Fig. 4: SHANK3 depletion triggers RAS-MAPK pathway hyperactivation and apoptosis in KRAS-mutant cells.
From: SHANK3 depletion leads to ERK signalling overdose and cell death in KRAS-mutant cancers
a ERK activity in PANC-1 and A549 cells post SHANK3 silencing (3 days). Samples blotted on duplicate membranes, m#1 and m#2. SHANK3 mRNA levels (fold change) indicated below [mean ± s.d.; PANC-1, n = 8 (siCTRL), 5 (siSHANK3_2) or 6 (siSHANK3_7); A549, n = 5 (siCTRL) or 3 (siSHANK3_2 and siSHANK3_7) independent experiments; Kruskal-Wallis one-way ANOVA and Dunn’s post hoc tests]. b Confocal images of ERK-KTR-mRuby2-expressing siCTRL and siSHANK3 cells (nuclei outlined by yellow dashed lines). C/N, ERK-KTR-mRuby2 cytoplasmic/nuclear ratio [mean ± s.d.; n = 61 (siCTRL, 48 h), 38 (siSHANK3_7, 48 h), 185 (siCTRL, 72 h) or 59 (siSHANK3_7, 72 h) cells from three independent experiments; unpaired two-tailed Student’s t-test with Welch’s correction]. c Immunoblotting analysis of cleaved-PARP1 from 4a [mean ± s.d., normalized to siCTRL; PANC-1, n = 6 (siCTRL, siSHANK3_7) or 3 (siSHANK3_2); A549, n = 5 (siCTRL, siSHANK3_7) or 3 (siSHANK3_2) independent experiments; Kruskal-Wallis and Dunn’s post hoc tests]. d, e siSHANK3 PANC-1 cell death in 2D (annexin V-FITC flow cytometry analysis, d) and 3D (annexin-positive spheroid area, e) [mean ± s.d.; five independent experiments; unpaired two-tailed Student’s t-test with Welch’s correction (d); one-way ANOVA with Holm-Sidak’s multiple comparison test at endpoint (e)]. f Cleaved caspase-3-postive cells in siSHANK3 A549 tumours (CAM assay) (mean ± s.d.; n = 10 tumours from two independent experiments; two-tailed Mann–Whitney test; no/residual siSHANK3 PANC-1 tumours detected). g ERK activity in siSHANK3 PANC-1 cells ± MEK inhibitor (trametinib) (two independent experiments). h siSHANK3 PANC-1 cell proliferation (confluence % at day 5) ± trametinib (mean ± s.d; n = 4 technical replicates; representative of three independent experiments). i siSHANK3 PANC-1 cell proliferation ± 300 nM trametinib over time (mean ± s.e.m.; n = 4 technical replicates; representative of three independent experiments). j, k siSHANK3 PANC-1 cell viability ± selumetinib (MEK inhibitor) (j) or SCH772984 (ERK inhibitor) (k) [mean ± s.d; n = 4 (j, k siSHANK3) or 3 (k, siCTRL) technical replicates; representative of three independent experiments]. Source data are provided as a Source Data file.
