Article Figures & Data
Figures
- Figure 1.
Silencing of HuR alters glioma growth and sensitizes cells to chemotherapeutic induction of apoptosis. A, the growth of U251 is constrained compared with controls over a 4-day time course. B, HuR silencing in U251 induces activation of the intrinsic pathway of apoptosis with cleavage of caspase-3 and PARP. The time course is after the addition of the siRNA oligos. C, colony formation in soft agar is markedly reduced for HuR knockdown compared with control. Quantitation of colony number in soft agar experiment is represented by the bar graph (*, P = 0.0016). D, the graph depicts a sensitization of glioma cell line to cytotoxic agents with HuR knockdown for common types of chemotherapy.
- Figure 2.
Inducible overexpression of HuR enhances bcl-2 expression and chemoresistance. A, the Flag-HuR inducible clones (n = 2) show a specific and marked increase in bcl-2 family interaction with HuR following induction (dox treated is normalized by the untreated) and immunoprecipitation with a control IgG or HuR antibody (experiment done in triplicate). The error bars represent standard deviation. An inset reveals robust expression of the Flag-tagged HuR with dox. B, the IC50 curves with standard cytotoxic agents in a cell viability assay show chemoresistance after the induction of HuR, a shift to the right is seen for all agents.
- Figure 3.
A specific molecular interaction exist between HuR and the bcl-2 family RNA. A, lane 1 shows the UVX pattern of glioma protein extract and 32P-labeled riboprobes for the indicated bcl-2 member 3′-UTR; lane 2 is an IP of the UVX with a HuR Ab to define the HuR band; and lane 3 is a control IP with normal mouse IgG. A (right), UVX using 4 riboprobes representing various regions of the mcl-1 3′-UTR (Supplementary Table S1 for definition of regions). The UVX lanes are overexposed to better visualize the HuR IP in lane 2. B, qRT-PCR for the bcl-2 family from total RNA isolated by HuR IP confirms an interaction. The bcl-2 family can be quantified from HuR IP compared with the control IP with IgG. C, a schematic illustrating the location of riboprobes (P) relative to bcl-2 family 3′-UTR and the open reading frame.
- Figure 4.
The knockdown of HuR primarily effects the degradation kinetics of the bcl-2 family. A, a significant difference was not noted in RNA expression levels for the bcl-2 family following knockdown with shHuR or shControl (replicates of 3 for 2 shHuR clones and 6 for single shControl clone). B, the degradation kinetics of the bcl-2 family was markedly reduced after silencing for HuR (replicates of 3)
- Figure 5.
Impact of HuR knockdown on bcl-2 family RNA distribution within the polyribosome and protein expression. A, the silencing of HuR in multiple clones with the concurrent reduction in protein expression of all bcl-2 family members in select clones (*) which were used for phenotypic studies. Band density was normalized to tubulin and then compared with U251 to generate percent expression (shown below the blot). B, Western blot analysis from protein samples of 12 fractions collected following polyribosome isolation. S6 and elf4E are ribosomal components and used for control. HuR is reduced in the polysome fractions of shHuR cells compared with controls. C, the quantitation of RNA from each fraction for GAPDH and the bcl-2 family. Overall, the patterns differ depending on the target with a slight increase in distribution to later fractions (8–12) following HuR knockdown. The data are presented so that each bar represents the percentage of RNA from that fraction divided by the total for all 12 fractions.
- Figure 6.
Lentivirus-mediated HuR silencing in established and primary GBM lines. A, fluorescent microscopy showing robust infectivity of control (V-GFP) and 3 shHuR GFP-lentiviral constructs (V1–3-shHuR) in U251. B, the lentivirus constructs robustly knockdown HuR. B, the quantitation of HuR RNA levels by TaqMan and a following Western blot show effective knockdown of HuR with each of the shHuR lentiviruses (V1–3) but not with control (V-GFP). The most consistent and robust was V2, which was carried forward in animal experiments. C, the 4 primary GBM lines available are shown in phase contrast (top) and after transduction with the GFP lentivirus (bottom). D, all lines were uniformly infected with 100% cellular transduction by 20 viral units per cell. E, Western blotting of protein extracts from D456 confirms knockdown of HuR in V2shHuR cells compared with shControl. Similar results were seen for the other primary GBM lines.
- Figure 7.
The GFP marker in the lentivirus facilitates in vivo assessment of GBM growth. A, whole brain dissection after sacrifice showing extensive and bilateral green fluorescence in the shControl mouse versus limited signal in the shHuR mouse. B, a background coronal section is shown at the top followed by a GFP image and then a subtraction. The subtraction allows for quantification of tumor volume and extent of infiltration and confirms the extensive growth and infiltration of the shControl-infected cells.
- Figure 8.
HuR knockdown reduces GBM tumor growth in vivo. A, coronal sections of mouse brains injected with shControl or V2shHuR-infected D456 GBM cells are shown. V2shHuR cells produced a smaller and less infiltrative tumor compared with shControl (arrows). B, representative tumor sections immunostained for HuR (red) and counterstained with DAPI (blue) showing reduced expression of HuR in the V2shHuR-infected GBM cells.
Tables
- Table 1.
Data for the 3 sets of animal experiments with each primary GBM line
Primary GBM line shControl Mice SEM shHuR Mice SEM P Primary endpoint is tumor area mm2 D456 98 5 11 62 5 1.5 0.0116 GBM12 31 5 8 12 5 4.7 0.10 GBM6 1.0 5 0.06 0.04 5 0.02 0.0001 Primary endpoint is number of distinct tumor foci GBM6 41 5 9 2.4 5 4 0.0002 NOTE: When tumor size is the primary endpoint, a consistent size difference is seen following silencing of HuR (shHuR). GBM6 shows a very migratory behavior and a difference in tumor foci is quantified as a second primary endpoint.
Supplementary Data
Supplementary Figures S1-S2.
Files in this Data Supplement:
Volume 9, Issue 5
