1. Fig. 4. JAK2 knockdown abrogates tumorsphere expansion after chemotherapy.
JAK2 knockdown abrogates tumorsphere expansion after chemotherapy. (A) HCC-38 cells stably transduced with doxycycline-inducible shRNA targeting JAK2 (two independent sequences) or shNTC were grown in 10% FBS and treated with vehicle (control), IC50 of paclitaxel (50 nM) ± doxycycline (100 ng/ml), or doxycycline for 4 days and allowed to recover in fresh medium for 3 days; cells were then trypsinized and assessed for their ability to form mammospheres. Colony numbers are expressed relative to untreated controls. (B) On day 7, colonies were stained with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and photographed. Scale bars, 200 μm. (C and D) Experiments identical to (A) and (B) were carried out in MDA-436 cells (paclitaxel IC50, 150 nM). (E) Immunoblot analysis demonstrating doxycycline-inducible knockdown of JAK2 at 72 hours in HCC-38 and MDA-436 cells. (F) MDA-436 and HCC-38 cells were grown in 10% FBS and treated with vehicle (control), paclitaxel (at the cell line IC50) ± 5 μM BSK805, or 5 μM BSK805 alone for 4 days and then allowed to recover in fresh medium for 3 days. Colonies were quantitated as described above. All experiments were replicated at least twice (n = 3). (G) Limiting dilution assay results for MDA-436 or HCC-38 cells treated with shNTC + doxycycline or shJAK2 + doxycycline, or parental cells treated with BSK805 (1 μM) or vehicle control. Cells were pretreated for 1 week before inoculation, and treatment was maintained in the mice for 1 week after orthotopic (no. 4 mammary fat pad) injection. Data are presented as the number of tumors (palpable at 30 days for MDA-436 and either palpable or confirmed microscopically after biopsy at 60 days for HCC-38) formed out of the number of mice inoculated. P value represents the χ2 assay result. Statistics were performed using the protocol at
Justin M. Balko et al., Sci Transl Med 2016;8:334ra53
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