Journal: Nature Communications
Article Title: SHANK3 depletion leads to ERK signalling overdose and cell death in KRAS-mutant cancers
doi: 10.1038/s41467-024-52326-1
Figure Lengend Snippet: a Representative images of GFP-SHANK3 and mCherry-KRASG12V localisation in A549 cells (maximum projections shown; one experiment with this cell line). Insets and yellow arrows indicate colocalization of GFP-SHANK3 with mCherry-KRASG12V at membrane protrusions. b KRAS–SHANK3 SPN-ARR in an open conformation modelled by aligning the RBD and SPN domains of RAF and SHANK3, respectively, on a membrane composed of POPC (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)/ Phosphatidylinositol 4,5-bisphosphate/ Cholesterol. c Structural alignment between KRAS–SHANK3 SPN-ARR (model) and nanodisc-bound KRAS–RAF complex (PDB:6PTW). d Analysis of RAF-RBD–KRAS binding in the presence of the SHANK3 SPN domain using the depicted pulldown assay. Samples were resolved on SDS-PAGE gel and stained with Coomassie. A representative gel is shown (three independent experiments). e Quantification of relative FRET efficiency between GFP-KRASG12V (FRET donor) and mRFP-RAF-RBD (FRET acceptor) in siCTRL or siSHANK3 (smartpool SHANK3 siRNA) HEK293 cells. Shown are the individual data points [mean ± s.d., n = 79 (siCTRL) or 87 (siSHANK3) from three independent experiments. Unpaired two-tailed Student’s t -test with Welch’s correction]. f A representative immunoblot and quantification of ERK activation levels (phospho-ERK1/2 (Thr202/Y204) / total ERK relative to loading) in HCT-116 cells expressing GFP-SHANK3 SPN WT or GFP-SHANK3 SPN R12E/K22D (data represent the individual values; mean ± s.d.; mean of control is set to 1.0 by definition; three independent experiments; Kruskal-Wallis one-way ANOVA and Dunn’s post hoc test). g Representative confocal images (middle plane) and quantification of nuclear ERK (indicating ERK activity) in MIA PaCa-2 cells. Yellow arrowheads point to representative nuclei. N/C, nuclear to cytoplasmic ratio. Shown are the individual data points and the population average of each biological replicate (mean ± s.d.; three independent experiments; one-way ANOVA with Holm-Sidak’s multiple comparison test). h Representative images and quantification of tumour growth of HCT-116 cells, transiently expressing GFP-SHANK3 SPN WT or GFP-SHANK3 SPN R12E/K22D, on CAMs. Tumours are delimited by the yellow circles [mean ± s.d.; n = 21 (GFP, SPN WT) or 19 (SPN R12E/K22D) tumours from two independent experiments; Kruskal-Wallis one-way ANOVA and Dunn’s post hoc test]. Source data are provided as a Source Data file.
Article Snippet: The KRAS pulldown assays were done by incubating the indicated recombinant proteins with RAF-RBD glutathione affinity beads from the Ras Pull-Down Activation Assay Biochem Kit (Cat. no. BK008, Cytoskeleton) according to the manufacturer’s instructions.
Techniques: Membrane, Binding Assay, SDS Page, Staining, Two Tailed Test, Western Blot, Activation Assay, Expressing, Control, Activity Assay, Comparison
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![a Representative images of GFP-SHANK3 and mCherry-KRASG12V localisation in A549 cells (maximum projections shown; one experiment with this cell line). Insets and yellow arrows indicate colocalization of GFP-SHANK3 with mCherry-KRASG12V at membrane protrusions. b KRAS–SHANK3 SPN-ARR in an open conformation modelled by aligning the <t>RBD</t> and SPN domains of <t>RAF</t> and SHANK3, respectively, on a membrane composed of POPC (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)/ Phosphatidylinositol 4,5-bisphosphate/ Cholesterol. c Structural alignment between KRAS–SHANK3 SPN-ARR (model) and nanodisc-bound KRAS–RAF complex (PDB:6PTW). d Analysis of RAF-RBD–KRAS binding in the presence of the SHANK3 SPN domain using the depicted pulldown assay. Samples were resolved on SDS-PAGE gel and stained with Coomassie. A representative gel is shown (three independent experiments). e Quantification of relative FRET efficiency between GFP-KRASG12V (FRET donor) and mRFP-RAF-RBD (FRET acceptor) in siCTRL or siSHANK3 (smartpool SHANK3 siRNA) HEK293 cells. Shown are the individual data points [mean ± s.d., n = 79 (siCTRL) or 87 (siSHANK3) from three independent experiments. Unpaired two-tailed Student’s t -test with Welch’s correction]. f A representative immunoblot and quantification of ERK activation levels (phospho-ERK1/2 (Thr202/Y204) / total ERK relative to loading) in HCT-116 cells expressing GFP-SHANK3 SPN WT or GFP-SHANK3 SPN R12E/K22D (data represent the individual values; mean ± s.d.; mean of control is set to 1.0 by definition; three independent experiments; Kruskal-Wallis one-way ANOVA and Dunn’s post hoc test). g Representative confocal images (middle plane) and quantification of nuclear ERK (indicating ERK activity) in MIA PaCa-2 cells. Yellow arrowheads point to representative nuclei. N/C, nuclear to cytoplasmic ratio. Shown are the individual data points and the population average of each biological replicate (mean ± s.d.; three independent experiments; one-way ANOVA with Holm-Sidak’s multiple comparison test). h Representative images and quantification of tumour growth of HCT-116 cells, transiently expressing GFP-SHANK3 SPN WT or GFP-SHANK3 SPN R12E/K22D, on CAMs. Tumours are delimited by the yellow circles [mean ± s.d.; n = 21 (GFP, SPN WT) or 19 (SPN R12E/K22D) tumours from two independent experiments; Kruskal-Wallis one-way ANOVA and Dunn’s post hoc test]. Source data are provided as a Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3128/pmc11393128/pmc11393128__41467_2024_52326_Fig3_HTML.jpg)