Fig. 4: RSK1 alters FLT3 protein dynamics through deubiquitinase USP1. | Blood Cancer Journal

Fig. 4: RSK1 alters FLT3 protein dynamics through deubiquitinase USP1.

From: RSK1 dependency in FLT3-ITD acute myeloid leukemia

Fig. 4

A Immunoblot of MV4-11 and MOLM-13 cells treated with 2 µM PMD-026 for 24 h. B Immunoblot of MV4-11 and MOLM-13 cells after knockdown with two RPS6KA1 shRNAs or a non-targeting pLKO control. C Immunoblot of MV4-11 cells following knockdown with shRPS6KA1 #1 or control vector and treated with 50 µg/mL CHX for the indicated duration. Line plot shows the half-life of FLT3 following RPS6KA1 inhibition normalized to FLT3 expression from each genetically altered group at time 0 and normalized to HSP90 control. D Pearson correlation of gene KO gene dependency scores with FLT3 KO gene dependency scores across 26 AML cell lines from DepMap. E USP1 KO gene dependency score across 26 AML cell lines from DepMap. F Immunoblot following immunoprecipitation (IP) of HA in 293 T cells co-transfected with FLT-ITD and HA-Ub. Following 36 h co-transfection, cells were treated with 10 µM PMD-026 and 10 µM MG-132 for 18 h. G Immunoblot following IP of FLT3 in Ba/F3 cells transduced with FLT3-ITD and treated with 10 µM PMD-026 for 18 h. H Immunoblot of MV4-11 and MOLM-13 cells treated with 2 µM PMD-026 for 24 h. I Immunoblot of MV4-11 cells after knockdown with two RPS6KA1 shRNAs or a non-targeting pLKO control. J Immunoblot of MV4-11 and MOLM-13 cells treated with the indicated doses of SJB3-019A for 24 h. K Immunoblot of MV4-11 and MOLM-13 cells after knockdown with two USP1 shRNAs or a non-targeting pLKO control. L Cell viability assay upon knockdown of USP1. Cells were plated at n = 6 for each condition and grown for 96 h with viability normalized to the control. Data are presented as mean values ± SD. Statistics were assessed by two-tailed Student’s t-test. M Immunoblot of MV4-11 cells following knockdown with shUSP1 #1, shUSP1 #2, or control vector and treated with 50 µg/mL CHX for the indicated duration. Line plot shows the half-life of FLT3 following USP1 inhibition normalized to FLT3 expression from each genetically altered group at time 0 and normalized to actin control. N Time course immunoblot of Ba/F3 FLT3-ITD cells treated with 5 µM SJB3-019A. O Immunoblot following IP of HA in Ba/F3 FLT3-ITD HA-Ub cells (left) or IP for FLT3-ITD in Ba/F3 FLT3-ITD cells (right) following treatment with 5 µM SJB3-019A for 2 h. P Immunoblot of Ba/F3 FLT3-ITD cells treated with 10 µM SJB3-019A and/or 10 µM MG-132 for 18 h.

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