human megakaryocytic leukemia cell line  (ATCC)

Journal: Blood Advances

Article Title: Thrombospondin-1 is an endogenous substrate of cereblon responsible for immunomodulatory drug–induced thromboembolism

doi: 10.1182/bloodadvances.2023010080

Figure Lengend Snippet: IMiDs upregulate intracellular THBS-1 expression in a proteasome-dependent manner. (A) Immunoblot analysis for THBS-1 and IKZF1 protein levels in MEG01 cells treated for 24 hours with DMSO or 10 μM lenalidomide. (B) Time course analysis of THBS-1 protein expression in MEG01 cells treated with 10 μM lenalidomide and pomalidomide for the indicated times. (C) Immunoblot analysis for THBS-1 in MEG01 cells treated with thalidomide, lenalidomide, and pomalidomide at indicated concentrations for 24 hours. (D) Relative mRNA expression levels of THBS1 in MEG01 cells. Cells were treated with DMSO, 10 μM lenalidomide, or pomalidomide for 24 hours. Data are from 9 independent experiments and are expressed as means ± standard error of the mean (SEM). P values were determined by 2-tailed Student t test. (E) Immunoblot analysis for THBS-1 in MEG01 cells treated with 10 μg/mL cycloheximide for the indicated times. (F) Immunoblot analysis for THBS-1 in MEG01 cells treated with 10 μM IMiDs and/or 1 μM MG132 for 24 hours. Results are representative of 2 (panel A) and 3 (panels B, C, E, and F) independent experiments. Cont, control; IB, immunoblotting; Len, lenalidomide; Pom, pomalidomide; Thal, thalidomide.

Article Snippet: Human megakaryocytic leukemia cell line MEG01, human monocytic cell line THP-1, transfectable human embryonic kidney cells 293T, and human umbilical vein endothelial cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Expressing, Western Blot, Control

Journal: Blood Advances

Article Title: Thrombospondin-1 is an endogenous substrate of cereblon responsible for immunomodulatory drug–induced thromboembolism

doi: 10.1182/bloodadvances.2023010080

Figure Lengend Snippet: IMiDs interfere with the direct binding of THBS-1 and cereblon. (A and B) Protein expression of THBS-1 (A) and relative mRNA expression levels of THBS-1 (B) in cereblon-depleted MEG01 cells. In panel B, data are from 3 independent experiments and expressed as means ± SEM. P values were determined by 2-tailed Student t test. (C) Immunoblot analysis for THBS-1 in cereblon-depleted MEG01 cells treated with DMSO, 10 μM lenalidomide, or 10 μM pomalidomide for 24 hours. (D) Lysate mixture of MEG01 cells and 293T cells transfected with FLAG-tagged cereblon were incubated with or without 10 μM lenalidomide and subjected to immunoprecipitation with antibody against FLAG. (E) Purified recombinant His-tagged THBS-1–loaded beads were incubated with glutathione S-transferases–tagged cereblon in the presence of pomalidomide at indicated concentration. Mixture was immunoprecipitated with antibody against His. Results are representative of 3 independent experiments (panels A, C, D, and E). (F) Homologous sequences in THBS-1 and cereblon binding site of IKZF1 are aligned. The identical residues are highlighted in red. Residues of IKZF1 composing backbone of zinc-finger structure are shown in bold characters. Arrows represent indicated interacting residues between IKZF1 and cereblon in previous report. Sequence similarity with each protein was analyzed using the protein-protein BLAST program on The National Center for Biotechnology Information. CRBN, cereblon; Cont, control; GST, glutathione S-transferases; IB, immunoblotting; IP, immunoprecipitation; Len, lenalidomide; Pom, pomalidomide; Thal, thalidomide.

Article Snippet: Human megakaryocytic leukemia cell line MEG01, human monocytic cell line THP-1, transfectable human embryonic kidney cells 293T, and human umbilical vein endothelial cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Binding Assay, Expressing, Western Blot, Transfection, Incubation, Immunoprecipitation, Purification, Recombinant, Concentration Assay, Sequencing, Control