Fig 1: Circ_0000326 promotes CDK4 expression by functioning as a sponge of miR-338-3p. (A) CDK4 expression in cervical cancer tissues predicted by TCGA database. (B) Representative images of immunohistochemistry (× 200) and positive-CDK4 ratio. (C) RT-qPCR analysis of CDK4 expression in 60 paired cervical cancer tissues and adjacent normal tissues. (D) The binding relationship between CDK4 and miR-338-3p detected using dual-luciferase reporter assay. (E, F) CDK4 mRNA expression in SiHa and Hela cells after alteration of miR-338-3p and silencing of circ_0000326 measured by RT-qPCR. (G–I) CDK4 protein expression in SiHa and Hela cells after treatment of circ_0000326 detected by Western blot analysis. Data were expressed as mean ± standard deviation. The data between two groups were analyzed by unpaired t-test with independent sample while the data among multiple groups was analyzed by ANOVA followed by Tukey’ s post hoc test.
Fig 2: Regulation of the c-Myc-CDK4/6-phosphoRb axis by UHRF1 in B-ALL and T-ALL. A knockdown of UHRF1 led to reduced c-Myc protein in (A) T-ALL and (B) B-ALL cells. The ALL cells were transfected with siRNA-targeting UHRF1 and a control siRNA. After being incubated for 48 h, the UHRF1, c-Myc, CDK4/6, and p-RB/RB protein levels were analyzed using Western blot and densitometry. The uncropped blots are shown in Figure S1.
Fig 3: Inhibition of YAP induces cell cycle arrest in RPMI 2650 cells. (A) RPMI 2650 cells treated with VP (0, 10, 15, 20 µM) after 24 h and subjected to cell cycle detection. (B) The proportion of cells in each phase was shown (n = 3 for each group, one-way ANOVA). (C) Representative Western blotting analysis of cell cycle progression proteins cyclinD1 and CDK4 in RPMI 2650 cells treated with VP for 24 h (n= 3 for each group). (D) RPMI 2650 cells were treated with VP (0, 10, 15, 20 µM) for 24 h and the apoptosis was detected (n= 3 for each group). (E) The proportion of apoptotic cells treated with VP of different concentrations after 24 h was shown (n = 3 for each group, one-way ANOVA). Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4: TRAF3 is a direct target of miR-361-3p. (A) Venn diagram of predicted miR-361-3p targets by five programs (miRDB, DIANA, miRwalk, TargetScan and miRTarBase). (B) The seed regions of miR-361-3p, the seed-recognising sites in the TRAF3 3' UTR, and the nucleotides mutated in TRAF3 mutant 3' UTR are shown. (C) Luciferase reporter assay was conducted to verify that miR-361-3p bound to the 3'-UTR region of TRAF3 directly. miR-361-3p overexpression significantly suppressed, while miR-361-3p loss increased the luciferase activity that carried wild-type (WT) but not mutant (MUT) 3'-UTR of TRAF3. (D) RIP assays confirmed the binding status between miR-361-3p and TRAF3 in CRC cell lines, respectively. (E) miR-361-3p overexpression decreased the level of TRAF3 mRNA and protein in CRC cells. (F) qRT-PCR analysis indicated that the mRNA expression of TRAF3 in low-miR-361-3p tumours was significantly higher than that in high-miR-361-3p tumours. (G) Pearson's correlation analysis showed the negative correlation of TRAF3 with miR-361-3p. (H) Protein levels of HIF-1a, TRAF3, ki-67, CDK4, Cyclin D1, Bcl-2 and Bax in CRC tissues were determined by IHC. Scale bar = 200 µm. FISH was used to detect the miR-361-3p in CRC tissues. Scale bar = 50 µm. (I) The relative expression of exosomal miR-361-3p in plasma in different tumour size. (J) Comparison of overall survival (OS) and disease-free survival (DFS) between patients with high expression of exosomal miR-361-3p in plasma and low expressing cases in CRC via Kaplan–Meier analysis. Data represent the mean ± SD. Student's t-test was used to determine statistical significance: *p < 0.05 and ***p < 0.001
Fig 5: Overexpression of miR-486 suppresses the expression of CDK4 and BCAS2. (A) Predicted consequential pairing of miR-486 and CDK4/BCAS2. (B) Dual-Luciferase assay (**p<0.01). (C) Expression of CDK4/BCAS2 was suppressed in the miR-486 mimic-transfected EC9706 cells (***p<0.001, **p<0.01).
Supplier Page from Abcam for Anti-Cdk4 antibody [EPR4513-32-7]