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. 2024 Sep 20;14:21926. doi: 10.1038/s41598-024-72990-z

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — AGR2-dependent changes in PD-L1 expression. (A) RT‒qPCR analysis of PD-L1 mRNA levels in SW620 scr cells and its two AGR2-knockout clones B3 and C6 before and after induction with INF-γ alone or in combination with TNF-α after 4 h of treatment. The values were normalised to those of scr IFN-γ and are presented as 2^−ddCt mean values ± SD of four biological replicates. GAPDH served as an endogenous control. The results for HPRT1 endogenous control are presented in Supplementary Fig. S7 (A) (B) The surface level of PD-L1 in SW620 cells measured by flow cytometry before and after 16 h of IFN-γ/TNF-α induction. MFI values represent the median fluorescence intensity and are presented as the mean ± SD of three biological replicates. On the right is a representative histogram showing the difference in fluorescence intensities of the analysed samples. The histogram including the respective negative controls is available in Supplementary Fig. S7 (B) (C) PD-L1 mRNA levels in SW480 pcDNA3 and AGR2 cells treated with INF- γ or INF- γ/TNF-α after 4 h. The values were normalized to those of pcDNA3 INF- γ and are presented as 2^−ddCt mean values ± SD of three independent replicates. GAPDH served as an endogenous control. The results for the HPRT1 endogenous control are presented in Supplementary Fig.S7 (C) (D) Surface PD-L1 levels in SW480 cells are shown as the mean MFI ± SD of three independent replicates, with a representative histogram on the right. The histogram including the respective negative controls is shown in Supplementary Fig. S7 (D) (E) RT‒qPCR analysis of PD-L1 mRNA levels in SW620 scr cells and its two AGR2-knockout clones B3 and C6 after NPM3 siRNA silencing and 4 h INF-γ/ TNF-α treatment. The values were normalised to those of scr siCTRL INF-γ/ TNF-α and are presented as 2^−ddCt mean values ± SD of three biological replicates. GAPDH served as an endogenous control. The results for HPRT1 endogenous control are presented in Supplementary Fig. S7 (E) (F) Representative WB showing the changes in PD-L1 protein levels in SW620 clones and control cells after siRNA-mediated NPM3 and 16 h of INF- γ/TNF-α induction. GAPDH served as a loading control. The graph showing mean ± SD densitometry is presented in Supplementary Fig. S7 (F) The figure shows cropped blots, the originals are presented in Supplementary Fig. S7 (G) * p 0.05, ** p 0.01, *** p 0.001, **** p 0.0001. (G) Correlation analysis of AGR2 and PD-L1 expression in colorectal patient samples represented by Pearson and Spearman correlation coefficients extracted from the cBioPortal database.