Fig 1: Resistance to indisulam is modulated through reduced RBM39 degradation and CAND1 loss.(A) Resistance screen was performed in A549 cells treated with 3 µM indisulam. Volcano plot of indisulam-treated samples compared with untreated. X axis shows log2 fold change of normalized read counts and Y axis shows false discovery rate (FDR). Each dot represents an individual gene and hits are highlighted. (B) Proliferation assay of A549 control (sgCTRL) and sgCAND1 cells treated with 1 µM indisulam. Mean of three technical replicates is shown and error bars indicate SD. (C) Long-term colony-formation assay of A549. Wild-type, control, and two individual sgCAND1 cells were treated with indicated doses of indisulam for 10 d. (D) Western blot analysis of RBM39 and CAND1 in A549 cells. Wild-type, control and sgCAND1 cells were treated with 0.5 µM of indisulam for 8 d. GAPDH was used as loading control. (E) Long-term colony-formation assay of Panc10.05. Wild-type, control (sgCTRL), and two individual sgCAND1 cells were treated with indicated doses of indisulam for indicated number of d. (F) Western blot analysis of RBM39 and CAND1 in Panc10.05 cells. Wild-type, control, and sgCAND1 cells were treated with 0.5 µM of indisulam for 8 d. GAPDH was used as loading control. (G) CUL4-DCAF15 E3 ubiquitin ligase (CRL) complexes get activated by neddylation (N8) which allows ubiquitination of the substrate (RBM39). Neddylation is reversed by NEDD8-activating enzyme (NAE), which can be inhibited by a small molecular inhibitor MLN4924 leading to inactive CRL complex and reduced substrate degradation. (H) Western blot analysis of RBM39 and CUL4A in HCT-116 and A549 cells pretreated for 2 h with 1 µM MLN4924 or 5 µM MG-132 followed by a 6-h treatment with 0.5 µM indisulam. GAPDH was used as loading control. The upper CUL4A band (arrow) represents neddylated CUL4A, whereas the lower band represents the deneddylated CUL4A. (I) Western blot analysis of RBM39 and CUL4A in HCT-116 (62.5, 125 and 250 nM MLN4924) and A549 (125, 250, and 500 nM MLN4924) cells treated with 0.5 µM indisulam and increasing doses of MLN4924 for 24 h. The upper CUL4A band (arrow) represents neddylated CUL4A, whereas the lower band represents the deneddylated CUL4A. (J) Long-term colony-formation assays of HCT-116 (62.5 nM MLN4924), HCC-1806 (62.5 nM MLN4924), and A549 (125 nM MLN4924) treated with indicated doses of indisulam and a fixed concentration of MLN4924 for 8–13 d depending on the cell line.Source data are available for this figure.