Fig 2: Activation of CBX4 reduced cellular senescence markers in WI-38. CBX4 was activated using the CRISPR/Cas9 Synergistic Activation Mediator (SAM) lentiviral particle system in presenescent WI-38 (CPD47-50). (a) Representative Western blot of CBX4 protein expression in CBX4 activation (ACT) and lentiviral control (Ctrl). (c) CBX4 protein (n = 6) and gene expression (n = 5) both increased in response to CBX4 ACT compared with Ctrl (Ctrl: white, CBX4 ACT: light red). (c) SA-ßgal, p16, and EdU proliferation expression in Ctrl and CBX4 ACT in representative flow cytometry. (d) By flow cytometry, CBX4 activation resulted in decreased protein expression of senescence markers SA-ßgal, p16, and DPP4 and nonsignificantly increased EdU (n = 7).

Fig 3: Regulation of senescence-related genes by CBX4. A selected panel of senescence-related genes was analyzed by qPCR microarray. Two serial culture experiments were performed, and the resulting Log2FC values were computed (normalized with YWHAZ and calculated relative to the earliest CPD of serial culture). (a) and (b) Heatmaps of standardized gene expression at 10 time points of WI-38 serial culture performed in two biological replicates. (c) the Rmcorr correlation (and 95% confidence intervals) of CPD with expression (Log2 FC) of 37 senescence-related genes from serial culture. Green: positive correlation of gene expression with CPD. Red: negative correlation gene expression with CPD. Black: insignificant correlation of gene expression with CPD. (d) Heatmap of pairwise Spearman correlations of genes measured in the serial culture experiment. Based on expression pattern and consistent with (a) and (b), two groups of genes were highly clustered. (e) Volcano plot of gene expression changes in response to CBX4 KD vs. Ctrl showing increased expression of DPP4, CDKN2A, and SLC52A1 and decreased expression of CXCL8, PCNA, DNMT1, E2F1, and PARP1. (f) Volcano plot of gene expression changes in response to CBX4 ACT vs. Ctrl showing decreased expression of DPP4, CDKN2A, and HDAC1 and increased expression of CBX4, SIRT1, and MDM4. Red: increased expression, green: decreased expression.

Fig 4: Flow cytometry. Histogram overlays of unstained controls (dotted lines for all three cell lines) and measurements for HROLu55 (green), HROLu22 (blue), and HROBML01 (red) for the epitopes CD326, PD-L1, EGFR, CD26, LYPD3, DSG3, CCD59, CD27, and CD90 are shown.

Fig 5: Mapping effects of CBX4 (wild-type and mutants) on primary human chondrocytes. Expression of senescence markers and cell proliferation of primary human chondrocytes transduced by CBX4 wild-type (WT) or mutants. A CBX4 gene expression (n = 6) and B protein expression by Western blot (representative blot with quantification of n = 5 biological replicates in dot plot) in cells transduced by the empty vector Control (Ctrl), CBX4 WT, or its mutants. The expected molecular weights of CBX4 WT, CBX4 CDM, CBX4 ΔSIMs, CBX4 ΔC-box, and CBX4 CDMΔSIM were 75 kDa, 75 kDa, 74 kDa, 72 kDa, and 74 kDa, respectively. C Protein-level expression of senescence markers DPP4, SA-β-gal activity, and p16.INK4A and cell proliferation marker EdU quantified by flow cytometry and plotted as a dot bar graph. Repeated measures with Dunnett’s post hoc test were performed to compare the effects of CBX4 WT and mutants. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001