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. 2023 Apr 27;14:2421. doi: 10.1038/s41467-023-38160-x

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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. 4 — a Schematic of the identification of POLD1 positively correlated genes in the TCGA-BLCA database (the blue circle, statistical significance was determined by Jarque-Bera test) and genes that may be positively regulated by POLD1 in 5637 cells (the brown circle, statistical significance was determined by two-tailed Wald test). b A Heatmap of genes with significant changes in the Hallmark_MYC_Targets_V1 gene set upon POLD1 knockdown in 5637 cells (n = 3). Statistical significance was determined by two-tailed Wald test. c POLD1 related gene set enrichment analysis (GSEA). Statistical significance was determined by two-tailed Fisher’s exact test. d After POLD1 knockdown in T24, 5637, and UM-UC-3 cells with specific siRNA, Western blot analysis showed that the protein levels of MYC were significantly reduced. e Western blot analysis of endogenous ubiquitination after MYC-IP under denaturing conditions in DOX-inducible shPOLD1 T24 cells. f Representative images of Western blot analysis of the effect of POLD1 depletion on MYC degradation in T24 cells incubated with CHX (50 μg/mL) or CHX plus MG132 (10 μM) for the indicated times (left panel) and statistical diagram of protein half-life assays (right panel, n = 3 biologically independent experiments in each group). g qPCR analysis of MYC and its target genes in DOX-induced shPOLD1 T24 cells (Graph shows mean ± SD, n = 3 biologically independent experiments in each group). h Representative images of Western blot analysis of the expression changes of POLD1 and MYC after POLD1 knockdown induced by DOX at gradient time point (n = 3 biologically independent experiments in each group). The protein levels of POLD1 and MYC were quantified by ImageJ software and standardized to GAPDH. i Cell cycle and j proliferation analysis after POLD1 knockdown induced by DOX at gradient time points (Graph shows mean ± SD, n = 3 biologically independent experiments in each group). k Timeline of all assay results of DOX-induced POLD1 knockdown in this section. l Western blot analysis of POLD1 and MYC expression in different BLCA cell lines. For Western blot experiments, GAPDH was used as a loading control (d–f, h, l). Statistical significance was determined by two-tailed Student’s T-test (g, h, j). NES: Normalized Enrichment Score; ns: not significant. Source data are provided as a Source Data file.