Fig. 1: RB− and RB+ palbociclib-resistant cell lines have distinct signalling and transcriptional profiles.

A Western blot profiling of PalboR cells for cell cycle, ER-regulated and PI3K pathway markers. Cells cultured long-term with continuous palbociclib (CP) 3 μM T47D, 1 μM MCF7, palbocilib withdrawn (PW) for 1 (MCF7) or 2 (T47D) weeks. Parental cells treated for 24 h with DMSO (C) or palbociclib (T) 3 μM T47D, 1 μM MCF7. B Effect of palbociclib on cell proliferation in parental versus PalboR cells plated without palbociclib overnight and then treated for 5 days with palbociclib at concentrations indicated. Mean ± SD of three independent experiments performed with duplicates represented. C Stability of resistance phenotype measured by removing palbociclib for 1 and 7 days, then retreating palbociclib for 5 days. Mean duplicate cell counts ± SD representative of three independent experiments. D PalboR cells in continuous palbociclib compared to parental cell lines treated with DMSO. Pathway heatmaps of the top 30 pathways ordered by combined log p-value across treatment groups as indicated; upregulated pathways red (top heatmap), downregulated pathways blue (bottom heatmap). Shade represents log p-value. Numbers to the right of the heatmap represent the total number of genes in the pathway signature, yellow numbers in boxes DEG found in signature. RET respiratory electron transport, PROD production, GF growth factor. E, F Heatmap showing E T47D and F MCF7 mRNA z-scores of (i) differentially expressed genes in T47D RB− and MCF7 RB− following palbociclib withdrawal compared to continuous treatment, (ii) differentially expressed genes in T47D CDK6H and MCF7 PacqR following palbociclib withdrawal compared to continuous treatment. Each row represents a gene with significant differential expression between groups in one or both resistant cells (−1 > log2FC > 1 and p-value (FDR) < 0.01). No genes were found in common between (i) and (ii) for T47D or MCF7-resistant cell lines.