Fig 2: Model of neuronal activity-triggered stabilization of Mmp-9 mRNA. (A) In the non-depolarized hippocampal neurons, a few molecules of HuB and HuR are bound to the Mmp-9 mRNA 3'UTR (HuB is attached to ARE1 and HuR is associated with ARE1 and ARE4). As a result, Mmp-9 mRNA decay greatly enhances Mmp-9 mRNA stabilization, and Mmp-9 mRNA is expressed at low levels. (B) After seizure induction (2 h), there is increased binding of HuB to ARE1 as well as HuR to ARE1 and ARE4 motifs of the Mmp-9 mRNA 3'UTR. This leads to significant augmentation in Mmp-9 mRNA stability and, consequently, in its cellular abundance. (C) The interactions of HuR with ARE1 and ARE4 are reduced at 4 h after seizure induction, whereas HuB binding to ARE1 remains stably enhanced, which maintains elevated Mmp-9 stabilization and generates further accumulation of Mmp-9 transcripts.

Fig 3: (A) Vemurafenib dose response of A375 cells comparing noninfected cells (NI, dotted line) with the control adenovirus expressing GFP (aG, gray line) or the T7 epitope-tagged HuR adenovirus (aH, blue line) infected cells: a to b comparison indicates an inverse dose effect of vemurafenib on paradoxical proliferation. b to c comparison (performed in the same experiment) indicates a dose-suppressive effect of HuR overexpression on paradoxical proliferation. (B) Vemurafenib dose response for various BRAFV600E-sensitive melanoma cell lines (A375, Malme-3M) and BRAFV600E-resistant melanoma (MEL-CLS-3) and colon carcinoma (HT-29) cell lines: paradoxical proliferation is observed at low dose in A375 and to lower extend in MEL-CLS-3 cells. (C) Top panel: Western blot analysis using a mouse monoclonal antibody (3A2) on A375 whole-cell extracts infected with aG or aH at the indicated multiplicity of infection (m.o.i.). Bottom panel: Western blot analysis of HuR expression in A375 cytoplasmic (C) and nuclear (N) compartments following aG or aH infection (shown for the highest m.o.i. used). Note the slight tag-induced shift in aH samples. An additional second upper band (tagged HuR) is clearly visible especially in cytoplasmic extracts in aH samples. (D) WST-1 cell proliferation assay: at both indicated m.o.i. values, the proliferation rates of the aG- or aH-infected A375 cells are not significantly affected compared with noninfected (NI) cells. (E) GFP fluorescence in aG-infected cells and T7 epitope tag (Alexa 568) staining in aH-infected cells at the indicated m.o.i.: more than 90% of the cells are stained homogenously even at the lower m.o.i. value. Cell nuclei were stained with DAPI (blue). Note that HuR (T7 epitope tag) stains primarily in the nucleus. (F) Cell death effects of HuR overexpression in low-dose vemurafenib-treated A375 melanoma cells: flow cytometry analysis of double-stained (propidium iodide and Annexin V) A375 cells infected with aG or aH at the indicated m.o.i. and treated with either excipient (DMSO) or vemurafenib (100 nmol/L). A total of 25,000 events were analyzed for each sample. Black portion of the histograms represents the percentage of Annexin V-positive cells within the percentage of dead cells (gray plus black). Their percentages are indicated in the black portion of the histograms. (G) A375 cells infected with aG and aH at m.o.i. 25 and treated with vemurafenib at the indicated concentrations were stained with propidium iodide for cell cycle analysis gated on live cells (blue: G1 phase, yellow: S phase, green: G2/M phase). Corresponding percentage of cells in each phase is indicated with histograms. (H) viSNE and SPADE single-cell mass cytometry analysis of the Ki67 proliferation marker shown for noninfected (NI) excipient-treated A375 cells. The color scale line gives an indication of the fold change in expression. For the Sp1 fast-proliferating cells, the distribution of Ki67 expression is shown for NI cells (gray), aG cells (green, at the indicated m.o.i. values), and aH cells (blue). Hatched curves correspond to vemurafenib-treated cells (20 nmol/L). Note that, although not detected in the initial proliferation assay (D), a small vector-induced inhibitory effect is detectable in aG cells (at both m.o.i. values, compared with each other and to the NI cells). This inhibitory effect does not prevent the occurrence of paradoxical proliferation. The aH-infected cells at m.o.i. 25 are not shown (See Fig. 2 legend). The dashed line helps the comparison.

Fig 4: Nucleolin, CAS and HuR identify the nucleolus in HeLa cells treated with actinomycin D.HeLa cells were incubated with (A) the solvent DMSO or (B) 100 nM actinomycin D according to ref. [21]. Following treatment, samples were processed as in Fig. 2, and confocal images were used to identify nucleoli. Comparison of the segments and their overlay reveals that in control and actinomycin D-treated cells, all of the three proteins served as appropriate markers for nucleoli. Size bar is 20 µm.

Fig 5: CAS and HuR, but not nucleolin, delimit nucleoli in DRB-treated HeLa cells.Cells were incubated with (A) DMSO or (B) DRB essentially as described [21] and processed as in Fig. 2. Individually, the marker proteins CAS and HuR detected nucleoli upon DRB incubation, although some nucleoli were missed (indicated by arrow heads). The identification of nucleoli was improved by combining the information from CAS and HuR images with the add function [21]. Nucleolin was redistributed by DRB throughout the nucleoplasm. Based on the nucleolin image, neither the “detect light holes” nor “detect dark holes” filter could identify nucleoli. Size bar is 20 µm.