Fig 2: Bach1-KO promotes mesendodermal differentiation by activating Nodal signaling.(A) mRNA levels of Nodal and the Nodal receptors ALK4 and ACVRIIB were evaluated in WT and Bach1-KO hESCs on day 3 of differentiation via qRT-PCR, and the results were normalized to measurements in WT cells (n = 3). **P < 0.01 compared with WT; t test. (B) Protein levels of Nodal, ACVRIIB, phosphorylated Smad2 and Smad3 (p-Smad2 and p-Smad3, respectively), and total Smad2/3 were evaluated in WT and Bach1-KO hESCs on day 3 of differentiation via Western blot. (C) DoxBach1-transfected WT hESCs were transfected with plasmids containing luciferase reporter constructs controlled by WT or mutated versions of the Nodal promoter; then, luciferase activity was measured in cells that had been treated with (Dox+) or without Dox (Dox-) on day 3 of differentiation, and results were normalized to measurements in Dox- cells (n = 3). *P < 0.05 compared with Dox-; t test. (D) p-Smad2, total Smad2/3, and histone 3 protein levels were evaluated in the nucleus and cytoplasm of WT and Bach1-KO hESCs on day 3 of differentiation via Western blot. (E) The binding of promoter sequences for mesendodermal genes and for the signaling molecules Wnt3 and Nodal was evaluated in WT and Bach1-KO hESCs and in DoxBach1-transfected WT hESCs that had (Dox+) or had not (Dox-) been treated with Dox on day 3 of differentiation via ChIP-qPCR; results were normalized to measurements in WT or Dox- cells (n = 3). *P < 0.05; **P < 0.01 compared with WT or Dox-; t test. (F and G) mRNA levels of mesendodermal genes were evaluated in WT and Bach1-KO hESCs and in WT and Bach1-KO hESCs that had been treated with SB-431542 (20 µM), which inhibits TGF-ß receptors (F) or transfected with adenoviruses coding for ß-catenin shRNA and treated with SB-431542 (G), and assessments were performed on day 3 of differentiation via qRT-PCR; results were normalized to measurements in WT cells (n = 3). **P < 0.01 compared with WT, DMSO (F) or WT, DMSO, Ad-shCtrl (G); ##P < 0.01 compared with KO, DMSO (F) or KO, DMSO, Ad-shCtrl (G); one-way analysis of variance. Data were collected from three independent replicates and are shown as means ± SD. (H) Model showing that Bach1 plays an important role in the maintenance of hESC self-renewal and suppression of mesendodermal differentiation. Bach1 interacts with Nanog, Sox2, and Oct4 and facilitates deubiquitylation of these pluripotency factors by recruiting DUB Usp7 and therefore stabilizes pluripotency factors and maintains the stem cell self-renewal. Bach1 also impedes mesendodermal differentiation via recruitment of PRC2, which leads to the deposition of H3K27me3 and gene silencing, as well as inhibiting Wnt/ß-catenin and Nodal/Smad2/3 signaling.

