Fig. 8: Clonal heterogeneity contributes to acquired resistance to anti-HER2 treatments.

a Representative RT-qPCR (n = 2) assessing expression of the indicated PP2A members in the indicated single cell–derived clones from BT474. Data are expressed as mean ± s.d. of technical triplicates. b Left panel: representative images (n = 6) where the cells are pseudo-colored red from drug-tolerant cell assay with the ten single cell–derived clones from BT474 treated with lapatinib (lap; 100 nM) for three weeks. Scale bar: 150 µm. Right panel: quantification of the assay on the top. Data are expressed as mean ± s.d. of technical triplicates and representative of two independent experiments. c Representative images (left; n = 6) where the cells are pseudo-colored red and quantification (right) of drug-tolerant cell assay with BT474 carrying shNC or shRNAs against PPP2R2B and treated with trastuzumab (trast; 20 µg/ml) or lapatinib (lap; 100 nM) for three weeks. Data are expressed as mean ± s.d. of two independent experiments performed in triplicate (n = 6). Scale bar: 150 µm. P values were determined with two-tailed Student’s t-test, and corrected with Bonferroni adjustment. **P = 0.0032 (trast-treated shNC vs. shPPP2R2B #2), ***P = 0.0008 (trast-treated shNC vs. shPPP2R2B #1), ***P < 0.0001 (lap-treated shNC vs. shPPP2R2B #1, or vs. shPPP2R2B #2). d ChIP-qPCR of EZH2 (left) and H3K27me3 (right) enrichments on PPP2R2B promoter in the indicated single cell–derived clones from BT474, using the four pairs of ChIP primers flanking the promoter. Data are expressed as mean values of technical triplicates. P values were determined using two-way ANOVA. e RT-qPCR assessing expression of PPP2R2B and PPP2R2A in single cell–derived clone #3 and #4, treated with or without EPZ. Data are expressed as mean ± s.d. of technical triplicates. f Top panel: schematic showing how the cell regrowth assay was performed. Briefly, the cells were treated with trastuzumab (trast.) at 1 µg/ml alone or the combination of EPZ (2 µM) and trast. for 25 days, followed by 20 days’ recovery in drug-free medium. Imaging and quantification were performed after the treatment and recovery. Bottom panel: representative images (left) and quantification (right) of the cell regrowth assay shown in the top panel with BT474. Data are expressed as mean ± s.d. of technical triplicates and representative of two independent experiments.