Fig. 4: Identifying upstream drivers of oncogenic Ras activity inside EML4-Alk granules. | Nature Biotechnology

Fig. 4: Identifying upstream drivers of oncogenic Ras activity inside EML4-Alk granules.

From: Computationally designed sensors detect endogenous Ras activity and signaling effectors at subcellular resolution

Fig. 4: Identifying upstream drivers of oncogenic Ras activity inside EML4-Alk granules.The alternative text for this image may have been generated using AI.

a, Representative pseudocolored FRET ratio image of a Beas2B cell transfected with Ras-LOCKR-S localized to EML4-Alk (Key fused to EML4-Alk, Cage untargeted). b, Schematic of strategy for localizing Ras-LOCKR-PL to EML4-Alk granules. c, Representative epifluorescence images from three biologically independent experiments of Beas2B cells transfected with YFP-fused EML4-Alk variant 1 (v1) and GFP nanobody (GFPnb)-fused V5-tagged TurboID or GFPnb-fused Key of Ras-LOCKR-PL. Cells were treated with 500 μM biotin for 3 h and then probed for biotin labeling. d, Left, representative epifluorescence images from three biologically independent experiments of Beas2B cells expressing YFP-tagged EML4-Alk v1, and treated with 1 μM YB-0158 or DMSO for 1 h and immunostained for SAM68. Arrows indicate co-localization of EML4-Alk with SAM68 puncta. Right, co-localization analysis (n = 3 biologically independent experiments per condition). e, Table of inhibitors used in this figure. f, Representative pErk immunoblots from three biologically independent experiments of H3122 (EML4-Alk v1 positive) or H2228 (EML4-Alk v3 positive) cancer patient cells treated with indicated concentrations of inhibitors. g, Representative images of crystal violet staining of Beas2B, H3122 and H2228 cells incubated for 1 week with inhibitors to Alk (Cz: 1 μM Crizotinib, Ce: 1 μM Ceritinib), SAM68 (Y: 1 μM YB-0158) or DMSO. h, Left, normalized to minimum FRET ratio timecourses of Beas2B cells transfected with Ras-LOCKR-S localized to EML4-Alk v1 and incubated with 10 μM of the inhibitors shown (n = 21 cells per condition). Puncta and diffuse regions were analyzed separately (**P = 0.0022 and ****P = 1.6 × 10−18). Normalized to minimum FRET ratios are calculated by normalizing the dataset to the condition with the largest decrease in FRET ratios (Ce + Y in both cases), where 0 represents the lowest FRET ratio out of the entire dataset. Right, raw FRET ratios of puncta and diffuse EML4-Alk regions after Alk inhibition for 1 h. Bar graphs represent mean ± s.e.m. ****P < 0.0001 and **P < 0.01, one-way ANOVA. Scale bars, 10 μm.

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