Fig. 3: EcSTH causes growth inhibition and cell death by inducing NADH accumulation.

a Western blots for the expression of inducible Tet-on EGFP, EcSTH, LbNOX or EcSTH-LbNOX in HeLa cells induced by Dox for 12 h. b The ratios of cellular NADH/NAD⁺ and NADPH/NADP⁺ in HeLa cells expressing Tet-on EGFP, EcSTH, LbNOX, or EcSTH-LbNOX cultured with different concentrations of Dox for 12 h (n = 3 biologically independent samples). c Effects of the expression of EGFP, EcSTH, LbNOX or EcSTH-LbNOX on the proliferation of HeLa cells cultured with different concentrations of Dox as indicated (n = 3 biologically independent samples). From top to bottom: **P < 0.0001, **P < 0.0001, **P < 0.0001, two-way ANOVA. d Effects of EGFP, EcSTH, LbNOX, or EcSTH-LbNOX expression on colony formation in HeLa cells cultured with Dox, as indicated, for 10 days (n = 3 biologically independent samples). From left to right: *P = 0.013, **P = 0.0001, **P = 6.58e-07, two-tailed Student’s t-tests. e The in vivo tumor growth of HeLa cells expressing inducible Tet-on EcSTH or EcSTH-LbNOX (n = 5 biologically independent animals). 2 mg/mL Dox was added in drinking water on day 10 and updated every two days. **P < 0.0001, two-way ANOVA. f Effects of α-KB treatment on proliferation of HeLa^{Tet-on EcSTH} cells. Cells were counted after treatment with Dox (1 µg/mL) and α-KB (1 or 2 mM) for 48 h (n = 4 biologically independent samples). From left to right: **P = 2.44e-06, **P = 1.85e-06, two-tailed Student’s t-tests. g Effects of inhibitors on proliferation of HeLa^{Tet-on EcSTH} cells. Cells were pretreated with the indicated inhibitors for 2 h, and cells were counted after treatment with Dox (1 µg/mL) for 48 h (n = 3 biologically independent samples). z-VAD (z-VAD-FMK, 20 µM), NSA (necrosulfonamide, 10 µM), Ferr-1 (Ferrostain-1, 5 µM). From left to right: **P = 1.74e-06, **P = 9.64e-06, two-tailed Student’s t-tests. h Effects of α-KB treatment on cell apoptosis. Cell apoptosis was measured by Flow cytometry after treatment with Dox (1 µg/mL) and α-KB (2 mM) for 24 h (n = 3 biologically independent samples). From left to right: **P = 0.00023, **P = 0.00024, **P = 0.00013, **P = 5.51e-05, **P = 3.22e-06, **P = 0.00021, two-tailed Student’s t-tests. i Western blots for the cleavage of PARP-1 and Caspase 3. Cells were treated with Dox (1 µg/mL) and α-KB (2 mM) for 24 h. All experimental data were verified in at least three independent experiments. Error bars represent mean ± SD. *P < 0.05, **P < 0.01. Source data are provided as a Source Data file.