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. 2024 Jul 5;15(7):484. doi: 10.1038/s41419-024-06870-9

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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. 5 — A Heatmap exhibiting expression levels of glycolysis-related genes between CCHuh7R and Huh7R. B As in (A) but between CCHuh7R and NCCHuh7. C UpSet plot displaying overlapping glycolysis-related genes based on (A) and (B). D Flow cytometry analysis of 2-NBDG-FITC-A and cell proportion at 48 h after cell attachment in Huh7R (Alone group) /CCHuh7R (CC group) transfected with shRNA against ENO2 (shENO2), ENO2 overexpression plasmid (oeENO2) or oeENO2+shENO2. E Mountain map of 2-NBDG-FITC-A in (D). * CCHuh7R-oeENO2 vs CCHuh7R-Control (p < 0.05); # CCHuh7R-shENO2 vs CCHuh7R-Control (p < 0.05); & CCHuh7R-shENO2+oeENO2 vs CCHuh7R-shENO2 (p < 0.05); $ Huh7R-oeENO2 vs Huh7R-Control (p < 0.05); ¥ Huh7R-shENO2 vs Huh7R-Control (p < 0.05); % Huh7R-shENO2+oeENO2 vs Huh7R-shENO2 (p < 0.05). F Quantitative statistics of ratio m + /m- in (D). G Cellular lactic acid production levels at 48 h after cell attachment in Huh7R (Alone group) /CCHuh7R (CC group) transfected with shENO2, oeENO2 or shENO2+oeENO2 by flow cytometry cell sorting. H Protein levels of LC3B, TOMM20, BNIP3, ENO2, GLUT1, HK1, HK2, MCT1, MCT4 and PFKP at 48 h after cell attachment in Huh7R (Alone group) /CCHuh7R (CC group) transfected with shENO2, oeENO2 or shENO2+oeENO2 detected by western blot. I Quantitative statistics of protein levels in (H). J (Left) High content immunofluorescence imaging of colocalization of autophagosomes (Cy5.5-LC3: purple) and mitochondria (TOMM20: green) at 48 h after cell attachment in Huh7R (Alone group) /CCHuh7R (CC group) transfected with shENO2, oeENO2 or shENO2+oeENO2 using 63X water immersion objective lens. (Right) Quantification of colocalization between LC3 (purple peak) and TOMM20 (green peak) in the above groups. Purple/green peak height represents fluorescence intensity of LC3B/TOMM20; overlapping peaks indicate colocalization numbers between LC3B and Tomm20. Three independent experiments were conducted, and the values are represented by means ± SEM using a two-way ANOVA with Tukey’s multiple comparisons test (E, G and I) or Ordinary one-way ANOVA with Sidak’s multiple comparisons test (F). *p < 0.05, #p < 0.05, $p < 0.05, ¥p < 0.05, &p < 0.05, %p < 0.05, ns = non-statistically significant. See also Fig. S5.