Fig. 7: Downregulation of PPP2R2B in acquired resistance to HER2-targeted therapies.

a Representative images (left) and quantification (right) of soft agar assay with BT474 and BT474LR (lapatinib resistant) treated with increasing concentrations of lapatinib (lap). b Representative images (left) and quantification (right) of soft agar assay with BT474 and BT474TR (trastuzumab resistant) treated with increasing concentrations of trastuzumab (trast). Data in a and b are expressed as mean ± s.d. of technical triplicates, and representative of three independent experiments. c Expression of the indicated PP2A members assessed by RT-qPCR in BT474, BT474LR, and BT474TR. Values represent mean ± s.d. of two independent experiments performed in triplicate (n = 6). d ChIP-qPCR of EZH2 (left) and H3K27me3 (right) enrichment on PPP2R2B promoter in BT474 and BT474LR, using the four pairs of ChIP primers flanking the promoter. Data are expressed as mean ± s.e.m. of technical triplicates. P values were calculated with two-way ANOVA. e RT-qPCR (top) and western blot analysis (bottom) with BT474LR treated with the indicated compounds. Values represent mean ± s.d. of technical triplicates. f RT-qPCR assessing expressions of the indicated genes with BT474LR carrying shNC or shRNAs against EZH2. Values represent mean ± s.d. of two independent experiments performed in triplicate (n = 6). P values in e and f were determined with two-tailed Student’s t-test, and corrected with Bonferroni adjustment. g Representative images (left) and quantification (right) of clonogenic assay with BT474TR treated with the solvent, EPZ (2 µM), trastuzumab (10 µg/ml), or in combination at the indicated concentrations. Values represent mean ± s.d. of technical triplicates. Data are representative of two independent experiments. P value was determined with two-tailed Student’s t-test. The parental cell line is labeled in black, whereas the resistant ones are in red.