Supplementary Figure 6: Notch1 is upregulated during in vivo Ras-induced senescence (RIS) and mediates lateral induction of senescence and Notch signalling.
From: NOTCH1 mediates a switch between two distinct secretomes during senescence
(a) Pancreatic tissue from p48-cre; LSL-KrasG12D and matched control mice was analysed by immunohistochemistry (IHC) for Dec1 and Notch1; insets demonstrate the staining pattern within normal pancreatic ducts (upper, arrowhead), pancreatic intraepithelial neoplasia (PanIN) (middle) and acinar-ductal metaplasia (ADM) (lower). Scale bar 200 μm. (b) Time series analysis of serial sections of murine liver tissue 9 days after HDTV-injection of NRASG12V-IRES-mVenus or NRASG12V-IRES-dnMAML1-mVenus stained by IHC for NRAS and Hes1; quantitation of positivity for Hes1 in NRAS-positive cells; bars are means; unpaired t-test; n = 3 mice per time point; ∗P ≤ 0.05. (c) Representative images of two-colour IHC staining of murine liver sections 9 days after HDTV-injection of NRASG12V-IRES-mVenus for NRAS (brown) and Hes1 (red) demonstrating Hes1-positive/ NRAS-negative cells (asterisk) adjacent to NRAS-positive hepatocytes. Arrows demonstrate Hes1-negative cells for comparison. Scale bar 50 μm. (d,e) IHC for NRAS and p21 on serial murine liver sections at indicated time points after HDTV injection of NRASG12V-IRES-mVenus or NRASG12V-IRES-dnMAML1-mVenus. Representative IHC images; Insets, magnified picture of dotted rectangular areas. Asterisk demonstrates p21-positive/NRAS-negative cells adjacent to NRAS-positive hepatocytes (d). Scale bar 200 μm. Quantitation of p21 expression in NRAS-positive and negative hepatocyte populations (e). Hepatocytes were dichotomised into NRAS-positive (top) or negative (bottom) populations by automated image analysis and then p21 expression analysed in these 2 populations; values are mean ± s.e.m. from automated image analysis of ≥105 cells per liver section; for NRASG12V injected animals at D6, 9 & 12, n = 3, 3 & 4 mice respectively; for NRASG12V-IRES-dnMAML1 injected animals at D6, 9 & 12, n = 4, 3 & 5 mice respectively; unpaired t-test. (f) Representative immunohistochemistry (upper) and immunofluorescence (lower) of CD3-expressing T-lymphocytes within the livers of mice injected with either NRASG12V-IRES-mVenus or NRASG12V-IRES-dnMAML1-mVenus. Arrows indicate CD3+ T-lymphocytes. (See also Fig. 6c) Scale bar upper panels 200 μm, lower panels 25 μm. (g) Quantification of B220 (B-lymphocyte marker) positive cells within indicated livers as in Fig. 6c. Values are mean ± s.e.m. from automated image analysis of ≥105 cells from liver section; for NRASG12Vinjected animals at D6, 9 & 12, n = 3, 3 & 4 mice respectively; for NRASG12V-IRES-dnMAML1 injected animals at D6, 9 & 12, n = 4, 3 & 5 mice respectively. Statistics source data for b,e and g are provided in Supplementary Table 2.
