Rb inactivation does not change the histology and differentiation status of MYC-induced HCC.

<p><b>A.</b> Histology of non-tumor liver tissue from <i>MYC</i> mutant mice (left) and tumor tissue from <i>MYC</i> (center) and <i>MYC/Rb</i> (right) mutant mice. <b>B.</b> RT-qPCR analysis of <i>Albumin</i> (a marker of hepatocytes), <i>CK19</i> (a marker of bile ducts), and <i>Afp</i> (a marker of HCC) in control livers (white, n = 2), <i>MYC</i> mutant tumors (black, n = 3), and <i>MYC/Rb</i> mutant tumors (grey, n = 3). Note that the p-value for <i>Afp</i> between control livers and <i>MYC/Rb</i> mutant tumors is 0.06, just below the significance threshold.</p

Rb inactivation and MYC activation cooperate to increase ploidy in hepatocytes.

<p><b>A.</b> Genomic PCR analysis for the deleted allele of <i>Rb</i> (<i>Rb</i>^Δ) using DNA from control, <i>MYC</i>, <i>Rb</i>, and <i>MYC/Rb</i> mutant livers before the development of tumors in <i>Rosa26^+/CreER</i> mice. <i>Actin</i> serves as a positive PCR control. <b>B.</b> Quantification of immunostaining experiments for the number of Ki67⁺ liver cells per field on control, <i>MYC</i>, <i>Rb</i>, and <i>MYC/Rb</i> mutant liver sections 5 weeks after the deletion of <i>Rb</i> and the induction of <i>MYC</i>. The number of positive cells in eight 20× fields was counted in 2 mice for each treatment group. The differences observed are not significant in a t-test. <b>C.</b> Representative microphotographs of hepatocytes from a mouse injected with corn oil alone in the presence of doxycycline (wild-type for RB and MYC) and a mouse injected with tamoxifen in the absence of doxycycline are shown (<i>MYC/Rb</i> mutant liver). Arrows show a small nucleus in the control mouse and a large nucleus in the mutant mouse. <b>D.</b> Quantification of ploidy by FACS of hepatocyte populations from control (n = 5), <i>MYC</i> (n = 8), <i>Rb</i> (n = 5), and <i>MYC/Rb</i> (n = 8) mutant livers 5 months after <i>Rb</i> deletion (<i>Rosa26^+/CreER</i> mice) and MYC activation. Only statistically significant differences are shown.</p

Rb inactivation does not change the mRNA expression or protein levels of cell cycle markers in MYC-induced HCC.

<p><b>A.</b> RT-qPCR for several cell cycle genes on RNA from <i>MYC</i> (black, n = 3) and <i>MYC/Rb</i> (grey, n = 3) mutant HCC. <i>Rb^lox/lox MYC^Off</i> non-tumor liver is used as a control (CTRL) (white, n = 2). There is not statistical difference for any of these gene expression levels between <i>MYC</i> and <i>MYC/Rb</i> tumors. <b>B.</b> Representative immunoblot analysis for several cell cycle markers on protein extracts from <i>MYC</i> and <i>MYC/Rb</i> mutant HCC compared to wild-type liver. The concentration of the protein extracts was quantified using a Bradford assay and similar amounts of proteins were loaded in each lane. Actin serves as a loading control; note that CTRL liver cells express less Actin per µg of protein extract than tumor cells.</p

Rb inactivation does not affect the degree of proliferation or cell death of MYC-induced HCC.

<p><b>A–C.</b> Immunofluorescence staining for Ki67 (<b>A</b>), BrdU (<b>B</b>), and cleaved caspase 3 (CC3) (<b>C</b>) on <i>MYC</i> mutant non-tumor liver tissue and tumor tissue from <i>MYC</i> and <i>MYC/Rb</i> mutant mice. DAPI nuclear staining is used to indicate the density of cells on the sections. The pictures shown are representative of each group. <b>D.</b> Quantification of staining shown in A. The number of positively stained cells for each antibody over the number of DAPI stained cells expressed as a percent was determined using the BioQuant software. For each antibody and genotype combination the average of n = 2 mice was calculated where each n is the average percent of positive cells from three fields containing at least 250 cells each.</p

MYC activation and Rb deletion drive HCC development in the liver of adult mice.

<p><b>A.</b> Strategy to produce <i>MYC</i> and <i>MYC/Rb</i> mutant tumors using <i>Rb^lox/lox</i> and TRE-<i>MYC</i> LAP-<i>tTA</i> mice. MYC is activated in the liver by the removal of doxycycline from the drinking water while <i>Rb</i> is specifically deleted in the liver by splenic injection of Ad-Cre. Ad-GFP is used as a control. <b>B.</b> RT-qPCR analysis of <i>MYC</i> RNA levels in control livers (<i>Rb^lox/lox MYC^Off</i>, n = 2) and in <i>MYC</i> and <i>MYC/Rb</i> mutant tumors (n = 3). <b>C.</b> Immunoblot analysis of MYC protein levels in control livers (<i>MYC^Off</i>) and in <i>MYC</i> and <i>MYC/Rb</i> mutant tumors (two independent tumors each, T1 and T2). Actin serves as a loading control. <b>D.</b> Genomic PCR analysis for the deleted allele of <i>Rb</i> (<i>Rb^Δ</i>) using DNA from <i>MYC</i> and <i>MYC/Rb</i> mutant tumors. <i>Actin</i> serves as a positive PCR control. <b>E.</b> RT-qPCR analysis of <i>Rb</i> RNA levels in <i>MYC</i> (black, n = 3) and <i>MYC/Rb</i> (grey, n = 3) mutant tumors.</p

Survival analysis of Myc and Myc/Rb mutant mice.

<p><b>A.</b> Kaplan-Meier survival analysis of <i>MYC</i> (n = 27) and <i>MYC/Rb</i> (n = 19) mutant mice when both tumorigenic events were triggered simultaneously (time 0) in adult mice (8–12 weeks after birth) by removal of doxycycline and Ad-Cre infection. <b>B.</b> Survival analysis of <i>MYC/Rb</i> mutant mice developing HCC when both tumorigenic events were triggered simultaneously in adult mice (8–12 weeks after birth) (n = 19) and when <i>Rb</i> deletion by Ad-Cre was performed 4 weeks before MYC activation (8–12 weeks and 12–16 weeks after birth, respectively) (n = 15); time 0 is the time of MYC activation.</p