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. 2020 Sep 9;5:183. doi: 10.1038/s41392-020-00230-7

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© The Author(s) 2020

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. 5 — The acidic tumor microenvironment inhibits VDR aggregation in the nucleus and suppresses the expression of VDR through PPARD. a Immunofluorescence staining of VDR (green) and DAPI (blue) in CC tissue-adherent cells under pH 7.4 and 6.8. Scale bars: 10 μm. b Immunoblotting of VDR in the cytoplasm and nucleus in CC tissue-adherent cells cultured under pH 7.4 and 6.8. α-Tubulin is mainly expressed in the cytoplasm. Lamin B is a fibrous protein that exhibits a structural function and performs transcriptional regulation in the cell nucleus. c Schematic representation of the NES site in the LBD domain of VDR and the mutated amino acids in NES mutant. NES, nuclear export signal. LBD, ligand-binding domain. DBD, DNA-binding domain. d Immunofluorescence staining of VDR (green) in CC tissue-adherent cells treated with ethanol or LMB for 1 h at 37 °C under pH 7.4 and 6.8. LMB, leptomycin B (a nuclear export protein inhibitor). Scale bars: 10 μm. e Immunofluorescence staining of VDR (green) and DAPI (blue) in CC tissue-adherent cells transfected with pLV-CMV-VDR or pLV-CMV-VDR-mut (the wild-type or mutated VDR shown in c, respectively). Scale bars: 5 μm. f Tumor sphere formation assays of CC tissue CSCs transfected with pLV-CMV-VDR or pLV-CMV-VDR-mut under pH 7.4 and 6.8. Student’s t-test. g Correlation of PPARD expression with VDR expression in CRC samples from TCGA. h qPCR of PPARD in CC tissue adherent, HCT8, DLD1, SW480, SW620, and RKO CRCs cultured under pH 7.4 and 6.8. Student’s t-test. i Immunofluorescence staining of PPARD (green) and DAPI (blue) in CC tissue-adherent cells under pH 7.4 and 6.8. Scale bars: 10 μm. j ChIP was used to assess PPARD binding to the promoters of VDR in CC tissue-adherent cells (upper). A polyclonal anti-PPARD antibody or a mouse IgG antibody was used. The immunoprecipitated DNA was quantified by qPCR (lower). Student’s t-test. k qPCR of PPARD and VDR in CC tissue-adherent cells transfected with siRNA for PPARD. Student’s t-test. l, m Immunoblotting of PPARD, VDR, SOX2, and the stemness markers NUMB, CD133, and OCT4 in CC tissue-adherent cells transfected with siRNA for PPARD. NUMB is an endocytic adaptor protein that has a crucial role in asymmetrical cell division. Three independent experiments were performed to obtain the data in f, h, j, and k. The data are shown as the mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001