Figure 2. Knockdown of p16 at later timepoints decreases IL6 and CXCL8 expression without bypassing oncogene-induced senescence. IMR90s expressing BRAFV600E alone or in combination with shRNAs targeting p16 (shp16 hairpin #1 and #2). An empty pBabe retroviral vector and a shRNA targeting GFP lentiviral vector were used as controls. See Supplementary Figure 2A for an experimental timeline. (A) Immunoblot of BRAF and p16. β-actin was used as 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) 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 3 experiments is shown. *p<0.05. ns = not significant.