Fig. 3.

Inhibition of the IGFBP-3 interaction with NONO and SFPQ. a Western blots of NONO and SFPQ in MDA-MB-468 cell lysates immunoprecipitated with IGFBP-3-Fab beads, showing that incubation overnight with 20 µM NU7026 blocked the formation of IGFBP-3 complexes with NONO and SFPQ formed in response to 20 µM etopside. b Similar experiment in HCC1806 cells. c Quantitation of IGFBP-3–NONO and IGFBP-3–SFPQ complexes in MDA-MB-468 cells: mean relative band density (normalized to time 0 controls) ± SEM from 4 experiments. *P < 0.05 vs. the corresponding time 0 by post hoc Fisher’s LSD test after ANOVA. NS, not significant. d, e Proximity ligation assays in MDA-MB-468 cells, after 2 h etoposide treatment showing that NONO-IGFBP-3 complexes (red dots) are inhibited in cells preincubated overnight with DNA-PKcs inhibitor NU7026, 10 µM (d), or 48 h after DNA-PKcs knockdown with siRNA (e). Blue = nuclei (DAPI). Bar 50 µm. f Quantitation of inhibition by NU7026 of IGFBP-3 interaction with NONO measured by PLA; 5 fields (~ 20 nuclei/field) counted for each condition and each time-point in each experiment. Means ± SEM for 3 replicate experiments. *P < 0.05 vs. the corresponding time 0 by post hoc Fisher’s LSD test after ANOVA. NS, not significant. g, h Western blots of NONO and SFPQ in MDA-MB-468 and HCC1806 cell lysates, respectively, IP’d with IGFBP-3-Fab beads, showing that incubation overnight with 10 µM gefitinib blocked the formation of IGFBP-3 complexes with NONO and SFPQ in response to 20 µM etopside. i Western blots showing NONO, SFPQ, IGFBP-3, and pEGFR (Y1068) in HCC1806 cell lysates immunoprecipitated with NONO Ab. NONO bands are partly obscured by strong IgG heavy-chain bands. j Quantitation of pEGFR immunoprecipitated in NONO complexes in HCC1806 cells: mean relative band density (normalized to time 0 controls) ± SEM from 4 experiments. *P < 0.05 vs. time 0 values by post hoc Fisher’s LSD test after ANOVA