Supplementary Data 6 from Abemaciclib Is Effective Against Pancreatic Cancer Cells and Synergizes with HuR and YAP1 Inhibition

Supplemental figure 6: (A) Pico Green assay assessing cell viability in Mia PaCa2 HuR CRISPR knock out cells treated with abemaciclib. Assay was performed for 5 days prior to collection. (B) Western blot of Mia PaCa2 HuR CRISPR knock out cells treated with abemaciclib for 3 days and probed for pRb and HuR. Samples were quantified and normalized to untreated parental cell type. (C) Mia PaCa2 HuR CRISPR KO clone validation, both by western demonstrating no HuR product (top) and by Sanger sequencing. Clone 8 has a 476 base pair insertion, and clone 10 has a single base pair deletion in one allele and a two base pair deletion in the other allele, both leading to a frameshift mutation. (D) Pico Green assays in Mia PaCa2 cells assessing cell viability in si negative control, siHuR or siYAP1 transfected cells treated with abemaciclib. Assay was performed for 5 days prior to collection. (E) Western blot in Mia PaCa2 cells of si negative control, siHuR or siYAP1 transfected cells treated with abemaciclib for 3 days. Samples were probed for pRb, cyclin D1, HuR, YAP1 and α-tubulin. Samples were quantified and normalized to untreated si negative control. (F) qPCR results from RNA immunoprecipitation (RIP) and probed for HuR targets cyclin D1, p27 and PIM1 (positive control). Cytoplasmic extraction samples normalized to IgG. Significance is denoted as ***p<0.0001. (G) Pico Green assays in Mia PaCa2 cells assessing growth rate inhibition in si negative control or si cyclin D1 transfected cells treated with abemaciclib. Assay was performed for 5 days prior to collection. (H) Western blot in Mia PaCa2 cells of si negative control or si cyclin D1 transfected cells treated with abemaciclib for 3 days. Samples were probed for pRb, cyclin D1 and α-tubulin. Samples were quantified and normalized to untreated si negative control.