Fig 2: BACH1 inhibits PDAC cells proliferation and angiogenesis. (A) Stable overexpression or knockdown of BACH1 in CFPAC-1 and BXPC-3 cells. (B) Effect of BACH1 overexpression or knockdown on colony formation of CFPAC-1 and BXPC-3 cells. Results represent colony formation ability relative to Control or shControl set to 100% from three experiments. P-values are for Student's t-test. *, P<0.05; **, P<0.01 compared with Control or shControl. (C) Overexpression of BACH1 substantially reduced proliferation of both CFPAC-1 and BXPC-3 cells determined by CCK-8 assay. Results are mean ± SEM from three experiments and each experiment had six replicates. *, P<0.05 compared with control. (D) Knockdown of BACH1 significantly enhanced proliferation of both CFPAC-1 and BXPC-3 cells. Results are mean ± SEM from three experiments and each experiment had six replicates. *, P<0.05 compared with shControl. (E-F) Xenograft tumor formation and growth of CFPAC-1 and BXPC-3 cells with BACH1 overexpression or knockdown in nude mice. Results are mean ± SEM from 6 animals in each group. *, P<0.05 compared with Control or shControl. (G) Effect of BACH1 expression on VEGF levels quantified by ELISA in the culture medium of CFPAC-1 or BXPC-3 cells. Results are mean ± SEM from three measurements. *, P<0.05; **, P<0.01 compared with Control or shControl. (H) Effects of culture medium of CFPAC-1 or BXPC-3 cells with BACH1 overexpression or knockdown on HUVEC tube formation. Results are mean ± SEM from three measurements. *, P<0.05 compared with Control or shControl.

Fig 3: BACH1 occupies the CFTR gene promoter or its CREs in Calu-3 and 16HBE14o− cells, respectively.(A) UCSC genome browser graphic showing the CFTR locus within the −80.1 kb to +48.9 kb TAD boundaries. Below sites of BACH1 occupancy determined by ChIP-seq are shown in Calu-3 (blue) and 16HBE14o− (purple). The ATAC-seq profiles of the same region are shown below each ChIP-seq track for Calu-3 (light blue) and 16HBE14o− (light purple) cells, respectively. Orange vertical bars denote IDR peak calls. (B) ChIP-qPCR analysis of BACH1 occupancy at the −44 kb, −20.9 kb and +6.8 kb CREs and at the CFTR promoter in Calu-3 cells under conditions of 20% or 8% oxygen, normalized to the chromatin input levels. HMOX1 and GCLM serve as positive controls for BACH1 occupancy. (C) Transcript abundance of genes regulated by BACH1 at 20% and 8% oxygen, measured by RT-qPCR. PGK1 is the control and data are normalized to the 20% levels. (D) CFTR transcript levels under 20% or 8% oxygen measured by RT-qPCR as in (C). N ≥ 3 for all experiments. Error bars represent SEM and data were analysed by the Student's unpaired t-test, *P < 0.01 **P < 0.001 ***P < 0.0001.

Fig 4: Gemcitabine sensitivity in PDAC cells depends on BACH1 in an allele-specific manner. (A) Effects of BACH1 on gemcitabine sensitivity in CFPAC-1 and BXPC-3 cells. Results represent mean ± SEM from three independent experiments and each had four replications. *, P<0.05 and **, P<0.01. (B-C) Effect of HO-1 expression on gemcitabine sensitivity in CFPAC-1 and BXPC-3 cells with overexpression (B) or knockdown (C) of BACH1. Results are mean ± SEM from three independent experiments and each had four replications. *, P<0.05; **, P<0.01 and ***, P<0.001. (D-F) Effect of miR-1257 on gemcitabine sensitivity in BXPC-3 (D), CFPAC-1 (E) and Capan-2 (F) cells carrying the rs372883 CC, CT or TT genotype, respectively. Cells transiently transfected with miR-1257 or its inhibitor were exposed to gemcitabine at final concentrations ranging from 10-3 to 103 nM. Cells were enumerated with the CCK-8 assay at 48h after drug exposure. IC50 represents gemcitabine concentration that inhibits cell proliferation by 50%. Results are mean ± SEM from three experiments and each had four replicates. *, P<0.05 and ***, P<0.001.

Fig 5: Expression of BACH1 in PDAC. (A) BACH1mRNA levels determined by qRT-PCR were significantly lower in PDAC compared with paired adjacent normal tissues (N=75). Results represent mean ± SEM normalized to GAPDH and P-values are for Student's t-test. (B) BACH1 protein levels determined by Western blot were significantly lower in PDAC (T) than in paired normal tissues (N). Shown is a representative picture selected from 35 pairs of clinical specimens. (C) Representative IHC images (200×) showing BACH1 protein expression in normal pancreatic tissues (left) and PDAC (right). Bar scale, 100 μm. (D-E) Expression of BACH1 in published data sets of Pei et al.27 and Badea et al. 28 (Oncomine, https://www.oncomine.org/resource/main.html). The line in the middle of the box represents the median; bars represent 10th and 90th percentiles and greater values were plotted as individual points. P-values are for unpaired Wilcox rank-sum test.