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. 2022 Nov 2;13:6575. doi: 10.1038/s41467-022-34251-3

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© The Author(s) 2022, corrected publication 2022

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. 7 — Western blotting (a) and quantification (b) following DNA affinity purification for FOXA1 (n = 3) and IRF1 (n = 2) in parental UM-UC-1 treated in the presence and absence of IFNɣ. In the absence of IFNɣ, FOXA1 exhibits significantly greater binding to the wild-type CD274 probe relative to scrambled negative control DNA probe (Student’s t-test; p = 0.0319) or mutant (Student’s t-test; p = 0.0256) CD274 probes. In the presence of IFNɣ, FOXA1 still showed significantly greater binding to wild-type CD274 promoter relative to scrambled DNA probe (Student’s t-test; p = 0.0319) and FOXA1-binding mutant (Student’s t-test; p = 0.0256) CD274 probes. There were no significant differences in FOXA1 binding relative to IFNɣ treatment. Following IFNɣ treatment, IRF1 shows increases in binding to wild-type CD274 relative to scrambled and mutant probes. Q-RT-PCR data are expressed as the mean ± S.D. from independent experiments of FOXA1 (n = 3), IRF1 (n = 2), respectively. Source data are provided as a Source Data file (c) and western blotting (d) shows that FOXA1 KO in UM-UC-1 results in significant increases in IRF1 expression at the mRNA and protein levels, respectively. Data of Q-RT-PCR are expressed as the mean ± S.D. from independent experiments (n = 3). Source data are provided as a Source Data file. Western blotting (e) and quantification (f) following DNA affinity purification for FOXA1 and IRF1 in parental UM-UC-1 (Ctrl) and FOXA1 KO UM-UC-1 (FOXA1 KO) cell lines. IRF1 purified from FOXA1 KO UM-UC-1 exhibited a 26-fold increase in binding to the CD274 promoter fragment relative to parental UM-U-C1. IRF1 was unable to be purified from parental and FOXA1 KO UM-UC-1 cells with scrambled negative control oligo (n = 2). Ectopic expression of IRF1 in parental UM-UC-1 cells followed by Q-RT-PCR (n = 4). Data are expressed as the mean ± S.D. from independent experiments (g) and western blotting (h). Source data are provided as a Source Data file. Overexpression of IRF1 significantly increased expression of i CD274 (n = 4, Student’s t-test; p = 0.0344), j STAT2 (n = 4, Student’s t-test p = 0.0321), k ISG15 (n = 4, Student’s t-test p = 0.0008), l IFIT2 (n = 3 Student’s t-test; p = 0.0338), m IFIT3 (n = 4, Student’s t-test; p = 0.0009) and n IFI35 (n = 4, Student’s t-test; p = 0.0406). Data are expressed as the mean ± S.D. from independent experiments. *p < 0.05 was considered as a statistically significant. ns not significant. All tests are unpaired two-sided Student’s t-test.