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. 2023 Dec 9;14:8177. doi: 10.1038/s41467-023-44031-2

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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 Representative immunofluorescent staining of GR in HEK293 cells treated with KH-103, or DEX, or cortisol, or DEX + KH-103, or cortisol + KH-103. Scale bars are 100 μm. (each group n = 3) b Representative immunofluorescent staining of GR in HEK293 cells that were pretreated with DEX for 1 h followed by KH-103 treatment. Scale bars are 50 µm. c Representative immunoblots and quantification of GR levels upon KH-103 treatment in the absence or presence of Lenalidomide and/or DEX in HEK293 cells. All groups n = 4. Ordinary one-way ANOVA showed a significant difference (F(7,24) = 105.6, p = <0.0001). Follow-up Dunnett’s multiple comparisons showed significant differences for DMSO vs. all other groups (p = <0.0001) except Len which was not significant, p = 0.0919). Len: Lenalidomide d Representative immunoblot of fraction-specific markers in cytosolic, membrane, and nuclear fractions obtained from HEK293 cells. *unspecific bands. d Representative immunoblots of GR in cytosolic, membrane, and nuclear fractions obtained from HEK293 cells treated under various conditions. U: untreated, A: DMSO, B: 2 h pretreatment with DMSO followed by 16 h treatment with KH-103 (DMSO/KH-103), C: DEX/DEX, D: DEX/DEX + KH-103, E: DEX/DMSO, F: DEX/KH-103. E and F conditions included an extra wash step after the pretreatment with DEX. e Band quantification of GR in the cytosolic fraction. Two-way ANOVA showed a significant main effect for KH-103 (F(1, 12) = 38.84, p = <0.0001) and a significant interaction between KH-103 and DEX (F(2, 12) = 7.53, p = 0.0076). There was no significant main effect for DEX (F(2,12) = 0.43, p = 0.6595). f Band quantification of GR in the membrane fraction. Two-way ANOVA showed a significant main effect for KH-103 (F(1,12) = 35.41, p = <0.0001) and a significant interaction between KH-103 and DEX (F(2,12) = 7.21, p = 0.0088). There was no significant main effect for DEX (F(2, 12) = 2.06, p = 0.1698). g Band quantification of GR in the nuclear fraction. Two-way ANOVA showed a significant main effect for KH-103 (F(1,11) = 106.30, p = <0.0001) and DEX (F(2, 11) = 129.20, p = <0.0001). There was also a significant interaction between KH-103 and DEX (F(2,11) = 9.88, p = 0.0035). Follow-up Fisher’s LSD tests for all three fractions are summarized in Supplementary Tables 1–3. n = 3. P-values # <0.1, *<0.05, **<0.01, ***<0.001, ****<0.0001. Data are presented as mean values ± SEM. Source data are provided as a Source Data file.