Fig. 4

An attenuated TCA cycle is involved in the increased ROS production. a A metabolic model describing the flow of [U6]-¹³C-glucose into the TCA cycle. b–e MCF-7 cells were seeded in 90 Pa soft 3D fibrin gels for 4 days under normoxic or hypoxic conditions, and then incubated with ¹³C-glucose for another 24 h, LC-MS were performed for m0, m + 2 or m + 4 labeled citrate, α-KG, fumarate and malate detection. f CS, IDH3G and OGDH expression in hypoxic or normoxic MCF-7 TRCs were analyzed by western blotting. g, h MCF-7 cells were transfected with MDH2 siRNA and then cultured in 90 Pa soft 3D fibrin gels under hypoxic condition (1% O₂). The colony size was analyzed (g) and ROS levels were detected by flow cytometry (h). i, j MCF-7 cells were transfected with IDH3G siRNA and then cultured in 90 pa soft 3D fibrin gels under hypoxic condition (1% O₂). The colony size was analyzed (i) and ROS levels were detected by flow cytometry (j). k MCF-7 cells were transfected with IDH3G siRNA and treated with different concentrations of H₂O₂ in 90 Pa soft 3D fibrin gels under hypoxic condition (1% O₂). The colony size was presented photographically and graphically. 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 bar, 20 μm