Fig. 3: Downregulation of IL-6 in palbociclib-resistant hormone receptor–positive breast cancer cells re-sensitizes them to palbociclib and endocrine therapy.

A Relative gene expression of IL-6 by quantitative (q)RT-PCR analysis in MCF7 parental (Par) and resistant (Res) cell lines after shRNA knockdown of IL-6 by one of two clones (shIL-6 #1 and shIL-6 #2) or control scrambled shRNA (shSCR). Cells were grown in culture for 3 days. B Relative gene expression of STAT3 by qRT-PCR analysis. C Secreted IL-6 levels measured by ELISA and normalized based on the number of cells (relative concentration [conc]). D Western blot analysis of pSTAT3 (Y705) and total STAT3 after 3 days in culture. E Dose-response curves of treatment with 0.01–12 μM palbociclib for 6 days followed by 6 days of recovery compared with MCF7 Par shSCR. Number of cells/growth was assessed using the crystal violet assay. Data were normalized to DMSO (100%), plotted, and analyzed using nonlinear regression on GraphPad Prism 9. Dashed line and bar graphs depict half-maximal inhibitory concentration (IC₅₀) values. F Dose-response curves after 24 h of estrogen deprivation followed by re-addition of 10 nM beta-estradiol and treatment with varying concentrations (0.01–12 μM) of fulvestrant for 2 days compared with MCF7 Par shSCR. Number of cells/growth was assessed using the crystal violet assay as in panel E. G Dose-response curves with 0.01–12 μM TTI-101 for 3 days followed by 9 days of recovery. Number of cells/growth was assessed using the crystal violet assay as in panel (E). H qRT-PCR analysis of estrogen responsive genes—ER and PgR. I qRT-PCR analysis of epithelial-mesenchymal transition (EMT) markers N-cadherin and Vimentin. J qRT-PCR analysis of transcription factors related to breast cancer stem cell-like markers CD44 and ALDH1. K Western blot analysis of estrogen receptor (ERa), PgR, EMT markers, and G1/S transition proteins. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.