Fig. 2

miR-125b dichotomizes via NF-κB signaling into altruistic fitness benefits and disadvantage. A Schema showing cell cycle process and regulators. Those indicated with red boxes are involved in G1-S transition and which were selected for further investigation as inhibitory targets of miR-125b. B Cell cycle analysis of miR-125b^m or Control^m-transfected MCF7 or MDA-MB-468 cells, or control LNA or miR-125b LNA inhibitor-transfected MDA-MB-231 cells. C Box plots of fold change in enrichment of indicated gene transcripts pulled-down using biotinylated miR-125b. D Relative luciferase activities of HEK293T cells following transfection of wild-type or mutant reporter construct for indicated genes and mimics. E Homogeneous and heterogeneous mixtures of MCF7 (Left) or MDA-MB-468 (Right) were transfected with siRNA against indicated genes, exposed to sublethal 0.8 nM docetaxel, and the RF_α measured. Blue or red dotted line/box plot indicate respective levels of RF_α for homogenous mixture or heterogenous mixture with control KD. IKBKB gene codes for IKKβ. F Immunoblotting to detect for indicated proteins extracted from indicated cell lines transfected with combination of indicated mimics and siRNAs for IKBKB. G, H Conditioned media was harvested from cell culture of miR-125b^m or Control^m-transfected MCF-7 cells and analyzed using cytokine array kit (G) and iTRAQ (H). I Immunoblotting to detect CCL28 and IGFBP2 in conditioned media from mimic or LNA inhibitor-transfected, or EGFP reporter-sorted cell fractions of indicated cell lines. Ponceau S was used to visualize protein load. Quantification of band intensities (relative to Control^m/ Control LNA / Mi/EGFP^Low) is shown. See Fig. S6F for results of plasmid-transfected MCF7 cells. J Viability of indicated cell lines with indicated exposure to docetaxel and/or neutralizing antibodies to IGFBP2 and/or CCL28. K Percentage change in size of MDA-MB-231 xenografted tumors in NSG mice with indicated treatment of docetaxel and/or neutralizing antibodies to IGFBP2 and/or CCL28. L Immunoblotting to detect for CCL28 and IGFBP2 in conditioned media from indicated cell lines transfected with combination of indicated mimics and siRNA. Ponceau S for protein load normalization. Quantification of band intensities (relative to first band of each set) is shown. M RS_α of homogeneous and heterogeneous mixture of MCF7 (Left) or MDA-MB-468 (Right) cells transfected with indicated siRNA and exposed to indicated docetaxel concentration. N Schema showing proposed mechanism of how miR-125b dichotomizes into fitness benefit and disadvantage of cancer cell altruism. O Schematic showing the different components of the NF-κB signaling pathway and how the oncogenic (fitness benefits) and tumor suppressive (fitness disadvantage) events may be mediated by different parts of the same signaling pathway. Experiment repeated two times, representative result shown (B,F,L). Data are mean ± s.d. from three independent biological sets of triplicates (C,D,E,M). Experiment performed once (G), and results validated in (I). Representative blots from three independent replicates (I). Data are mean ± s.d. from three independent biological duplicates (H) or quadruplicates (J). n = 8-9 independent animals (K). Statistical analysis was performed using two-tailed one sample t-test against 1 (C,M) or 100 (D) or one-way ANOVA with post-hoc Tukey HSD (J,K). NT: no treatment; DTX: docetaxel treatment. Schematic created using Biorender (A,O). Exact P values are shown.