Research Paper Volume 13, Issue 3 pp 3290—3312

Suppression of p16 alleviates the senescence-associated secretory phenotype

Knockdown of p16 in melanoma cells decreases IL6 and CXCL8 expression. The melanoma cell lines SKMel28, RPMI-7951, and Hs 600T expressing wildtype p16 were infected with lentivirus expressing a shRNA targeting p16 (shp16 hairpin #1). An shRNA targeting GFP lentiviral vector was used as control. (A) Immunoblot of p16. Vinculin was used as loading control. (B–D) mRNA expression of IL6 and CXCL8 (fold change relative to control mean) in SKMel28 (B), RPMI-7951 (C), and Hs 600T (D) melanoma cells. Expression of target genes was normalized against multiple reference genes. Data normalized against MRPL9 are shown. n=3/group and mean±SD. 1 out of 3 experiments is shown. (E–H) p16 was stably knocked down in SKMel28 melanoma cells with a shRNA (shp16 hairpin #1). An shRNA targeting GFP lentiviral vector was used as control. Cells were treated with 1μM etoposide for 6 days. (E) Representative images of senescence-associated β-galactosidase (β-GAL) staining and colony formation (CF). (F) Quantification of β-GAL in (E). (G) Quantification of CF in (E). (H) IL6 and CXCL8 mRNA expression (fold change relative to control mean). Expression of target genes was normalized against multiple reference genes. Data normalized against PMSC4 are shown. n=3/group and mean±SD. 1 out of 2 experiments is shown. *p

Figure 3. Knockdown of p16 in melanoma cells decreases IL6 and CXCL8 expression. The melanoma cell lines SKMel28, RPMI-7951, and Hs 600T expressing wildtype p16 were infected with lentivirus expressing a shRNA targeting p16 (shp16 hairpin #1). An shRNA targeting GFP lentiviral vector was used as control. (A) Immunoblot of p16. Vinculin was used as loading control. (BD) mRNA expression of IL6 and CXCL8 (fold change relative to control mean) in SKMel28 (B), RPMI-7951 (C), and Hs 600T (D) melanoma cells. Expression of target genes was normalized against multiple reference genes. Data normalized against MRPL9 are shown. n=3/group and mean±SD. 1 out of 3 experiments is shown. (EH) p16 was stably knocked down in SKMel28 melanoma cells with a shRNA (shp16 hairpin #1). An shRNA targeting GFP lentiviral vector was used as control. Cells were treated with 1μM etoposide for 6 days. (E) Representative images of senescence-associated β-galactosidase (β-GAL) staining and colony formation (CF). (F) Quantification of β-GAL in (E). (G) Quantification of CF in (E). (H) IL6 and CXCL8 mRNA expression (fold change relative to control mean). Expression of target genes was normalized against multiple reference genes. Data normalized against PMSC4 are shown. n=3/group and mean±SD. 1 out of 2 experiments is shown. *p<0.05.