Fig 1: Roquin2 destabilizes mRNAs of angiogenic factors via the ROQ domain. (A) The half-lives of indicated angiogenic mRNAs were shortened by Roquin2 in MDA-MB-231 cells. (B) Cell lysates were isolated from the MDA-MB-231 cells infected with Roquin2-overexpressing adenovirus, followed by western blot analysis with GFP, ENG, EDN1, VEGFB, PDGFC, and β-actin antibodies. (C) Schematic representation of the domains in Roquin2, and their truncation mutants. (D) The expression of Roquin2 and its truncation mutants were confirmed by immunoblotting with anti-GFP antibody. (E) The expression of indicated angiogenic factors mRNAs were measured by qPCR after overexpressed Roquin2 and its mutants in MDA-MB-231 cells. (F) Relative luciferase activities of the indicated reporters were determined by the luciferase reporter assay. (G) Quantification of the number of branching points of HUVECs treated with indicated tumor CMs in the tube formation assay. Results shown represent the mean ± SEM of three independent experiments; unpaired Student's t-test, *P < 0.05.
Fig 2: Roquin2 depletion increases angiogenic gene transcripts stability and promotes tumor angiogenesis. (A) Stable knockdown of Roquin2 protein expression by infecting Roquin2-shRNAs/lentivirus and scramble-shRNA/lentivirus in MDA-MB-231 and MDA-MB-468 cells, respectively, assessed by western blot. Western blot quantification was performed by ImageJ software. (B) The angiogenic factors mRNAs were measured in the MDA-MB-231 and MDA-MB-468 cells, respectively, after knockdown Roquin2 by qPCR. (C) The half-lives of indicated angiogenic genes were increased after Roquin2 knockdown in MDA-MB-231 cells. (D) ELISA quantification of EDN1 and PDGFC in serum-free culture medium of MDA-MB-231/shRoquin2 cells. Data are expressed in ng/100 µl of CM. (E) Quantification of open area of HUVECs treated with indicated tumor CMs in the wound-healing assay. (F) Quantification of the number of branching points of HUVECs treated with indicated tumor CMs in the tube formation assay. (G) MDA-MB-231/shRoquin2 cells were infected with scramble/lentivirus or shRNA-lentivirus targeting EDN1 and PDGFC, respectively. Total RNA extracted to measure mRNAs of EDN1 and PDGFC. (H) Quantification of open area of HUVECs treated with indicated tumor CMs. Unpaired Student's t-test, *P < 0.05, **P < 0.01.
Fig 3: Roquin2 targets the stem-loop structure in the 3'UTR of angiogenic genes for mRNA degradation. (A) The diagram of the luciferase reporter constructs of EDN1 and PDGFC containing truncated 3'UTRs without the stem-loop structure. (B) Relative luciferase activities of the indicated reporters were determined by the luciferase reporter assay. (C) Schematic representation of the luciferase reporter constructs of human β-actin 3'UTR containing the stem-loop structure of EDN1 (w/EDN1 stem-loop) or PDGFC (w/PDGFC stem-loop). (D) Relative luciferase activities of the indicated reporters were determined by the luciferase reporter assay. (E) The predicted stem-loop structures of EDN1 (top) and PDGFC (bottom) in their 3'UTRs and mutation strategy (asterisks indicate base substitution). Mutant1 becomes unable to form stem-loop structure (middle) and Mutant2 still forms a stem-loop structure (left). (F) Relative luciferase activities of the indicated reporters were determined by the luciferase reporter assay. (G, H) RNA-ChIP was conducted with genome fragments from MDA-MB-231/Roquin2-GFP cells. The pulled down 3'UTR sequences were amplified by PCR with primers flanking the putative stem-loop sequences. Results shown represent the mean ± SEM of at least three independent experiments. Unpaired Student's t-test, *P < 0.05.
Supplier Page from Thermo Fisher Scientific for Human PDGF-C ELISA Kit