Figure 7
Elevated FAM83A expression is essential for growth and tumorigenicity of murine KPC cells. (a) FAM83A mRNA expression in spontaneous pancreatic tumours of LSL-KRAS-G12D LSL-p53-R172H Pdx1-Cre (KPC) transgenic mice (n = 10) compared to normal murine pancreas from C57BL/6 (n = 5) and control Pdx1-Cre (n = 3) mice. Statistical significance of p < 0.01 as per the unpaired Mann-Whitney test. (b) Pancreatic ductal epithelial cells isolated from the pancreas of LSL-KRAS-G12D LSL-p53-R172H (KP) mice were infected in vitro with retroviruses encoding Cre or control vector (pB). Immunoblotting for mutant KRAS and p53 confirmed generation of KP-Cre cells in vitro and anchorage-independent growth in soft agar was assessed as indication of transformation. FAM83A expression in control KP-pB and KP-Cre cells upon 24 h serum stimulation (c) and in KP-Cre cells upon serum stimulation following pre-treatment with MEK and ERK inhibitors (d). (e–g) KP-pB and KP-Cre cells were subjected to FAM83A knockdown using lentivirus encoding shRNA targeting mouse FAM83A (shA4) or control (shGFP) and assessed for FAM83A expression by qRT-PCR (e; left), growth in culture (e; right). KP-pB and KP-Cre cells with FAM83A knockdown were also assessed for anchorage independent growth in soft agar (f) and for in vivo tumorigenicity upon subcutaneous injections (n = 4) in the flanks of immunocompromised mice (g). Images of the tumours were from day 21 post inoculation. All data in (b–g) are representative of two independent experiments. All bar graphs show mean ± SD. Student’s t-test was used to assess statistical significance with ** and *** representing p < 0.01 and p < 0.001 respectively.
