Fig 1: TWEAK increases proliferation in vivo. A) Representative staining and quantification of positive PCNA cells in injured femoral artery cross-sections from Wild type (N = 9), Tnfrsf12a-/- (N = 11) or Tnfsf12-/- (N = 10) mice. Data represent the mean ± SEM (One-way ANOVA with Bonferroni's post-test) ***p < .001 vs WT. Scale bars 50 µm. B) Relative CcnD1, Cdk4, Cdk6 and Cdkn2B mRNA expression levels normalized to GAPDH mRNA expression of WT, Tnfrsf12a-/- or Tnfsf12-/- femoral arteries after wire injury. Data represent the mean ± SEM (N = 8 per group) (One-way ANOVA with Bonferroni's post-test) *p < .05 vs WT, **p < .01 vs WT and ***p < .001 vs WT. C) Representative images of anti-Cyclin D1, CDK6, CDK4 and p15INK4B staining of cross-sections of injured femoral arteries from WT, Tnfrsf12a-/- or Tnfsf12-/- mice. Quantification of intimal and medial percentage of Cyclin D1, CDK6, CDK4 and p15INK4B staining respectively, in injured femoral artery cross-sections of WT (N = 11), Tnfrsf12a-/- (N = 11) or Tnfsf12-/- (N = 8) Scale bars 50 µm. Data represent the mean ± SEM (One-way ANOVA with Bonferroni's post-test) *p < .05 vs WT, **p < .01 vs WT and ***p < .001 vs WT.
Fig 2: Model illustrating the potential mechanism of TWEAK/Fn 14 axis in the development of neointimal formation after endovascular injury. Cartoon depicting TWEAK/Fn14 function in neointimal formation after wire injury. The interaction of TWEAK with its receptor Fn14 diminishes p15INK4B, increases cyclin D1, CDK4 and CDK6 expression and ERK1/2 and Akt activation in VSMCs, leading to an increase in VSMCs proliferation and migration. Therapeutic intervention with anti-TWEAK antibodies reduces neointimal formation.
Fig 3: Synergistic effects of genistein in combination with GLPG0634 or MK-2206 on the proliferation of Eca-109 cells. (A) A CCK-8 assay was conducted to assess the effects of the JAK1 pathway inhibitor GLPG0634 or the Akt pathway inhibitor MK-2206 on the proliferation of Eca-109 cells. (B) The effects of genistein (4 µM) in combination with the JAK1 pathway inhibitor GLPG0634 (16 nM) or the Akt pathway inhibitor MK-2206 (32 nM) on the proliferation of Eca-109 cells was measured through a CCK-8 assay. The effects of genistein (4 µM) in combination with GLPG0634 (16 nM) or MK-2206 (32 nM) on (C) apoptosis, (D) ROS levels and (E) the cell cycle in Eca-109 cells were measured through flow cytometry. (F) qPCR analysis was performed to quantify the expression of CyclinD1, CDK4, P53, STAT3 and MDM2 in Eca-109 cells co-treated with genistein (4 µM) and GLPG0634 (16 nM) or MK-2206 (32 nM) for 72 h. (G) The effects of genistein (4 µM) or MK-2206 (32 nM) or co-treatment on the protein levels of STAT3, p-STAT3, MDM2 and p-MDM2 in Eca-109 cells. (H) The effects of genistein (4 µM) or GLPG0634 (16 nM) or co-treatment on the protein levels of STAT3, p-STAT3, MDM2 and p-MDM2 in Eca-109 cells. (I, J) Genistein (5 mg/kg) treatment alone or in combination with MK-2206 (1 mg/kg) suppresses tumor growth and tumor volume in xenograft nude mice (n = 6 in each group). (J) Representative images of tumor volume. (K, L) Genistein (5 mg/kg) treatment alone or in combination with GLPG0634 (1 mg/kg) inhibits tumor growth and tumor volume in xenograft nude mice (n = 6 per group). (J, L) Representative images of tumor volume. All in vitro experiments were independently repeated three times. Data are analyzed using one-way ANOVA with Dunnett’s test and presented as the mean ± SD. *P<0.05; **P<0.01. Gen or G, genistein. OV, sodium orthovanadate. p-, phosphorylated.
Fig 4: FTO up-regulates MZF1/c-Myc axis to induce CRC cell proliferation. A, CCK-8 method determining the SW620 cell proliferative ability after sh-FTO, oe-MZF1 or sh-c-Myc treatment. B, Flow cytometry assessing the cycle distribution of SW620 cells after sh-FTO, oe-MZF1, or sh-c-Myc treatment. C, Flow cytometry examining the apoptosis of SW620 cells after sh-FTO, oe-MZF1 or sh-c-Myc treatment. D, Western blot analysis of the expression of CDK2, CDK4, Ki-67, PCNA, Bcl-2, and Bax genes in SW620 cells after sh-FTO, oe-MZF1 or sh-c-Myc treatment normalized to GAPDH. The data were expressed as mean ± standard deviation. The data among groups were compared using one-way ANOVA, and the data among multiple groups at different time were analysed by two-way ANOVA. * P < .05
Fig 5: CEP55 is required for the tumour-suppressive effects of overexpressed miR-144-3p in cervical cancer. A, Putative miR-144-3p binding sites in the 3'UTR of CEP55 mRNA in the bioinformatics website (http://mirdb.org/). B, miR-144-3p binding with the 3'UTR of CEP55 mRNA confirmed by dual-luciferase reporter gene assay. C, mRNA and protein expression of CEP55 was determined by RT-qPCR and Western blot analysis in SiHa cells, normalized to GAPDH. D, SiHa cell migration and invasion were detected by Transwell assay (scale bar = 50 µm). E, SiHa cell proliferation was detected by EdU assay (scale bar = 25 µm). F, Colony formation of SiHa cells was detected by colony formation assay. G, SiHa cell apoptosis was detected by flow cytometry. H, Representative Western blots of CDK4, Cyclin D1, E-cadherin, Vimentin, Bcl-2 and Bax proteins and their quantitation in SiHa cells, normalized to GAPDH. Data comparison was analysed by independent sample t test between two groups. *P < 0.05 versus SiHa cells treated with NC mimic or miR-144-3p mimic + NC. # P < 0.05 versus SiHa cells treated with NC inhibitor. Data are shown as the mean ± standard deviation of three technical replicates
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