Journal: Nature Communications
Article Title: Oncogenic EML4-ALK assemblies suppress growth factor perception and modulate drug tolerance
doi: 10.1038/s41467-024-53451-7
Figure Lengend Snippet: A Three common oncogenic variants of EML4-ALK (Variants 1–3) share a common ALK fragment but differ in the lengths of the EML4 domain. B Expression of mCh-EML4-ALK(V1/2/3) in GRB2:mNG2 Beas2B cells showed condensation of each variant as well as the propensity of the condensates to colocalize with GRB2. Scale = 10 µm. C Quantification of puncta per cell for each variant. D Quantification of the percent of EML4-ALK puncta that overlap with GRB2 puncta in each cell. Boxplots in ( C , D ) show median and upper/lower quartile, and whiskers extend to 1.5*IQR. C , D Data points represent 18 (V1), 28 (V2), and 21 (V3) cells. E Translocation was quantified by identifying the cell edge and defining a 10 pixel ring into the cytoplasm (“edge”). The remaining cell pixels beyond this ring wire designated as the cell “core”. Membrane localization was defined as the ratio of mean edge fluorescence to mean core fluorescence. Translocation was defined as the difference in adapter membrane localization after 1.5 min of EGF stimulation vs pre-stimulation. F , G Quantitation of translocation of GRB2 ( F ) or SOS ( G ) for cells transfected with one of the 3 EML4-ALK variants or for neighboring untransfected cells (wt). Due to small variations in imaging plane between acquisitions, the absolute magnitude of translocation differed between variants and drug conditions (note the differences in untransfected Beas2B responses, which are equivalent conditions between panels). However, cells with or without EML4-ALK (black vs. red in the same panel) were imaged in the same field of view and thus can be compared directly. Data points represent individual cells. For ( F ), n = 57(WT)/40(V1), 170(WT)/42(V2), 24(WT)/34(V3) cells. For ( G ), n = 46(WT)/50(V1), 50(WT)/27(V2), 67(WT)/40(V3) cells. Boxplots show median and upper/lower quartile, and whiskers extend to 1.5*IQR. H Definition of the magnitude of translocation. I , J Comparison of translocation suppression of GRB2 ( F ) or SOS1 ( G ) for each of the three variants. Data represent median translocation suppression from resampling of 1000 bootstrapped samples. Error bars show lower and upper quartiles. Significance determined by either one-sided T-test (panels F , G ) or one-sided bootstrap test (panels I , J ). Data for Variant 1 in ( F – J ) is reproduced from Fig. . K Beas2B cells were transfected with EML4-ALK-2A-H2B-miRFP constructs for one of 3 EML4-ALK variants (V1, V2, V3), or with an H2B-iRFP control, and ppERK levels were assessed after stimulation with EGF (15 min, 50 ng/mL) in the presence or absence of ALKi (1 µM crizotinib, 2 h), through immunofluorescence. L Quantification of ppERK immunostaining after EGF stimulation of Beas2B transiently expressing EML4-ALK in the presence or absence of ALKi. Significance assessed by Hsu MCB test. n = 8 biological replicates. M ppERK response in the presence and absence of ALKi pretreatment. Data points represent the mean ppErk intensity of 20–60 cells. Significance assessed by one-sided T-test. n = 8 biological replicates. Gray bars in ( M ) are reproduced from ( L ) for direct comparison to between non-treated and ALKi-treated cells.
Article Snippet: Membranes were blocked in 5% milk in Tris buffer saline with 0.5% Tween-20 (TBS-T) for 1 h and incubated overnight at 4 °C with primary antibodies against EGFR (CST #4267), SOS1 (CST #5890), SPRY2 (CST #14954), pERK1/2 (CST #4370), ERK (CST #4695), ALK (CST #3633) tubulin (CST #3873) or GRB2 (Thermo Fisher Scientific, PA1-10033).
Techniques: Expressing, Variant Assay, Translocation Assay, Membrane, Fluorescence, Quantitation Assay, Transfection, Imaging, Comparison, Construct, Control, Immunofluorescence, Immunostaining
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