Fig 3: Distinct roles of BACH1-induced ferroptosis in lymphatic and hematogenous metastasis in vivo.A Schematic diagram of the footpad implantation nude mouse model. Cells were injected into the footpads on the left side of BALB/c nude mice, and after 4 weeks, the ipsilateral draining popliteal lymph nodes (LNs) of the mice were dissected and examined. B Images of popliteal LNs removed from the indicated groups (n = 8). The weights of the popliteal LNs were measured and analyzed. C Histological examination of popliteal LNs with hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining. Representative results of the indicated groups are shown. Scale bars: upper, 500 μm; lower, 50 μm; Cytokeratin (CK), an epithelial marker; CD19, a B-lymphocyte marker. D Quantitative analysis of the metastatic positive percentage in all popliteal LNs in the indicated groups. LNM, lymph node metastasis. E Bioluminescence imaging and quantification of photon flux of lung metastatic lesions from NCG mice with intravenous injection of 2.5 × 105 KYSE150-vector or KYSE150-BACH1-OE cells for 20 days. F Representative H&E staining and quantitative analysis of lung metastatic foci of the dissected lungs from the NCG mice in panel (E). G Images of subcutaneous xenografts removed from the indicated groups. The ferroptosis inhibitor liproxstatin-1 (Liprox-1) promoted the growth of subcutaneous xenografts, whereas the ferroptosis inducer RSL3 had no significant effect. KYSE150-BACH1-OE cells (1 × 106) were subcutaneously transplanted into the flanks of nude mice (n = 18). After gross observation of tumor formation on day 12, tumor xenografts were established, and the mice were randomly divided into three groups by weight (6 mice per group). Each group of tumor-bearing mice was intraperitoneally injected with control (DMSO), Liprox-1 (30 mg/kg) or RSL3 (30 mg/kg) every two days for 20 days. Xenograft tumors were then removed for imaging and weighing. H Images of subcutaneous xenografts removed from the indicated groups (n = 18). Pretreatment with 1 µM RSL3 for 6 h significantly inhibited the subcutaneous growth of KYSE150-BACH-OE cells in nude mice compared with that in the vector groups. I Images of popliteal LNs removed from the indicated groups. The ferroptosis inducer RSL3 promoted the metastasis of popliteal LNs. A total of 5 × 105 KYSE150-BACH1-OE cells were inoculated into the footpads of nude mice (n = 18). After 10 days, the mice were randomly divided into three groups by weight (6 mice per group). DMSO, Liprox-1 (30 mg/kg) or RSL3 (30 mg/kg) was intraperitoneally injected into each mouse in the three groups every two days for 20 days. Popliteal LNs were then removed for imaging and weighing, which demonstrated that the LNs in the RSL3 group were larger and heavier than those in the DMSO and Liprox-1 groups. J Representative images of H&E and IHC staining of popliteal LNs and quantitative analysis of the metastatic positive percentage in the indicated groups. CK cytokeratin, LNM lymph node metastasis; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. not significant.

Fig 4: BACH1 promoted in vivo cell growth and angiogenesis in ESCC. (A‐C) KYSE170‐shBACH1 and KYSE170‐shCtrl cells were subcutaneously injected into the flanks of nude mice (n = 6). The xenograft tumors were obtained after 4 weeks, and (B) the tumor volume and (C) weight were measured. (D‐F) KYSE150‐BACH1 and empty vector control cells were subcutaneously injected into the flanks of nude mice (n = 6). The xenograft tumors were obtained after 4 weeks, and (E) the tumor volume and (F) weight were measured. (G‐H) Immunohistochemical staining for N‐cadherin, vimentin, and Slug in xenograft tumors derived from KYSE170‐shBACH1 and KYSE170‐shCtrl cells. Scale bar (40×), 50 μm. (I, J) Immunohistochemical staining for (I) CD31 and (J) VEGFC in xenograft tumors derived from KYSE170‐shBACH1 and KYSE170‐shCtrl cells. Scale bars (20×), 200 μm, scale bars (40×), 50 μm. (K) HE and immunofluorescence staining for CD31 in xenograft tumors derived from KYSE150‐BACH1 and KYSE150‐vector cells. Scale bars (20×), 100 μm, scale bars (40×), 50 μm. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student's t test.

Fig 5: Combination treatment of hemin and the MCT1 inhibitors is effective to suppress TNBC growth. (A) Viability (%) of BM1-wt BACH1 or mut Bach1-expressing cells treated with hemin, SR13800 (50 μM), hemin+SR13800, or AZD3965 (100 μM), hemin+AZD3965. After incubation of cells for 48 h, viable cells were stained using Calcein AM; mean (n = 6/cells) ± s.e.m., p-values by two-tailed t-test. (B) Tumor volumes from athymic nude mice orthotopically injected with control and BM1-shBACH1 cells (shCont n = 4, shBACH1 n = 6, shCont+ AZD3965 n = 6, shBACH1+AZD3965 n = 3) and treated with AZD3965 (100 mg/kg body weight) by oral gavage for 17 days. (C) Tumor weights from athymic nude mice orthotopically injected with BM1 cells and treated with hemin (50 mg/kg body weight) daily by intraperitoneal injection and/or SR13800 for 10 days (vehicle n = 9, hemin n = 8, SR13800 n = 9, hemin + SR13800 n = 8). Representative tumor images are shown (below). (D) Schematic diagram indicating lactate catabolic pathways regulated by BACH1 in TNBC cells.