HuR overexpression in HeLa cells promotes cell survival. (A) At 12 h after UVC irradiation (30 J/m²) of HeLa populations that had been transfected with either a control plasmid (Vector) or a plasmid overexpressing HuR (HuR-T), cell viability was assessed by Hoechst staining to monitor the presence of condensed and fragmented nuclei; representative fields are shown. (B) Graphs quantifying apoptotic cell numbers in cultures treated as described in panel A; shown are the means±s.e.m. from three independent experiments. (C) Western blot analysis to monitor the abundance of ectopically expressed HuR (HuR-T), endogenous HuR (HuR), cleaved caspase-9, cleaved caspase-3, cleaved PARP, and GAPDH in whole-cell lysates (20 μg) prepared from cells that were treated as described in panel A. (D) HuR downregulation-triggered apoptosis (elicited through an siRNA targetting the HuR 3′UTR, HuR(3) siRNA) was rescued by HuR-T overexpression. Left, quantification of apoptotic nuclei, shown as the means±s.e.m. from three independent experiments; right, Western blot analysis of the levels of HuR and HuR-T in each population (HuR(3) siRNA reduced endogenous HuR levels to 20% of the levels seen in control (Ctrl. siRNA) cells).

UVC- and HuR-dependent increase in cytoplasmic abundance of the ProTα mRNA. (A) The levels of mRNAs encoding ProTα, cyclin D1, and p21, as well as loading control 18S rRNA were assessed by Northern blotting following UVC irradiation of HeLa cells. % mRNA, percent ProTα mRNA signals relative to 18S signals. (B) The abundance of ProTα was monitored in whole-cell lysates (Materials and methods) that were prepared 8 h after 30 J/m² UVC irradiation, size-fractionated by SDS–PAGE, and visualized by staining the gels with Coomassie blue. (C) Changes in the levels of mRNAs encoding ProTα, p21, and GAPDH (as well as 18S rRNA) in the cytoplasmic (Cytopl.) or Nuclear fractions (Materials and methods) 6 h after UVC irradiation (30 J/m²) were assessed by either RT+real-time PCR (left) or by Northern blotting (right). (D) Cells expressing normal HuR levels (Vector) or overexpressing HuR (pHuR-T, described in the legend of Figure 2) were either left untreated or irradiated with UVC. After 6 h, Cytopl. and Nuclear fractions were prepared and ProTα mRNA levels detected by RT+real-time PCR. Graphs depict the means±s.e.m. from four independent experiments. Controls to monitor the adequate preparation of nuclear and cytoplasmic lysates are available (Supplementary data).

HuR promotes the translation and cytoplasmic accumulation of chimeric ProTα. HeLa cells expressing either normal (Ctrl. siRNA) or knocked-down HuR levels (HuR siRNA) were subsequently transfected with plasmids encoding EGFP or EGFP-ProTα; 24 h later, the expression levels of HuR, EGFP-ProTα, EGFP, and GAPDH were assessed by Western blotting (A) and the nascent translation rates of EGFP and EGFP-ProTα were examined as described for Figure 5E (B). HeLa cells expressing either higher (pHuR-T) or normal HuR levels (Vector) were transfected with plasmids encoding EGFP or EGFP-ProTα, and 24 h later the expression of HuR, HuR-T, EGFP-ProTα, EGFP, and GAPDH was assessed by Western blotting (C), and the nascent translation rates of EGFP and EGFP-ProTα were monitored as described in the legend of Figure 5E (D).

Downregulation of HuR expression in HeLa cells decreases cell survival. (A) At the times indicated after irradiation of HeLa cells with 30 J/m² UVC, cytoplasmic (Cyto., 10 μg) and whole-cell (Total, 20 μg) lysates were prepared and the abundance of HuR, cleaved PARP (a marker of apoptosis), and GAPDH (loading control) was assessed. (B) At 48 h after transfection with 20 nM of either a control siRNA (Ctrl. siRNA) or an siRNA targeting HuR (HuR siRNA), cells were either left untreated (Untr.) or were irradiated with UVC (UVC); cell viability 8 h later was assessed by Hoechst staining to monitor the presence of condensed and fragmented nuclei. Shown are representative fields from three independent experiments. (C) Graphs quantifying apoptotic cell numbers in cultures treated as explained in the legend of panel B; shown are the means±standard error of the means (s.e.m.) from three independent experiments. (D) Western blot analysis to monitor the abundance of HuR (with 10% remaining after siRNA-mediated knockdown), cleaved caspase-9, cleaved caspase-3, cleaved PARP, and GAPDH in whole-cell lysates prepared from cells that were treated as explained in the legend of panel B.

