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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