Fig. 2

CARM1–PI3KC2α axis determines intracellular tubulin levels. A and B Correlation analysis of tubulin versus CARM1 (A) or PIK3C2A (B) using Pearson correlation in 67 breast cancer cell lines, derived from DepMap Expression Public 24Q2 data. Each dot represents a cell line and the black line indicates a linear regression line. C Representative confocal images showing α-tubulin (red) and PI3KC2α (green) levels. DAPI (blue) was used to stain cell nuclei. D After CARM1 was rescued with a GFP-CARM1 plasmid, the cell lysates were analyzed by immunoblotting to detect the indicated proteins. E and F The levels of mature mRNA (E) and pre-mRNA (F) of each tubulin (TUBA1A or TUBB) were measured in PI3KC2α-knockdown MCF7 cells. The error bar indicates the standard deviation (n = 3). G After transfection with PI3KC2α siRNA for 60 h, MCF7 cells were treated with actinomycin D (act. D) for 1, 2, and 3 h. TUBA1A mRNA levels at each time point are presented as relative values to those of cells not treated. The error bar represents the standard deviation (n = 3). H and I After transfection with PI3KC2α siRNA for different durations (24, 48, and 72 h), soluble and insoluble fractions were immunoblotted to measure α-tubulin levels (H), and total α-tubulin and STMN1 levels in total lysates were measured (I). The level of each protein in the graphs was normalized to that of the loading control (actin). J and K The STMN1 protein (J) and mRNA (K) levels were measured in PI3KC2α-knockdown cells