Fig 1: Protein data from the Toronto cohort. Boxplot of p16 protein abundance with a molecular group (MG), b WHO grade, and c CDKN2A status (homozygous deletion, heterozygous deletion, CDKN2Ahigh, CDKN2Alow). d Correlation plot of p16 protein levels vs CDKN2A protein levels. CDK4 Protein abundance by e MG, f WHO grade, and g CDKN2A status. h Correlation plot of CDK4 protein levels vs CDKN2A mRNA expression levels. i Western blot of Rb phosphorylation (pRB) at S780 and S807/811 in representative samples from each CDKN2A group. j Left, chromosome 9p CNV plot showing depth of CDKN2A loss, or lack of loss in one representative case from CDKN2A homodel, CDKN2Ahigh, and CDKN2Alow group that IHC was performed on; right, representative p16 IHC from three representative samples from each CDKN2A group (CDKN2A homodel, CDKN2Ahigh, CDKN2Alow) in each row. k proportion of meningiomas with each degree of p16 positivity (based on p16 IHC scoring) in CDKN2A homodel, CDKN2Ahigh and CDKN2Alow cases. l p16 IHC positivity based on WHO grade split into CDKN2A homodel cases (above) and CDKN2A intact/wt cases (below). m CDKN2A mRNA expression in p16 IHC samples with each score denoting degree of positivity (0, +1, +2, +3). n Heatmap of mRNA expression of primary and established meningioma cell lines of cell cycling genes. o pRB western blot in representative cell lines. p Panel from tSNE of DNA methylation of primary meningioma cell lines along with DKFZ classifier (v11.4) reference meningioma cases and Toronto clinical meningioma samples. q Cell viability assay at increasing doses of CDK4/6 inhibitors abemaciclib and palbociclib in primary and established meningioma cell lines. Adj. P-value from Kruskal Wallis test and post-hoc Dunn multiple comparisons test or two-sample test for equality of proportions, and for cell viability data from two-way ANOVA and post-hoc Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001
Fig 2: Expression of proteins/genes involved in cell cycle and senescence regulation upon H-RAS induction in BJ-Ras cells. (a) Immunoblot analysis of H-RAS and cyclin A1 after treatment of the cells with DOX or vehicle at indicated time points. (b) Immunoblot analysis of H-RAS, MYC, p21CIP1, phosphorylated pRb, H3K9me3 after treatment with DOX or vehicle at indicated time points. β-actin was used as loading control. (c) RT-qPCR analysis of mRNA expression of H-RAS, p16INK4A, p21CIP1 and p14ARF at day 16 after treatment with DOX or vehicle. Relative fold changes of mRNA expression are presented as mean ± SEM, **p < 0.01, ***p < 0.001. (a–c) The cells were split and reseeded during the experiments as described in Materials and Methods and in the legend to Figure 1.
from Cell Signaling Technology for Rb Antibody Sampler Kit