Figure 1

APOBEC3G (A3) inhibits DND1 function. (a) Diagram of luciferase construct pGL3-P27-3’-UTR showing miR-221 (blue) and DND1 binding sites (red) [4]. (b) DND1 blocks the effect of miR-221. Luciferase activity of pGL3-P27-3’UTR (or luc-P27 ) alone (lane 1); in the presence of miR-221 (lane 2); in the presence of DND1 (lane 3); in the presence of miR-221 and DND1 (lane 4). (**) miR-221 inhibited luc-P27 expression (P = 0.005) and (#) DND1 rescued the inhibition (P = 0.011). LacZ constructs were co-transfected into cells and β-galactosidase used for normalization of transfection efficiencies. Results are the mean and standard error from 3 independent experiments. (RLU = relative luminescence unit is the ratio between luciferase and β-galactosidase level, adjusted to 100%). Vectors encoding 1 ng luciferase, 50 ng miR, 10 ng DND1 and 10 ng APOBEC3G were used in all assays. (c) APOBEC3G counteracts the effect of DND1. Luciferase activity of pGL3-P27-3’UTR in presence of miR-221, DND1 and APOBEC3G (lane 4). (**) miR-221 inhibited luc-P27 (P= 0.004) (lane 2), (##) DND1 rescued the inhibition (P= 0.008) (lane 3) whereas ($$) APOBEC3G (A3) opposed the effect of DND1 (P = 0.009) (lane 4). Differences between (**) lane 2 and ($$) 4 are not statistically significant (P=0.0859). (d) pGL3-Cx43-3’UTR (Luciferase-CX43-3’UTR or luc-CX43) was cotransfected with miR-206, DND1 and APOBEC3G. (*) miR-206 inhibited luc-CX43 (P= 0.017) (lane 2), (#) DND rescued the inhibition (P= 0.024) (lane 3) whereas ($) A3 opposes DND1(P =0.016) (lane 4). Differences between (*) lane 2 and ($) 4 are not statistically significant (P= 0.17). (e) pGL3-3’UTR-LATS2 (Luciferase-LATS2-3’UTR or luc-LATS2) was cotransfected with miR-372, DND1 and APOBEC3G. (**) miR-372 inhibited luc-LATS2 (P= 0.001) (lane 2), (##) DND rescued the inhibition (P= 0.001) (lane 3), ($$) whereas A3 opposes DND1 (P = 0.001) (lane 4). Differences between (**) lane 2 and ($$) 4 are not statistically significant (P=0.176). (f) Control experiments demonstrate no significant effect of DND1 (lane 2) (P = 0.2860) or APOBEC3G alone (lane 3) (P =0.4356) on pGL3-p27-3’UTR; (g) no significant effect of DND1 (lane 2) (P = 0.1426) or APOBEC3G alone (lane 3) (P = 0.4196) on pGL3-Cx43-3’UTR; (h) no significant effect of DND1 (lane 2) (P = 0.1779) or APOBEC3G alone (lane 3) (P = 0.4615) on pGL3-3’UTR-LATS2. (i) Control experiments demonstrate no significant effect when DND1 together with APOBEC3G (lane 2) are transfected with pGL3-p27-3’UTR (P = 0.3433), (j) or pGL3-Cx43-3’UTR (P= 0.3565), (k) or pGL3-3’UTR-LATS2 (P = 0.4183). (l) Expression of HA-tagged DND1 (arrow) and myc-tagged APOBEC3G (myc-A3) (arrow) in 293T cells (lane 1) and 293T transfected with DND1-HA and APOBEC3G-myc (lane 2). Immunoblotting was using anti-HA, anti-myc and anti-GAPDH. (m) Expression of endogenous DND1 (arrow) and APOBEC3G (arrow) in 293T cells (lane 1) and 293T transfected with DND1-HA and APOBEC3G-myc (lane 2). Immunoblotting was using anti-DND1, anti-APOBEC3G and anti-GAPDH antibodies.