Fig 2: String network and IPA pathway analysis of CBX4 activation and knockdown in senescence. (a) STRING analysis of interactions of proteins corresponding to the 37 analyzed and detected genes. Red: genes related to regulation of cell cycle (GO: 0051726, strength: 1.0, FDR: 1.57e-15); green: genes related to regulation of apoptotic process (GO: 0042981, strength: 0.91, FDR: 8.16e-15); purple: genes related to DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest (GO: 0006977, strength: 1.95, FDR: 4.18e-14); yellow: proteins related to replicative senescence (GO: 0090399, strength: 2.31, FDR: 2.15e-08). (b) and (c) CBX4 knockdown (KD, light blue) and activation (ACT, light red) were normalized with the control for the gene expression and analyzed for canonical pathways (b) and compared using biofunction pathway analysis and presented in the heat map ((c), orange: increase; blue: decrease).

Fig 3: CBX4 serves as a direct miR-515-5p target in MCF7 and ZR-75-30 cells. (A) The putative binding sites between CBX4 and miR-515-5p was predicted using StarBase. (B) Dual-luciferase reporter assay was performed to assess the relationship between miR-515-5p and CBX4 in ZR-75-30 and MCF-7 cells. (C) RT-qPCR was performed to measure the mRNA expression of CBX4. (D) Western blot analysis was performed to measure the protein expression of CBX4 in MCF7 and ZR-75-30 cells transfected with miR-515-5p mimic. (E) RT-qPCR was performed to measure the mRNA expression of CBX4 in different human breast cancer cell lines MCF7, ZR-75-30, MDA-MB-231 and SKBr-3 and in the normal breast cell line MCF10A. The data are presented as the mean ± SD. **P<0.01 vs. NC mimic and MCF10A. CBX4, polycomb chromobox 4; miR, miRNA; NC, negative control; Mut, mutant; WT, wild-type; RT-qPCR, reverse transcription quantitative PCR.

Fig 4: Activities of PI3K/Akt and MAPK/ERK pathways induced by maleate for regulating CBX4 expressionThe mRNA levels of (A) CBX4 and (B) GnRH and the extracellular GnRH secretion impacted with the inhibitors or agonists of PI3K/Akt, MAPK/ERK, and cAMP/PKA in GT1-7 cells. The activities of PI3K/Akt and MAPK/ERK pathways affected by maleate detected by (C) WB and (D and E) IF assay with 200× magnification.

Fig 5: CBX4 decreased with WI-38 serial culture. CBX4 protein and gene expression were investigated in each generation of the WI-38 serial culture. Protein expression was examined by Western blot and normalized with β-actin. Gene expression was analyzed by qPCR and normalized with YWHAZ. Decreased CBX4 protein expression with serial culture is shown in a representative Western blot (a). CBX4 protein expression (n = 3) and gene expression (n = 2) in serial culture were normalized to controls and depicted as an expression ratio relative to CPD 41 ((b) left, protein) and CPD 39 ((b) right, gene). The Rmcorr correlation coefficient (rrm) and confidence interval of CPD with CBX4 protein and gene expression are depicted (c) for repeated measurements. Green: negative correlation. Red: positive correlation.