Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2013 Jul 26;14:16. doi: 10.1186/1471-2199-14-16

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 2013 Ali et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PMC Copyright notice

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.