Fig 2: Contextual signal-responsive repression of luciferase expression by BACH2. (a) Luciferase activity in inducible-BACH2 reporter line after 6 h stimulation with the indicated concentrations of PMA/ionomycin, with or without pre-treatment with titrated tetracycline doses. Concentrations of tetracycline are indicated in the figure legend. (b) Luciferase signal repression at the indicated PMA/ionomycin concentrations with or without tetracycline (1 μg/ml) pre-treatment. (a, b) Data are representative of 2 independently repeated experiments with 3 culture replicates per condition. Bars and error represent mean (SD).

Fig 3: Bioluminescence imaging of BACH2-reporter cells. (a) Imaging of inducible-BACH2 reporter cells 6 h after PMA/ionomycin stimulation with or without tetracycline (1 μg/ml) treatment. Unstimulated cells were included (indicated as Vehicle). Images were captured after luciferase substrate addition using brightfield (left) and luminescence (right) channels. Each panel is a representative 300 μm × 300 μm cropped area from the overview image. (b) Frequency of positive luminescent cells in stimulated inducible-BACH2 reporter cells following pre-treatment with the indicated doses of tetracycline.

Fig 4: Design and generation of an inducible cell-based luciferase reporter assay for BACH2-mediated repression of AP-1-driven gene expression. (a) Analysis of known BACH2, JunD and p300 binding at the mouse Ifng locus as determined by ChIP-Sequencing of CD8+ and CD4+ T cells. A BACH2-bound putative enhancer of Ifng, Ifng + 18k, is indicated by the black triangle. (b) DNA sequence at Ifng + 18k containing a TPA response element (TRE; red letters). This sequence was concatenated three times and subcloned upstream of a minimal promoter sequence (minP, grey box) controlling expression of NlucP luciferase cDNA sequence in the pNL2.2 reporter vector. (c) Experimental schema for generation of clonally derived inducible-BACH2 reporter and control reporter lines. Jurkat cells stably transduced with pcDNA6/TR vector resulting in expression of the tetracycline repressor protein were co-transfected with luciferase reporter (pNL2.2 Ifng + 18k) and inducible expression (pcDNA4/BACH2) vectors. Stably transfected cells were selected with hygromycin and zeocin and then subjected to single-cell cloning resulting in the generation of a luciferase reporter line with the potential for inducible BACH2 expression and a control reporter line lacking BACH2-inducibility.

Fig 5: BACH2-mediated repression of AP-1 driven luciferase expression using the inducible reporter system. (a) Inducible expression system. Tetracycline repressor (TR) protein in its active form (indicated as a circle) binds to the TetO2 sequence upstream of BACH2 cDNA subcloned into the pcDNA4/BACH2 vector inhibiting transcription of BACH2. Tetracycline (Tet) addition changes the conformation and inactivates the tetracycline repressor (TR) protein (indicated as a square), which is subsequently not able to bind to TetO2 sequence, allowing BACH2 transcription to commence. (b) Western blot for indicated proteins of total lysates isolated from the clonally derived inducible-BACH2 and control reporter cell lines with or without tetracycline treatment. (c) Luciferase activity in the inducible-BACH2 and control reporter lines after 6 h PMA/ionomycin stimulation with or without pre-treatment with tetracycline (1 μg/ml). Unpaired two-tailed Student’s t test (c). Data are representative of 3 independent experiments with 3 culture replicates per condition. Bars and error represent mean (SD); ns not significant; ***P < 0.001; ****P < 0.0001.