Fig 1: Low expression of miR-301 promotes the expression of CPEB1, thus inhibiting MCF-7 cell proliferation, migration, and invasion(A) Expression of CPEB1 detected by qRT-PCR. (B) Detection of breast cancer cell proliferation by CCK-8. (C) Distribution of breast cancer cell cycle. (D) The relative number of migrating cells of breast cancer cells, as detected by Transwell. (E) The relative number of invasive cells of breast cancer cells. (F) Apoptosis rate of breast cancer cells. (G) The mRNA expression level of proliferation-related factor MCM2, migration- and invasion-related factors MMP2, and apoptosis-related factors caspase-3 was detected by qRT-PCR. (H) The protein expression of proliferation-related factor MCM2, migration- and invasion-related factors MMP2, and apoptosis-related factors caspase-3 was detected by western blot. The continuous variables were expressed by mean ± standard deviation. *p < 0.05 compared with the inhibitor-NC + si-NC group; #means compared with the inhibitor-NC + si-CPEB1 group. Paired sample t test, independent sample t test, or one-way ANOVA was used to compare the differences of continuous variables among different groups. Two-way ANOVA was selected to test the difference of cell activity in different groups at different times. The experiment was repeated three times.
Fig 2: RP11-59J16.2 target regulate the expression of MCM2 which was low expressed AD serum and cell model. (A) Bioinformatics analysis of the binding sites of RP11-59J16.2 and MCM2. (B) Double luciferase reporter gene assay verified the binding sites of RP11-59J16.2 and MCM2. (C,D) Low expression of MCM2 in serum of AD patients by qRT-PCR and Western blot. (E,F) MCM2 was low expressed and inhibited by RP11-59J16.2 in AD cell model. (G) qRT-PCR confirmed high expression of RP11-59J16.2 and low expression of MCM2 with positive correlation in AD cell model. The experiment was repeated three times in each group, * p < 0.05; ** p < 0.01, *** p < 0.001.
Fig 3: MCM2-7 pan-reduction accompanies senescence in primary MEFs, and both are accelerated by Chaos3.(A) Replicative lifespans of Chaos3 and WT primary MEFs. Cells were maintained under the indicated O2 levels. Error bar = SEM. (B) Senescence is accelerated in Chaos3 MEFs. The % of cells positive for SA-ß-gal (senescence-associated ß-galactosidase) was assessed at passage 5. Error bar = SEM. (C) Mcm2-7 and Pcna mRNA levels in WT MEFs decline precipitously with extended culture. The level of each gene was measured by qRT-PCR. The mRNA level of each at P2 is considered to be 100%. At least 3 biological replicated were performed for each data point. Error bar = SEM. P = passage. (D) Western blot analysis of total cellular MCM protein during passage of primary WT MEFs. Immunoblots were probed with the indicated antibodies. (E) Mcm2-7 mRNA and (F) protein levels decline more rapidly in primary Chaos3 MEFs than WT MEFs during culture. Note that PCNA was unchanged between genotypes at both time points, while MCMs decreased between P2 and P4.
Fig 4: The Chaos3 mutation disrupts MCM2-7 association with replication forks.(A) SV40 large T antigen immortalization of primary MEFs rescues MCM2-7 expression in Chaos3 (C3) cells. Shown are western blots of whole cell proteins extract (WCE) and chromatin-bound (chromatin) fractions isolated from the WT and littermate Chaos3 immortalized MEFs. The slightly lower level of chromatin-bound MCM2 & 6 in C3 cells likely reflects helicase destabilization or an excess amount of these subunits relative to the others. ß-Actin and histone H3 serve as loading controls for total and chromatin-bound proteins, respectively. (B) Decreased MCM2-7 association at unchallenged replication forks in Chaos3 mutant MEFs. SILAC labeling was performed in immortalized WT and Chaos3 littermate MEFs followed by Dm-ChP and mass spectrometry. The data represents two experiments, each involving two different cell lines for each genotype. The isotope labeling was reversed in the two SILAC experiments. Relative protein quantity ratios (WT/Chaos3 cells) were plotted for both experiments. The size of each circle represents the relative peptide enrichment overall in the MS analysis. Open circles are the six MCM2-7 proteins; filled circles are other indicated replication proteins. Error bar = SEM for each individual experiment. (C) The Chaos3 mutation causes MCM2-7 disengagement from replication forks under RS. Shown are western blots containing proteins isolated before (“Input”) or after Dm-ChP. Both WT and Chaos3 cells were SV40 T antigen-immortalized. Histone H3 signal in the Dm-ChP pull-down fraction represents amount of nascent DNA labeled during EdU incorporation, which serves as loading control. Accumulation of ?H2AX and loss of PCNA from EdU-labeled nascent DNA after HU treatment reflects fork stalling and collapse, respectively [41]. Numbers below the MCM7 panel shows MCM7 retention relative to untreated WT cells. The numbers are generated after normalizing MCM7 to H3 loading control signal strength. N.C. = no ‘Click’ reaction performed during Dm-ChP. (D) Chaos3 disrupts DNA replicative helicase function in vivo following polymerase arrest. This assay is designed to assess the fraction of cells bearing replication forks that have helicases capable of continued unwinding upon HU-induced stalling of the replicative polymerase. See S2 Fig for experimental design and examples of primary data. Immortalized WT and Chaos3 cultures did not significantly differ in the fraction of actively replicating (S-phase) cells. The percentage of WT cells stained positively for BrdU under non-denaturing conditions (“ssDNA positive”) represent those with continued helicase unwinding after HU induced replication fork stalling. N.S. = not significantly different by two-sided t-test; ** represents a significant difference (p˜0.003).
Fig 5: Restoration of SKI reverses the promoting effect of miR-552 on VSMCs.(A) The influence of miR-552 and SKI on the proliferation of HBVSMCs and mVSMCs was detected by MTT assay. (B, C) The effect of miR-552 and SKI on the invasion and migration of HBVSMCs and mVSMCs was measured according to transwell assay. (D) The effect of miR-552 and SKI on the protein level of MCM2 and PCNA in HBVSMCs and mVSMCs was assessed by western blot assay. (LSD post hoc test in conjunction with ANOVA). *P < 0.05. ns: No significance.
Supplier Page from Abcam for Anti-MCM2 antibody [EPR4120]