Fig. 4: TFG domains have a role in the TFG-RET heteromerization and oncogenicity. | Nature Communications

Fig. 4: TFG domains have a role in the TFG-RET heteromerization and oncogenicity.

From: Proteogenomics analysis unveils a TFG-RET gene fusion and druggable targets in papillary thyroid carcinomas

Fig. 4: TFG domains have a role in the TFG-RET heteromerization and oncogenicity.

a Structural comparison of TFG-PB1 vs Dvl2 DIX or p62 PB1 and the TFG-PB1 model showing the head region (deep blue) and tail region (teal) based on the DIX polymer of hDvl24WIP (head (slate), tail (aquamarine)). b Mutations in PB1 domain (top panel)—Comparison of PB1 sequences (not entire) of p62 and TFG-RET. Conserved residues Lys14, Arg22 and Arg23 were substituted with oppositely charged Glu to create PB1 domain mutant TFG-RET (TFG-RETK14ER22ER23E). CC domain (bottom panel)—Leu97 to Glu124 comprise the CC domain of TFG-RET. This domain was deleted to obtain the CC deletion mutant of TFG-RET (TFG-RET Δ97-124). c, d Lysates collected from HeLa cells transiently expressing indicated constructs were loaded on Superose-6 gel filtration column. Collected fractions (every second fraction) were subjected to western blotting. TFG-RET, TFG-RETK14ER22ER23E as well as TFG-RET Δ97-124 were detected using V5 tag. Most of TFG-RET was detected in high molecular weight fractions, while TFG-RETK14ER22ER23E shifted toward the lower molecular weight fractions. (Molecular weight corresponding to the elution volume are indicated by the arrows above). Shown are representative data from at least two independent experiments. e Protein expression of TFG-RET, TFG-RETK14ER22ER23E and TFG-RET Δ97-124 (pcDNA DEST V5.His vector) following transient transfection in HeLa cells was verified by Western blot analysis of the cell lysates (cytosolic fraction obtained after ultracentrifugation). Shown are representative data from at least two independent experiments. f Soft agar colony formation assay. Nthy-ori 3-1 cells stably expressing indicated constructs were cultured in soft agar for 2 weeks followed by staining with crystal violet. Error bars represent ± SEM (n = 3). Paired t-test, two-tailed. Three independent experiments were performed: one experiment with technical triplicates, and two experiments with technical duplicates. ***p-value < 0.0001, **p-value < 0.05. Shown are the fold changes in the number of colonies between the analyzed samples. g Immunoblot analysis of expression of TFG-RET, TFG-RET Δ97-124 and TFG-RETK14ER22ER23E in Nthy-ori 3-1 cells. Shown are representative western blots from the experiments presented in f.

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