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. 2024 Nov 13;14:27768. doi: 10.1038/s41598-024-75142-5

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. 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-nc-nd/4.0/.

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Fig. 2 — The expression level of 4.1R protein is regulated by protein expression and activation levels of PKR in HCC cell lines. (A) Three HCC cell lines, HuH7, HLE, and HepG2 cells, were seeded in a 6-well, flat-bottomed plate, and cell lysates from three independent wells of each cell line were loaded, followed by WB with anti-4.1R antibody. Overexpression (B) and knockdown (C) of PKR and C16 treatment (D) affected the expression level of 4.1R protein. (B) HuH7 cells were transfected with or without Flag-PKR, followed by WB with anti-PKR antibody (top panel), anti-4.1R antibody (middle panel), and anti-actin antibody (bottom panel). The quantified PKR:actin ratio (center panel) and the 4.1R at 130 kDa:actin ratio (right panel) of the WB are shown. (C) HuH7 cells were transfected with control siRNA and two PKR siRNAs, followed by WB with anti-PKR antibody (top panel), anti-4.1R antibody (middle panel), and anti-actin antibody (bottom panel). The quantified PKR:actin ratio (center panel) and the 4.1R at 130 kDa:actin ratio (right panel) of WB are shown. (D) HuH7 cells were treated with DMSO or 1, 2, or 3 μM of C16 for 24 h, respectively, followed by WB with anti-phospho-PKR antibody (top panel), anti-PKR antibody (second panel), anti-4.1R antibody (third panel), and anti-actin antibody (bottom panel). The quantified PKR:actin ratio (center panel) and the 4.1R at 130 kDa: actin ratio (right panel) of WB are shown. The values in the figure represent fold change to those of control. Means ± SD values of three independent experiments are shown (*p < 0.05, **p < 0.01, ***p < 0.001 compared to control groups by the two-sided Student’s t-test or Tukey’s test).