Fig 2: Generation of a genetically-defined, stepwise model of HMEC transformation.48R expressing shRNA targeting p16 (shp16), p53 (shp53), or both (shp16-shp53) were further infected with control virus (Vec), or viruses encoding RAS alone (RAS), MYC alone (MYC), or MYC and RAS together (M/R) and assessed at passage 19 for AIG. (A) The bar graph represents the average colony number per plate of quadruplicates. Error bars represent the standard deviation. (B) Pictorial representation of the HMEC transformation model and growth in soft agar. Normal cells (left) are transformed (right) by sequential inactivation of p16 and p53 using shRNA, and over-expression of MYC and oncogenic RAS.

Fig 3: Optimization of DC-PCR for drug screening.Effect of cytoplasmic enzymes on DC-PCR (A). KMS-12-PE cells were diluted at 1∶25 in hypotonic PCR buffer with or without added protease inhibitors and incubated for 20 min before PCR was run to simulate the time required for pipetting during a small molecule screen. Inclusion of protease inhibitors in the PCR mastermix decreased the number of amplifiable sites in DAC treated and untreated KMS-12-PE cells suggesting that digestion of DNA binding proteins can occur in PCR buffer although epigenetic differences between samples are maintained in the studied example. Detection of DC-PCR product via UV transillumination of PCR plates and sensitivity compared to gel electrophoresis (B). Ethidium bromide was added to the PCR mastermix before serially diluted cells were submitted to DC-PCR: untreated, DAC treated or fibroblasts. After completion of thermal cycling samples were first analyzed under UV light (Left panel), then via agarose-gel electrophoresis (Right panel). Using gel-based detection, the level of detection for epigenetic differences of CDKN2A was lower, down to about 1 cell for site #6 compared to about 300 cells with direct UV transillumination. The primer numbers correspond to sequences on the map described in Figure 1A. Three independent experiments yielded the same detection levels. Correlation between direct UV transillumination and gel electrophoresis was seen in all experiments (hundreds).

Fig 4: IFN-γ promotes CUL5-mediated HER2 degradation(A and B) BT-474 (A) and HCC1419 (B) cells were treated for 96 h with increasing concentrations of IFN-γ (0, 12.5, 25, 50, and 100 ng/mL) and expression levels of HER2, CUL5, and HSP90 were detected by western blotting. (C and D) Relative mRNA expression of CUL5 in (C) BT-474 and (D) HCC1419 cells treated with increasing concentrations of IFN-γ for 48 h. (E) Proteasome inhibitor MG132 treatment for 2 h reverses IFN-γ-induced HER2 downregulation (p = 0.0149) in HCC1419 cells. (F) siCUL5 treatment reverses Th1 cytokine-induced HER2 downregulation (p = 0.0010) in HCC1419 cells. (G) IFN-γ induced p16 upregulation in HCC1419 cells following CUL5 or Stat1 siRNA transfection. (H) Silencing CUL5 or Stat1 interferes with IFN-γ-induced cell senescence in HCC1419 cells (siNT versus siNT+IFN-γ, p < 0.0001). Bar graphs represent % of SA-β-gal-positive cells, identified by ImageJ counting; mean ± SD (n = 3).

Fig 5: HPV20 E6 does not compete for binding of cdk6 to cyclinD1 or to p16INK4a.Total protein extracts from pLXSN-flag20E6- or pLXSN-NIKS cells were immuno-precipitated onto cdk6 protein. Protein A agarose precipitated with cdk6 antibody alone served as negative control. Levels of input proteins flagHPV20E6, p16, cyclinD1 and cdk6 were visualized by western blot analyses. Histograms represent input protein expression normalized to β-actin serving as loading control.