Fig 1.
ASPP2 increases oncogenic H-ras, K-ras and N-ras nanoclustering.
(A) Top, scheme explaining nanoclustering-FRET analysis in HEK cells. Green and red ovals represent mGFP- and mCherry-tags, respectively. Bottom, examples of FLIM-FRET images of HEK cells from the different FRET samples as indicated. (B-D) Nanoclustering-FRET analysis in HEK cells coexpressing mGFP- and mCherry-tagged (B) H-rasG12V, (C) K-rasG12V or (D) N-rasG12V. The effect of Gal-1 or ASPP2 expression on nanoclustering-FRET was compared to control samples. Statistical significance of differences between controls and treated samples was examined using one-way ANOVA (mean ± SEM, n = 3; ns, not significant; *, p<0.05, ****, p< 0.0001). (E-G) Electron microscopic nanoclustering analysis of BHK cells expressing mGFP-tagged (E) H-rasG12V, (F) K-rasG12V or (G) N-rasG12V alone or together with ASPP2. mGFP was immunolabeled with 4.5 nm gold nanoparticles coupled to anti-GFP antibody. The spatial distribution of gold particles was evaluated using univariate K-function, where L(r)–r values indicate the extent of nanoclustering as a function of length scale, r, in nm. At least 15 images were analysed for each condition. Statistical significance between different conditions was evaluated using bootstrap tests. Averaged curves are shown for each condition.
Fig 2.
ASPP2 increases oncogenic H-ras, K-ras and N-ras-effector-recruitment, as well as ERK- and AKT-signalling.
(A) Left, scheme explaining effector-recruitment FRET analysis in HEK cells. Right, examples of FLIM-FRET images of HEK cells from the different FRET samples as indicated. (B-D) Effector-recruitment FRET analysis in HEK cells coexpressing (B) mGFP-H-rasG12V, (C) mGFP-K-rasG12V or (D) mGFP-NrasG12V and mRFP-RBD from C-Raf. The effect of Gal-1 or ASPP2 expression on effector-recruitment FRET was compared to control samples. (E) Representative Western blots from HEK cells expressing mGFP-H-rasG12V (left), K-rasG12V (middle) or N-rasG12V (right) without or with Gal-1 or ASPP2. Statistical significance of differences between controls and treated samples was examined using one-way ANOVA (mean ± SEM, n = 3; ns, not significant; *, p<0.05, **, p< 0.01, ***, p<0.001, ****, p< 0.0001).
Fig 3.
N- and C-terminal truncation mutants of ASPP2 can still promote Ras nanoclustering.
(A) Schematic of full-length ASPP2, as well as ASPP2(1–360) and ASPP2(123–1128) truncation mutants. ASPP2 domains from left to right: Ubl, ubiquitin-like domain; α-helical domain; Pro, proline-rich domain; Ank, Ankyrin repeats; SH3, SRC homology 3 domain. (B) Confocal microscopic images of HEK cells cotransfected with mGFP-H-rasG12V (green) and full-length or truncated ASPP2 (red). (C-E) Nanoclustering-FRET analysis of HEK cells coexpressing mGFP- and mCherry-tagged (C) H-rasG12V, (D) K-rasG12V or (E) N-rasG12V. Cells were analysed after overexpression of Gal-1, full-length ASPP2 or its truncation mutants. (C-E) Statistical significance of differences between controls and treated samples was examined using one-way ANOVA (mean ± SEM, n = 3; ns, not significant; ****, p< 0.0001). (F) Western blot of anti-GFP immunoprecipitation samples probed with anti-ASPP2- (top) or anti-GFP- (bottom) antibodies. Samples were lysates prepared from mGFP-H-rasG12V transfected HEK cells that were cotransfected with full-length ASPP2 or its truncation mutants or an empty plasmid (control), as indicated. In, input; Ft, flow-through; W1, wash; E, elution. Red boxes indicate the immunoprecipitated ASPP2 fragments.
Fig 4.
ASPP2 blocks Gal-1 dependent nanoclustering and halts oncogenic H-ras induced transformation.
Nanoclustering-FRET analysis in HEK cells coexpressing mGFP- and mCherry-tagged (A, B) H-rasG12V or (C, D) K-rasG12V. Cells were analysed after overexpression of either Gal-1 or ASPP2 plasmids, or both (1:1 ratio). Plotted are the means ± SEM, n = 3. (E) Representative Western blots from HEK cells expressing mGFP-H-rasG12V (left) or K-rasG12V (right) alone or together with Gal-1 and ASPP2. Statistical significance of differences was examined using t-test (n = 3; *, p<0.05, **, p< 0.01, ***, p<0.001). (F, G) Colony survival assay of NIH/3T3 cells stably expressing (F) H-rasG12V or (G) K-rasG12V and transiently expressing indicated constructs. Colony survival was graphed based on mean foci areas calculated from at least 4 independent biological repeats. (A-D, F-G) Statistical significance of differences between controls and treated samples was examined using one-way ANOVA (ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001).
Fig 5.
ASPP2 dominates over Gal-1 thus robustly inducing senescence and abrogating mammosphere formation.
(A) SA-β-gal assay of MCF-7 cells transfected with plasmids encoding H-rasG12V, ASPP2, Gal-1 or the combination of the latter two, as indicated. Cells were stained 7 days after transfection. On the left, percentages of SA-β-gal positive cells are shown in the graph (mean ± SEM, n = 3). On the right, representative images from the assay. (B) Mammosphere formation assay with MCF-7, MDA-MB-231 or HS-578T breast cancer cell lines. Mammospheres were transfected with Gal-1, ASPP2, or both (1:1 ratio) and cells were then grown under non-adherent conditions for 9 days. On the right, representative images of mammospheres are shown as indicated. (A, B) Statistical significance of differences between controls and treated samples was examined using one-way ANOVA (mean ± SEM n≥3; ns, not significant; ****, p<0.0001).