Fig. 6

PCK2 downregulation causes fumarate accumulation. a MCF-7 cells were seeded in 90 Pa soft 3D fibrin gels for 5 days in 21% (N) or 1% O₂ (H) condition and PCK2 expression was determined by western blotting. b Tet-PCK2 MCF-7 cells were cultured in 90 Pa soft 3D fibrin gels with or without doxycycline in hypoxia for 5 days. Cell extracts were prepared, fumarate and GSF were detected by LC-MS, ROS was detected by flow cytometry. c, d Tet-PCK2 MCF-7 TRCs were treated with or without doxycycline in hypoxia for 5 days. NADPH/NADP⁺ and GSH/GSSG ratios were analyzed. e, f Tet-PCK2 MCF-7 cells were cultured in 90 Pa soft 3D fibrin gels with or without doxycycline in hypoxia for 5 days and the colony size was analyzed. g Tet-PCK2 MCF-7 cells were cultured in 90 Pa soft 3D fibrin gels with or without doxycycline in hypoxia for 5 days, Aldh1a1, CD133, and Sox2 mRNA levels were analyzed by real-time PCR. h OAA levels of normoxic or hypoxic MCF-7 TRCs were analyzed by OAA assay kit as described in methods (left). Tet-PCK2 MCF-7 cells were cultured in 90 Pa soft 3D fibrin gels with or without doxycycline in hypoxia for 5 days and OAA levels were analyzed (right). i–l Tet-PCK2 MCF-7 cells were seeded in 90 Pa soft 3D fibrin gels for 4 days in hypoxia, and then incubated with ¹³C-glucose for another 24 h. M + 2 or m + 4 labeled citrate, α-KG, fumarate and malate were analyzed by LC-MS. m–s Tet-PCK2 MCF-7 TRCs were inoculated into mammary gland of nude mice and mice were treated with sunitinib and doxycycline as described in methods. Thirty-five days after tumor inoculation, ALDH1 + tumor cells were sorted by Aldefluor assay, and then cell extracts were prepared, and TCA cycle intermediates was detected by LC-MS (m), ROS level was detected by flow cytometry (n). Tumor tissue was detected by immunofluorescence staining of pimonidazole adducts, ALDH1A1, Ki67 in sections from control or doxycycline-treated animals as described in methods (o, p). The NADPH/NADP + ratios were analyzed as described in methods (q), tumor weight was measured (r) and Aldh1a1, CD133, and Sox2 mRNA levels were analyzed by real-time PCR (s). Data shown are representative of three independent experiments and error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bars, 100 μm