Fig 1: PAICS silencing obviously induced cell‐cycle arrest in EGFR‐WT NSCLC cells. (A) PAICS was primarily involved in the regulation of the cell cycle in NSCLC indicated by single‐cell analysis from CancerSEA. (B) PAICS mRNA level was positively correlated with cell cycle demonstrated by Data from Guillaumet‐Adkins A. Genome Biol. 2017 (PDX_LUAD) (n = 42). (C and D) KEGG pathway and GO‐biological process analysis indicated that the cell cycle, DNA repair, and DNA replication pathway were down‐regulated after knocking down PAICS. (E) MSigDB H (hallmark) analysis identified nine signatures (Q value ≤ 0.05) that were differentially expressed once silencing PAICS. (F) Cell cycle distribution of shNC‐ and shPAICS‐NSCLC cells. (G) CDK2, CDK6, Cyclin D1, Cyclin E, and p‐Rb protein levels in shNC‐ and shPAICS‐NSCLC cells.
Fig 2: Prenatal D4 regulated cell cycle regulatory protein and estrogen receptor expression in neural progenitor cells. (A) Cell cycle regulatory protein content were analyzed by using Western blotting (β-actin as loading control). (B,C) Quantification of (A). (B) At E17.5, D4 25 mg/kg and 50 mg/kg groups showed significant decreases in CDK6 compared to both vehicle and D4 50 mg/kg groups (F3,22 = 8.886, p = 0.0007). Moreover, compared to vehicle group, D4-treated groups exhibited increases in p27 (F3,22 = 3.374, p = 0.0465). n = 5–7 per group. (C) At adult stage, D4 50 mg/kg groups showed decreased in CDK6 (F3,20 = 3.318, p = 0.0418). p27 was markedly higher in the D4-treated groups (F3,20 = 11.39, p = 0.0002). D4-treated groups showed increases in ERa at both E17.5 (F3,27 = 9.612, p = 0.0002) and adult stage (F3,22 = 5.194, p = 0.0177). However, compared to vehicle groups, D4 25 mg/kg and 50 mg/kg group exhibited increases in ERb E17.5; F3,29 = 4.406, p = 0.0124; adult; F3,27 = 5.218, p = 0.0065). n = 5 per group. Data represent mean ± SEM. Statistical significance was determined by one-way ANOVA with Bonferroni correction. * p < 0.05 vs. vehicle, ** p < 0.01 vs. vehicle, # p < 0.05 D4 10 mg/kg vs. D4 50 mg/kg, + p < 0.05 D4 25 mg/kg vs. D4 50 mg/kg.
Fig 3: Silencing HPSE suppresses cell proliferation of BRAF V600E-mutant CRC cells through AKT/p27Kip1 pathway.A Expression levels of cell proliferation-related signaling proteins in BRAF V600E-mutant CRC cells with or without HPSE silencing. Red color represents upregulated proteins. Green represents downregulated proteins. The same trend was observed in three independent repeated experiments. Representative images and semi-quantification analysis are shown. Data are presented as the mean ± standard deviation. B IHC staining of p27Kip1 and phospho-AKTser473 in subcutaneous tumor tissues. Representative images and semi-quantification analysis are shown. Data are presented as the mean ± standard deviation. C–E Representative images and statistical analysis of colony formation assay. Cells were seeded in six-well plates at 1000 cells per well and cultured for 10 days. In the group treated with SC79, SC79 (S7863, Selleckchem, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO, Sigma) and diluted to 10 µM with the complete medium before use. Cells were incubated with DMSO/SC79 (10 µM) for 7 days. The same trend was observed in three independent repeated experiments. Data are presented as the mean ± standard deviation. F Proposed model for the mechanism of HPSE function on cell proliferation in BRAF V600E-mutant CRC cells. Red color represents upregulated or activated proteins. Green represents downregulated or inactivated proteins. The student’s t-test was used to determine the P-value in two-group comparisons. One-way ANOVA analysis and Tukey’s test were used for multiple comparisons. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns P > 0.05.
Fig 4: CDCA2 promoted HCC cell proliferation, migration, and G1/S phase transition of HCC cells in vitro.(A) Proliferation of CDCA2-knockdown Huh7 or SK-Hep1 cells and CDCA2-overexpressing HepG2 cells was measured by CCK-8 assay. (B) Colony formation assay showed increased clonogenic ability of HepG2 cells after overexpression of CDCA2. (C) Transwell migration assays showed that knockdown of CDCA2 attenuated the migratory ability of Huh7 cells. (D) Western blotting analysis of epithelial-mesenchymal transition marker proteins in HCC cells with CDCA2 knockdown or overexpression. (E) Flow cytometry analysis showed the promotion of the G1-S phase transition in CDCA2-upregulated HepG2 cells and the inhibition of the G1-S transition in CDCA2-downregulated Huh7 cells. (F) The protein levels of CCNDs and Cdks in HCC cells with CDCA2 knockdown or overexpression were measured by Western blotting. (G) mRNA expression of p21 and p53 in HCC cells with CDCA2 knockdown or overexpression was measured by qRT-PCR. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Fig 5: NR2F1 mediates the transition from dormancy to proliferation in ENZ-R cells. (A–C) IC50 assays detected the sensitivities of ENZ-R cells to ENZ following NR2F1 overexpression or depletion. Negative control for silencing NR2F1 was listed as si-NC, while silencing NR2F1 as si-NR2F1. Overexpression of NR2F1 was listed as oe-NR2F1. (D–F) CCK-8 assays elucidated the proliferation abilities of ENZ-R cells. (G-I) Colony formation and EdU assays demonstrated ENZ-R cell proliferation following NR2F1 depletion or upregulation. (J) Flow cytometry revealed the cell cycles (G1, G2, and S phases) of ENZ-R cells following NR2F1 expression modulation. (K) WB assays elucidated the expression of cell cycle-associated proteins following NR2F1 expression modulation. (L) Luciferase activity in subcutaneous tumor xenografts was measured after NR2F1 knockdown. (M) The dimensions and volume of tumor xenografts are shown. (N–P) IHC experiments demonstrated NR2F1 and p21 expression in xenografts after NR2F1 knockdown. Scale bar = 20 μm, 50 μm, or 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
from Cell Signaling Technology for Cell Cycle Regulation Antibody Sampler Kit