HuR binds endogenous and recombinant ProTα transcripts. Schematic of ProTα mRNA, depicting AREs (gray), as well as the transcripts (5′UTR, CR, and 3′UTR) used in biotin pulldown analysis. (A) Whole-cell lysates were prepared from HeLa cells and binding of equimolar amounts of the biotinylated transcripts (Supplementary data) to either HuR or hnRNP A1 was tested by biotin pulldown analysis; representative Western blots are shown. (B) The binding of endogenous HuR to endogenous target transcripts was detected by RT–PCR assay of material obtained by immunoprecipitation (IP) using either IgG1 or anti-HuR antibodies. Amplification of housekeeping transcripts encoding GAPDH and SDHA, bound at low levels with the IP material, showed equal loading of IP samples. PCR products are shown.

UVC-triggered increase in ProTα mRNA levels in polysomes, elevation of ProTα translation, and cytoplasmic accumulation of chimeric ProTα. (A) Representative polysome distribution profiles obtained after centrifugation of cytoplasmic lysates (prepared from either irradiated cells (6 h after receiving 30 J/m² UVC) or unirradiated cells) over sucrose gradients. From left to right, fractions lacked ribosomes or ribosome subunits (fractions 1, 2), contained ribosome subunits or single ribosomes (fractions 3–5), or spanned polysomes of increasing molecular weight (fractions 6–10). From each fraction, RNA was prepared for Northern blot analysis of ProTα mRNA and 18S rRNA, and protein was prepared for Western blot analysis of HuR and β-actin. (B) The polysome-associated levels of ProTα mRNA as well as those of control mRNAs encoding p21 and cyclin D1 (UVC-regulated) and those encoding UBC, GAPDH, and SDHA (not UVC-regulated) were quantified by RT+real-time PCR. (C) The abundance of HuR-bound mRNAs encoding either ProTα or GAPDH in the cytoplasmic fraction (left, 500 μg) or in pooled gradient fractions 1–5 and 6–10 (right) was investigated by HuR IP followed by RT+real-time PCR; data are shown as relative enrichment over the signals obtained using IgG1 IP in samples assayed by RT+real-time PCR. Graphs depict the means±s.e.m. from four independent experiments. (D) Schematic of plasmids used in transfections to express either EGFP (pEGFP) or chimeric EGFP-ProTα (pEGFP-ProTα). Gray, EGFP cDNA; black, ProTα CR; white, ProTα 3'UTR. (E) Newly translated EGFP and EGFP-ProTα were assessed by incubating unirradiated cells (Untr.) or cells irradiated 6 h earlier (UVC) with L-[³⁵S]methionine and L-[³⁵S]cysteine for 20 min. Following IP using either IgG or anti-EGFP antibodies, samples were resolved by SDS–PAGE and transferred onto membranes for visualization of ³⁵S-radiolabeled signals. (F) EGFP and EGFP-ProTα expression levels in cytoplasmic lysates (20 μg) were determined by Western blotting using an anti-EGFP antibody. Assessment of hnRNP C1/C2 (a nuclear protein) and α-tubulin (a cytoplasmic protein) levels served to monitor the adequate preparation and loading of the cytoplasmic fractions. Data are representative from three independent experiments. Lys., whole-cell lysate (20 μg).

HuR- and ProTα-engendered protection against UVC are mutually dependent. (A) HeLa populations that expressed either normal or reduced HuR levels (Ctrl. siRNA and HuR siRNA, respectively, described in the legend of Figure 1) were transfected with plasmids that expressed either chimeric ProTα (EGFP-ProTα) or EGFP (as explained in the legend of Figure 6) and irradiated with UVC; apoptotic nuclei were scored 8 h later. (B) Cells that either overexpressed HuR or expressed normal HuR levels (HuR-T and Vector, respectively, described in the legend of Figure 2) were transfected with plasmids that expressed chimeric ProTα (EGFP-ProTα) or EGFP (EGFP) (Figure 6), and apoptotic nuclei were scored 12 h after UVC irradiation. (C) HeLa cells were incubated either with an oligomer that inhibited ProTα translation (AS oligo) or with a control oligomer (S oligo) (Sburlati et al, 1991), whereupon the levels of ProTα expression were assessed by monitoring mRNA levels (Real-time PCR), protein abundance (Western), and protein biosynthesis (Nascent translation). (D) The cell populations described in panel B were incubated with either AS oligo or with S oligo; 8 h after UVC irradiation, the percentage of apoptotic nuclei were quantified (left), and protein lysates were prepared to assess the degree of PARP cleavage by Western blotting (right). GAPDH signals revealed even loading and transfer of samples; Western data are representative of three independent experiments. Graphs represent the means±s.e.m. from three independent experiments.