mean ± SE of three independent experiments. * Significantly different (p < 0.05). Results in (D) and (F) are representative of three independent experiments.

Fig 4: HOXA-AS2 epigenetically silences p21 and KLF2 transcription by binding to EZH2 and LSD1. (a) qRT–PCR analysis of HOXA-AS2 nuclear and cytoplasmic expression levels in DLD1 and HCT116 cells. U6 was used as a nucleus marker, and GAPDH was used as a cytosol marker. (b) RIP experiments were performed in DLD1 and HCT116 cells, and the coprecipitated RNA was subjected to qRT–PCR for HOXA-AS2. The fold enrichment of HOXA-AS2 in EZH2/SUZ12/LSD1 RIP is relative to its matched IgG control. (c) The levels of p15, p16, p21, p27, KLF2 and PTEN mRNA were determined by qRT–PCR when knockdown of HOXA-AS2. (d) The p21 and KLF2 protein levels were determined by western blot in HOXA-AS2 knockdown in DLD1 and HCT116 cells. (e) The p21 and KLF2 expression levels were determined by qRT–PCR when knockdown of EZH2 or LSD1 in DLD1 and HCT116 cells. (f) ChIP-qRT–PCR of EZH2/LSD1 occupancy and H3K27me3/H3K4me2 binding in the p21 and KLF2 promoters in DLD1 and HCT116 cells treated with si-HOXA-AS2 (48 h) or si-NC; IgG as a negative control. Error bars indicate mean±s.e.m. *P<0.05, **P<0.01. ChIP, chromatin immunoprecipitation; NC, negative control.

Fig 5: PPARG mediated apoptosis is inversely related to TP53 pathway. A549 cells were transfected with PPARG (PPARG (TT) and irradiated 24 h post-transfection, and processed 48 h after transfection. These samples which include CONTROL, RAD CONTROL (5 Gy), PPARG (TT), and RAD+PPARG (TT) were processed for Western blotting. (A) The effect of PPARG overexpression on the levels of TP53 was evaluated using Western blotting. Same GAPDH was used as a loading control as that for PPARG blot in Figure 1. The graph on the right represents the relative quantification of TP53. The protein level has been normalized with loading control. The error bar represents standard deviation in which ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. The blots were repeated three times. (B) The effect of PPARG overexpression on the levels of CDKN1A, the downstream target of TP53 was evaluated using Western blotting. GAPDH was used as a loading control. The graph represents the relative quantification of CDKN1A. The protein level has been normalized with loading control. The error bar represents standard deviation in which ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. The blots were repeated three times. (C). The effect of PPARG overexpression on the levels of CDKN1B was evaluated using Western blotting. GAPDH was used as a loading control. The graph on the right represents the relative quantification of CDKN1B. The protein level has been normalized with loading control. The error bar represents standard deviation in which ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. The blots were repeated three times.