Skip to main content
. 2021 Jun;35(11-12):870–887. doi: 10.1101/gad.348316.121

Figure 5.

Figure 5.

Perturbation of the neddylation pathway disrupts the neuroendocrine signature in high-grade neuroendocrine cancers. (A) Immunoblot analysis for NEDD8, showing the levels of neddylated cullins and for neuroendocrine regulators in the MLN4924-sensitive ex vivo PDX models JHU-LX108 and MSK-LX227C. Cells were treated for 48 h with DMSO or MLN4924 (100 nM). β-ACTIN was used as loading control. (B) Immunoblot analysis for the levels of neuroendocrine regulators and neddylation pathway members upon inducible knockdown of NEDD8 or RBX1 in the ex vivo PDX model MSK-LX227C. Samples were supplemented with 1 μg/mL doxycycline and collected at days 0, 3, and 4. GAPDH was used as loading control. (C–E) Proliferation assay results comparing on-dox and off-dox MSK-LX227C shGFP (C), shNEDD8 (D), or shRBX1 (E) knockdown cell lines. Samples were assayed at days 1, 4, and 7 with readout determined by CellTiter-Glo. (F) Immunoblot analysis for the levels of INSM1 upon the inducible knockdown of INSM1 in MSK-LX227C ex vivo cells. Samples were supplemented with 1 μg/mL doxycycline and collected at day 0, 6, and 10. GAPDH was used as loading control. (G) Proliferation assay results comparing on-dox and off-dox MSK-LX227C shINSM1 knockdown cells. Samples were assayed at days 1, 4, 7, and 10 with readout determined by CellTiter-Glo. (H) Immunoblot analysis for the levels of INSM1 in MSK-LX227C ex vivo cells constitutively overexpressing INSM1 (phINSM1) or an empty vector control (phEmpty). Cells were treated for 96 h with DMSO or MLN4924 (25 nM). GAPDH was used as loading control. (I) Dose response curve for MLN4924-treated ex vivo MSK-LX227C cells overexpressing INSM1 or empty vector control. Percent survivability determined at 72 h by CellTiter-Glo. Data are means ± SEM (n = 4 biological replicates). (J) Proliferation of ex vivo MSK-LX227C cells overexpressing INSM1 or empty vector control cultured with MLN4924 (25 nM). Samples were assayed at days 1, 4, and 7. Readout was determined at each time point by CellTiter-Glo. (K) Proliferation of ex vivo MSK-LX227C cells overexpressing INSM1 or empty vector control cultured in standard growth media. Samples were assayed at days 1, 4, and 7. Readout was determined at each time point by CellTiter-Glo. For C–E,G, J, and K, data are means ± SEM (n ≥ 3 biological replicates in all groups). Relative cell growth determined by normalizing average luminescence (RLU) values for the initial time point to 1. (ns) Not significant, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001, by unpaired Student's t-test.