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

Fig. 6. AMPK functions as a signaling bridge in BNIP3-ENO2 crosstalk in lenvatinib-resistant cells.

Fig. 6

A GSEA analysis of AMPK signaling pathway enrichment in BNIP3 high expression vs BNIP3 low expression group of TCGA-LIHC cohort. B As in (A) but in ICGC-LIHC cohort. C Protein levels of AMPK and p-AMPK at 48 h after cell attachment in Alone group, NCC group and CC group detected by western blot. D Quantitative statistics of protein levels in (C). E Correlation analysis of BNIP3 expression and AMPK signaling pathway in TCGA-LIHC cohort. F As in (E) but in ICGC-LIHC cohort. G Flow cytometry analysis of 2-NBDG-FITC-A and cell proportion at 48 h after cell attachment in Huh7R (Alone group) /CCHuh7R (CC group) treated with AMPK-activator-2, AMPK-IN-3 or AMPK-IN-3 + AMPK-activator-2. H Mountain map of 2-NBDG-FITC-A in (G). * CCHuh7R+AMPK-activator-2 vs CCHuh7R-Control (p < 0.05); # CCHuh7R-AMPK-IN-3 vs CCHuh7R-Control (p < 0.05); & CCHuh7R+AMPK-IN-3 + AMPK-activator-2 vs CCHuh7R+AMPK-IN-3 (p < 0.05); $ Huh7R-AMPK-activator-2 vs Huh7R-Control (p < 0.05); ¥ Huh7R+AMPK-IN-3 vs Huh7R-Control (p < 0.05); % Huh7R+AMPK-IN-3 + AMPK-activator-2 vs Huh7R+AMPK-IN-3 (p < 0.05). I Quantitative statistics of ratio m + /m- in (G). J Cellular lactic acid production levels at 48 h after cell attachment in Huh7R (Alone group) /CCHuh7R (CC group) treated with AMPK-activator-2, AMPK-IN-3 or AMPK-IN-3 + AMPK-activator-2 by flow cytometry cell sorting. K (Top) 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) treated with AMPK-activator-2 using 63X water immersion objective lens. (Bottom) 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. L Protein levels of AMPK, p-AMPK and ENO2 at 48 h after cell attachment in Huh7R (Alone group) /CCHuh7R (CC group) treated with AMPK-activator-2, shENO2 or shENO2 + AMPK-activator-2 detected by western blot. M Quantitative statistics of protein levels in (L). N Protein levels of AMPK, p-AMPK and BNIP3 at 48 h after cell attachment in Huh7R (Alone group) /CCHuh7R (CC group) treated with oeBNIP3, AMPK-IN-3 or oeBNIP3 + AMPK-IN-3 detected by western blot. O Quantitative statistics of protein levels in (N). Three independent experiments were conducted, and the values are represented by means ± SEM using a two-way ANOVA with Tukey’s multiple comparisons test (D, H, J, M and O) or Ordinary one-way ANOVA with Sidak’s multiple comparisons test (I). *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. S6.