Figure 7. Brd4 inhibition impairs immune surveillance of oncogenic N-Ras expressing senescent cells in vivo.
(A) A schematic diagram of the three sleeping beauty constructs used in the hydrodynamic transfection experiments. The blue triangles indicate the sleeping beauty direct and inverted repeats. SB: Sleeping Beauty transposase.
(B) Experimental strategy to address the impact of systemic Brd4 inhibition on clearance of senescent hepatocytes. Mice were injected with the indicated constructs through hydrodynamic transfection and treated with I-BET 762 (iBET) or vehicle orally every day (15mg/kg). Livers were harvested at days 6 and 12 and subjected to histological analyses to assess the extent of immune-mediated senescent hepatocyte clearance (see Figs. C–E). The same vector system and timeline was used for hepatocyte-specific Brd4 depletion using shRNAs against Brd4 (see Figs. F–L). Figure adapted from ref. 35.
(C) Representative immunohistochemistry (IHC) stainings for GFP on liver sections harvested from vehicle or iBET-treated mice at day 6 and 12 post-transduction of NRasG12D-IRES-GFP by hydrodynamic injection. Arrows point to clusters of immune infiltrates.
(D) Quantification of the number of GFP positive foci in livers from vehicle or iBET-treated mice at the indicated time points post-injection. Each dot represents the mean numbers of GFP+ foci per mouse (ten 10X fields quantified/mouse) and results are expressed relative to vehicle-day 6 mean value. Means ± SD values for each experimental group are also indicated.
(E) Representative H&E staining of livers from vehicle or iBET-treated mice harvested at day 12 post-injection. Arrows point to clusters of immune infiltrates.
(F) Representative immunofluorescence (IF) for N-Ras (green) on day 6 (top panels) or day 12 (bottom panels) liver tissue sections from mice injected with transposon-based vectors encoding for oncogenic N-Ras- and indicated shRNAs. Nuclei are counterstained with DAPI.
(G) Bar graphs show corresponding quantifications of the number of N-Ras+ cells per field, at day 6 or day 12 post-injections. Data are presented as mean + S.D. (n=4) from thirty 10X fields per mouse. Results are presented as number of GFP positive foci relative to control shRNA on day 6.
(H) Immunohistochemistry (IHC) staining for GFP on day 6 liver tissues from mice injected with transposon-based vectors encoding for oncogenic N-Ras and indicated shRNAs. Hematoxylin was used for counterstaining. GFP marks hepatocytes co-expressing N-Ras and the indicated shRNAs. Arrows indicate immune infiltrates surrounding N-Ras/shRen-expressing GFP positive foci.
(I) Co-immunofluorescence for N-Ras (green) and CD45 (red) on frozen liver tissue sections harvested on day 6. Nuclei are counterstained with DAPI.
(J) Bar graphs show corresponding quantifications of the percentage of N-Ras foci infiltrated by CD45+ cells at day 6. Data are presented as mean + S.D. (n=4) from thirty 10X fields per mouse.
(K) Triple-immunofluorescence for N-Ras (green), Cd45 (white) and the SASP factor Mcp1 (red) on frozen liver tissue sections harvested on day 6. Nuclei are counterstained with DAPI. Arrows point to N-Ras/shRen-expressing foci exhibiting induced levels of the secreted chemokine Mcp1.