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. 2014 Dec 1;111(50):E5429–E5438. doi: 10.1073/pnas.1421438111

Fig. 4.

Fig. 4.

Paclitaxel-induced IL-6/IL-8 expression and BCSC enrichment are HIF dependent. (A–C) MDA-MB-231 subclones, which were stably transfected with a vector encoding a nontargeting control shRNA (NTC) or vectors encoding shRNAs against HIF-1α and HIF-2α (DKD), were treated with either vehicle (V) or 10 nM paclitaxel (P) for 4 d and immunoblot (A and B) or Aldefluor assays (C) were performed (mean ± SEM; n = 3). *P < 0.001 compared with vehicle-treated NTC and #P < 0.001 compared with paclitaxel-treated NTC, by Student's t test. (D) NTC and MDA-MB-231 subclones stably transfected with vector encoding an shRNA against HIF-1α (shH1α) or HIF-2α (shH2α) were treated with 10 nM paclitaxel for 4 d and Aldefluor assays were performed (mean ± SEM; n = 3). *P < 0.001 compared with vehicle-treated NTC, and #P < 0.001 compared with paclitaxel-treated NTC, by Student's t test. (E) NTC and subclones stably transfected with vector encoding an shRNA against HIF-1α (shH1α), HIF-2α (shH2α), or both (DKD) were treated with 10 nM paclitaxel for 4 d and mammosphere assays were performed (mean ± SEM; n = 3). *P < 0.001 compared with vehicle-treated NTC, and #P < 0.001 compared with paclitaxel-treated NTC, by Student's t test. (F and G) MDA-MB-231 subclones were treated with V or P for 4 d and analyzed by RT-qPCR using primers specific for IL-8 (F) or IL-6 (G) mRNA (mean ± SEM; n = 3). *P < 0.001, **P < 0.01 compared with vehicle-treated NTC, and #P < 0.001 compared with paclitaxel-treated NTC, by Student's t test. (H and I) MDA-MB-231 and SUM-149 cells were treated for 4 d with vehicle (V) or paclitaxel, either alone (P) or in combination with digoxin (P/D) or acriflavine (P/A). RT-qPCR was performed to assay JMJD1A (H) and JMJD3 (I) mRNA relative to 18S rRNA and normalized to V (mean ± SEM; n = 3). *P < 0.001 compared with V, and #P < 0.001 compared with P, by Student's t test.