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. 2023 May 25;19(7):887–899. doi: 10.1038/s41589-023-01344-z

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a, Experimental design for measuring and calculating invasiveness. b, Representative images of invading cells at the bottom of the inserts after 16 h–24 h invasion assays. Cell number was determined using nuclear staining and GFP fluorescence. Scale bar, 200 µm. c, Invasiveness of low-noise mNF-BACH1 (BL) and high-noise mNF-BACH1 (BH) clones over increasing dox concentrations (n = 3). d, Invasiveness of low-noise mNF-GFP (GL) and high-noise mNF-GFP (GH) clones at increasing dox concentrations (n = 3). e, Invasiveness at 0, 0.5 and 10 ng ml⁻¹ dox levels for mNF-BACH1 versus mNF-GFP clones (n = 3, one-way ANOVA for cross-comparison among four samples at each dose and unpaired two-tailed t-test for every comparison between low-noise and high-noise clones. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). f, Expression noise difference (ΔCV%, left y axis) and mean difference (ΔMean, right y axis) versus invasiveness difference (ΔInvasiveness) at different dox inductions between low-noise and high-noise mNF-BACH1 clones. Δ = BH − BL, n = 3, Pearson correlation coefficients for ΔCV% versus ΔInvasiveness, r = 0.6611, P = 0.0374; for ΔMean versus ΔInvasiveness, r = −0.3477, P = 0.3248. g, Invasiveness of low-noise mNF-BACH1 (BL) and high-noise mNF-BACH1 (BH) clones versus eGFP::BACH1 expression (fluorescence) level. Pink and cyan arrows indicate hemin concentrations needed to degrade BACH1 levels along an upslope, at 10 ng ml⁻¹ dox to resemble 0.5 ng ml⁻¹ dox induction, and along a downslope, at 0.5 ng ml⁻¹ dox to resemble 0.3 ng ml⁻¹ dox induction, respectively. h, Predictable, opposite shifts of invasiveness for low-noise mNF-BACH1 (BL) and high-noise mNF-BACH1 (BH) clones when treated with hemin to induce degradation along an upslope and a downslope, compared to control samples. Pink and cyan arrows indicate hemin-dependent BACH1 degradation corresponding to the upslope and downslope in g. Unpaired two-tailed t-test was used for each comparison, n = 3, P > 0.05. NS, not significant.

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