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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. 2 — a, Representative dose responses of fluorescence intensity histograms from low-noise mNF-GFP (GL), mNF-BACH1 (BL) and high-noise mNF-GFP (GH), mNF-BACH1 (BH) MB231 clones measured at 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10 and 100 ng ml⁻¹ dox levels, respectively. b, Dose responses of mean fluorescence intensity (MFI) for low-noise mNF-GFP (GL), mNF-BACH1 (BL) and high-noise mNF-GFP (GH), mNF-BACH1 (BH) MB231 clones (n = 3). c, Dose responses of CV for low-noise mNF-GFP (GL), mNF-BACH1 (BL) and high-noise mNF-GFP (GH), mNF-BACH1 (BH) MB231 clones (n = 3). d, Left: BACH1 mRNA level dose responses of both low-noise mNF-BACH1 (BL) and high-noise mNF-BACH1 (BH) MB231 clones. Relative mRNA levels were calculated between each individual replicate and the corresponding uninduced control (n = 3). Right: correlation between BACH1 and GFP mRNA levels in mNF-BACH1 clones (linear regression slopes of 0.9965 and 0.9778 for MB231-1.1-BL and MB231-1.8-BH, respectively; R² goodness-of-fit values of 0.9992 and 0.9971 for MB231-1-BL and BH, respectively). e, Left: protein-level dose responses of total (endogenous + ectopic) BACH1 protein in both low-noise mNF-BACH1 (BL) and high-noise mNF-BACH1 (BH) MB231 clones (n = 3). Right: total BACH1 protein level noise assessed from immunofluorescence measurements for both low-noise mNF-BACH1 (BL) and high-noise mNF-BACH1 (BH) MB231 clones (n = 3). f, Comparison of total BACH1 protein level at uninduced (0 dox) and fully induced (10 ng ml⁻¹ dox) conditions for low-noise mNF-BACH1 (BL) and high-noise mNF-BACH1 (BH) MB231 cell populations to native BACH1 protein level in low-noise mNF-GFP (GL) and their parental LP cell populations. n = 3, one-way ANOVA with Tukey’s multiple comparison correction at 0 dox and 10 µg ml⁻¹ dox with respect to LP sample, **P < 0.01, ****P < 0.0001. g, Western blot examination and quantitation of ectopic BACH1 protein-level dose response in low-noise mNF-BACH1 and mNF-GFP MB231 samples. BACH1 levels were normalized to corresponding internal β-tubulin levels using grayscale quantitation.

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