Research Paper Volume 13, Issue 3 pp 3290—3312

Suppression of p16 alleviates the senescence-associated secretory phenotype

Knockdown of p16 decreases IL6 and CXCL8 expression in oncogene-induced senescent cells. IMR90s expressing either BRAFV600E or HRASG12V alone or in combination with a shRNA targeting p16 (shp16 hairpin #1). An empty pBabe retroviral vector and a shRNA targeting GFP lentiviral vector were used as controls. See Supplementary Figure 1A for an experimental timeline. (A) Immunoblot of BRAF and p16. Vinculin was used a loading control. (B) Representative images of senescence-associated β-galactosidase (β-GAL) staining and colony formation (CF). (C) Quantification of β-GAL in (B). (D) Quantification of CF in (B). (E) Immunoblot of RAS and p16. β-actin was used as loading control. (F) Representative images of β-GAL staining and colony formation (CF). (G) Quantification of β-GAL in (F). (H) Quantification of CF in (F). (I, J) IL6 and CXCL8 mRNA expression (fold change relative to control mean). 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. *p

Figure 1. Knockdown of p16 decreases IL6 and CXCL8 expression in oncogene-induced senescent cells. IMR90s expressing either BRAFV600E or HRASG12V alone or in combination with a shRNA targeting p16 (shp16 hairpin #1). An empty pBabe retroviral vector and a shRNA targeting GFP lentiviral vector were used as controls. See Supplementary Figure 1A for an experimental timeline. (A) Immunoblot of BRAF and p16. Vinculin was used a loading control. (B) Representative images of senescence-associated β-galactosidase (β-GAL) staining and colony formation (CF). (C) Quantification of β-GAL in (B). (D) Quantification of CF in (B). (E) Immunoblot of RAS and p16. β-actin was used as loading control. (F) Representative images of β-GAL staining and colony formation (CF). (G) Quantification of β-GAL in (F). (H) Quantification of CF in (F). (I, J) IL6 and CXCL8 mRNA expression (fold change relative to control mean). 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. *p<0.05.