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Sequence of primers.

Transcriptomics of AF and NP cells in response to PDGF-AB/BB treatment.

A + B) Volcano plots showing the differentially expressed genes (DEGs) in AF (A) and NP (B) cells treated with rhPDGF-AB/BB for 5 days (cutoff: |FC| > 1.5 and FDR < 0.05). DEGs were annotated in the plot according to FDR. C+D) Heatmap plots showing the top 50 DEGs in rhPDGF-AB/BB treated AF (C) and NP (D) cells based on fold change. NP: n = 5 samples. AF: n = 6 samples.

Functional analysis of AF cells treated with PDGF-AB/BB.

A + B) Gene ontology (GO) analysis for biological process using upregulated (A) and downregulated (B) DEGs in AF cells. Y-axis label represents GO terms, and x-axis represents geneRatio (geneRatio refers to the proportion of genes that are annotated in specific GO term within all the input genes). The size of bubble represents the gene counts enriched in a particular GO term and the color indicates the FDR of GO terms. C) Gene set enrichment analysis (GSEA) on the entire transcriptome profile demonstrated an upregulation of cell cycle, cytosolic DNA-sensing pathway, ErbB signaling pathway, and proteasome and a downregulation of drug metabolism-cytochrome P450 pathway in rhPDGF-BB treated NP cells. D) The leading-edge genes associated with these pathways were shown using a chord diagram. E) Protein-protein interaction network using DEGs was constructed in STRING and visualized in Cytospace.

Functional analysis of NP cells treated with PDGF-AB/BB.

A + B) Gene ontology (GO) analysis for biological process using upregulated (A) and downregulated (B) DEGs in NP cells. Y-axis label represents GO terms, and x-axis represents geneRatio (geneRatio refers to the proportion of genes that are annotated in specific GO term within all the input genes). The size of bubble represents the gene counts enriched in a particular GO term and the color indicates the FDR of GO terms. C) Gene set enrichment analysis (GSEA) on the entire transcriptome profile demonstrated an upregulation of Wnt signaling pathway and a downregulation of oxidative phosphorylation and ribosome in rhPDGF-BB treated NP cells. D) The leading-edge genes associated with these pathways were shown using a chord diagram. E) Protein-protein interaction network using DEGs was constructed in STRING and visualized in Cytospace. F+G) The changes in the gene expression of PDGFRA and IL6 were verified in NP (F) and AF (G) cells treated with rhPDGF-AB (40ng/ml) or -BB (20ng/ml) for 5 days. *p< 0.05. **p< 0.01. ***p< 0.001.

Cellular senescence induction by irradiation.

A) SA-b-gal staining in healthy NP cells under different doses of irradiation at day 7. Images were captured under 10X magnification using Echo microscope. ROI: region of interest. B) MTT assay showed decreased cell proliferation after irradiation at day 7. C) Immunocytochemistry staining on Lamin B1 and P21 in irradiated cells at day 7. D) Quantification of P21 positive cells. E) Immunocytochemistry staining on P16 in irradiated cells at day 7. F) Quantification of P16 positive cells. G) Changes in the gene expression levels of P21, P16, and CASP3 in NP cells under 10 Gy of irradiation compared to the non-irradiated group at day 7 and 10 timepoints. H) The gene expression of PDGFRA was reduced under 10 Gy of irradiation compared to the non-irradiated group at day 10. *p< 0.05. **p< 0.01. ***p< 0.001. ****p< 0.0001.

PDGF-AB/BB treatment inhibited the progression of senescence in healthy NP and AF cells exposed to irradiation.

A-F) Cell cycle analysis by DAPI staining in irradiated healthy NP (A-C) and AF (D-F) cells treated with rhPDGF-AB(40ng/ml) or BB (20ng/ml) for 10days. The upper panel shows the data obtained from NP cells, and lower panel shows the data from AF cells. Representative graphs (A+D) showing the changes in cell cycle progression in NP and AF cells after treatment. B+E) Quantification of cell cycle analysis showed the cell percentage in each phase. Two-way ANOVA with Dunnett post hoc testing was performed. n = 4 each group. C+ F) The cell percentage in the S phase was shown to examine the effects of treatment on cell proliferation. One-way ANOVA with Dunnett post hoc testing was performed. n = 4 each group. G) SA-b-gal staining in irradiated healthy NP (upper panel) and AF cells (lower panel) showing the reduced SA-b-gal positive cells in after treatment. Images were captured under 10X magnification using Echo microscope. ROI: region of interest. The data is presented as mean with SD. *p< 0.05. **p< 0.01.

PDGF-AB/BB treatment suppressed the expression of senescence associated regulators.

A-B) Changes in the gene expression of PDGFRA, P21, P16, CASP3, and IL6 in irradiated NP (A) and AF (B) cells treated with rhPDGF-AB/BB. C+D) Protein expression levels of P21 and NF-κB in irradiated NP (C) and AF (D) cells treated with rhPDGF-AB/BB. n = 4 each group. One-way ANOVA with Dunnett post hoc testing was performed. The data is presented as mean with SD. *p< 0.05. **p< 0.01. ****p< 0.0001.

Principle component analysis (PCA) of NP and AF samples treated with rhPDGF-AB and BB.

A+B) PCA plot of NP (A) cells and AF cell (B) showed the distinct cluster between untreated and treated samples. The clusters of PDGF-AB and BB samples were overlapping. NP: n = 5 each group. AF: n = 6 each group.

Protein expression of PDGFRA was decreased in NP and AF samples treated with rhPDGF-AB and BB.

A) Representative images of western blot oof PDGFRA in NP (left) and AF (right) cells. B) Quantification of western blot results showing decreased PDGFRA expression in treated samples compared to the untreated group. NP: n=5; AF: n=6. One-way ANOVA with Dunnett post hoc testing was performed. The data is presented as mean with SD. *p< 0.05. **p< 0.01.