Journal: Cancers

Article Title: IGF2BP1 Promotes Proliferation of Neuroendocrine Neoplasms by Post-Transcriptional Enhancement of EZH2

doi: 10.3390/cancers14092121

Figure Lengend Snippet: IGF2BP1 is highly expressed in pNEN and is associated with worse prognosis. ( A ) IGF2BP1 mRNA expression in PDAC tumors matched with TCGA normal and GTEx data (left panel) and median overall survival in PDAC with low (blue line) or high (red line) IGF2BP1 expression (right panel). Blot generated using GEPIA2 database. ( B ) IGF2BP1 mRNA expression in primary pancreatic neuroendocrine tumors (pNEN) compared to corresponding lymph node and liver metastasis (left panel; data derived from Scott et al., 2020), as well as 5-year survival rate of pNEN patients (right panel) depending on cancer stage: Localized (tumor diagnosed at a localized stage), regional (tumor spreading to surrounding tissues and/or regional lymph nodes), distant (tumor with distant metastases). Data from Cancer.Net. ( C , D ) Western blots of IGF2BP1 protein levels in pooled benign murine pancreatic Islet (BL6) and individually isolated insulinomas of RIP1-Tag2 mice ( C ), as well as whole cell lysates from BON1, Colo320 and NCIH727 NEN cell lines ( D ). Actin served as loading control. The uncropped western blot figures were presented in .

Article Snippet: To characterize the functional role of IGF2BP1 in NEN cells we used homolog-specific siRNA pools to knock-down IGF2BP1 expression in BON1, Colo320, and NCIH727 cells ( A, ).

Techniques: Expressing, Generated, Derivative Assay, Western Blot, Isolation, Control

Journal: Cancers

Article Title: IGF2BP1 Promotes Proliferation of Neuroendocrine Neoplasms by Post-Transcriptional Enhancement of EZH2

doi: 10.3390/cancers14092121

Figure Lengend Snippet: IGF2BP1 knockdown affects the transcriptional landscape of NEN cells. ( A ) Volcano plots of differentially expressed (DE) genes (threshold: FDR < 0.05) determined by RNA-seq in BON1 cells upon IGF2BP1 knockdown compared to siC transfected cells after 72 h. ( B ) KEGG- pathway analysis of mRNAs downregulated upon IGF2BP1 knockdown in BON1 cells (threshold: FDR < 0.01). Pathways are ranked by Bonferroni-corrected significance. First number indicates counts mapped to the respective pathway and second number gives percentage of these counts related to total pathway counts. ( C ) Expression of potential PRC2 components as well as EZH2 transcriptional, post-translational regulators and targets in BON1 cells after IGF2BP1 (I1) knockdown compared to siC ( C ) evaluated by RNA-seq.

Article Snippet: To characterize the functional role of IGF2BP1 in NEN cells we used homolog-specific siRNA pools to knock-down IGF2BP1 expression in BON1, Colo320, and NCIH727 cells ( A, ).

Techniques: Knockdown, RNA Sequencing, Transfection, Expressing

Journal: Cancers

Article Title: IGF2BP1 Promotes Proliferation of Neuroendocrine Neoplasms by Post-Transcriptional Enhancement of EZH2

doi: 10.3390/cancers14092121

Figure Lengend Snippet: The IGF2BP1-MYC-EZH2 network is a target for pharmacological inhibition. ( A ) Representative Western blot of EZH2, IGF2BP1, and MYC knock-down in transfected BON1 cells. Actin served as loading control. ( B ) Proliferation of BON1 cells upon transfection with indicated siRNAs after 72 h. ( C ) Schematic strategy of IGF2BP1-MYC-EZH2 inhibition with commercially available inhibitors. DZNep targeting methylation activity of EZH2/PRC2 complex, BTYNB inhibiting IGF2BP1 binding to MYC mRNA and OTX015 inhibiting BRD2/3/4 transcription factors leading to reduced MYC expression. ( D ) Cell viability determined by Cell Titer Glo in BON1 and NCIH727 cells treated with indicated inhibitor concentrations for 72 h. Viability of DMSO treated cells served as control and was set to one. ( E ) Analysis of cell proliferation by cell counting at indicated time points upon treatment with indicated inhibitor concentrations. ( F ) Cell cycle analysis of NEN cells treated with indicated inhibitor concentrations for 72 h. Percentage of cells in indicated cell cycle phases were determined by propidium iodide staining. ( G ) Representative Western blot analysis of respective target proteins after treatment with indicated inhibitors for 72 h. Untreated (mock) and DMSO treated cells were used as expression controls. Actin served as loading control. Statistical significance was determined by Student’s t -test: * p < 0.05; ** p < 0.01; *** p < 0.001. The uncropped western blot figures were presented in .

Article Snippet: To characterize the functional role of IGF2BP1 in NEN cells we used homolog-specific siRNA pools to knock-down IGF2BP1 expression in BON1, Colo320, and NCIH727 cells ( A, ).

Techniques: Inhibition, Western Blot, Knockdown, Transfection, Control, Methylation, Activity Assay, Binding Assay, Expressing, Cell Counting, Cell Cycle Assay, Staining