Figure 3

Effect of pRb phosphorylation status on integrin α10 transcription. (a) Three integrin α10 promoter deletion constructs (Δ590, Δ463, Δ163) and a p27 promoter construct control were co-transfected separately with either pEGFPc2 (control), pRb wild type large pocket (Rb wt LP), or pRb non-phosphorylatable large pocket (Rb 7 LP) into MC3T3 pRb-null cells. Promoter activation was measured by luciferase activity. p27 control activity is set to one. (b) Western blot of control (pEGFPc2), pRb wild type large pocket (Rb wt LP), and pRb non-phosphorylatable large pocket (Rb 7 LP) transfected into MC3T3 pRb-null cells. Blot was probed for pRb (9309; Cell Signaling, Boston, MA, USA) and β-Actin (A5441; Sigma). (c) Expression of endogenous integrin α10 mRNA in MC3T3 wild-type cells treated with the CDK 4/6-specific drug, PD0332991 at two different doses (200 nM and 500 nM). RNA extraction was performed 48 h post drug treatment followed by qRT-PCR with integrin α10-specific primers to determine expression levels relative to GAPDH. The untreated MC3T3 pRb wild type expression levels were set as one. Asterisks represent significant P-values as follows: *P<0.05, **P<0.01, and ***P<0.001