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. 2022 Mar 15;82(6):1110–1127. doi: 10.1158/0008-5472.CAN-21-1397

Figure 5.

Figure 5. MAPK pathway is dynamically regulated in RET-rearranged cells and may impinge upon RET inhibition. A, HMSC, HMSC-RET, and SR-Sarc-0001 cells were serum-starved for 24 hours and then whole-cell extracts were prepared for Western blotting. s.e, shorter exposure; l.e., longer exposure. One representative immunoblot from two independent experiments is shown for each protein. B, Densitometry signals were quantified and normalized to corresponding loading controls (total SHP2, MEK1/2, ERK1/2, and P90RSK for corresponding phosphoproteins; GAPDH for DUSP4 and DUSP6). The resulting values were normalized to HMSC-RET. C, Cells were treated with 100 nmol/L selpercatinib for the indicated times and then whole-cell extracts prepared for Western blotting. One representative immunoblot from two independent experiments is shown for each protein. D, Densitometry signals were quantified and phosphoproteins were normalized to their corresponding totals while DUSP4 and DUSP6 were normalized to GAPDH. E, HMSC-RET cells were treated with 100 nmol/L selpercatinib or 100 nmol/L trametinib for 1 and 24 hours, and then cytoplasmic and nuclear extracts prepared for Western blotting. GAPDH represents a predominantly cytoplasmic protein control and HDAC1 represents a predominantly nuclear protein control. Selperc, selpercatinib 100 nmol/L; Tram, trametinib 100 nmol/L; CICs, short CIC isoform; CICL, long CIC isoform. F, Schematic illustration of the proposed model of RET–MAPK–CIC signaling.

MAPK pathway is dynamically regulated in RET-rearranged cells and may impinge upon RET inhibition. A, HMSC, HMSC-RET, and SR-Sarc-0001 cells were serum-starved for 24 hours and then whole-cell extracts were prepared for Western blotting. s.e, shorter exposure; l.e., longer exposure. One representative immunoblot from two independent experiments is shown for each protein. B, Densitometry signals were quantified and normalized to corresponding loading controls (total SHP2, MEK1/2, ERK1/2, and P90RSK for corresponding phosphoproteins; GAPDH for DUSP4 and DUSP6). The resulting values were normalized to HMSC-RET. C, Cells were treated with 100 nmol/L selpercatinib for the indicated times and then whole-cell extracts prepared for Western blotting. One representative immunoblot from two independent experiments is shown for each protein. D, Densitometry signals were quantified and phosphoproteins were normalized to their corresponding totals while DUSP4 and DUSP6 were normalized to GAPDH. E, HMSC-RET cells were treated with 100 nmol/L selpercatinib or 100 nmol/L trametinib for 1 and 24 hours, and then cytoplasmic and nuclear extracts prepared for Western blotting. GAPDH represents a predominantly cytoplasmic protein control and HDAC1 represents a predominantly nuclear protein control. Selperc, selpercatinib 100 nmol/L; Tram, trametinib 100 nmol/L; CICs, short CIC isoform; CICL, long CIC isoform. F, Schematic illustration of the proposed model of RET–MAPK–CIC signaling.