Fig 1: Analysis of HK2 expression, HK activity and ATP content of αMHC/miR-143/145TG (L9) mice and characterization of αMHC/miR-143/145/HK2TG (L9/HK2) mice. a Whole cell extracts from the hearts of 3-month old male L3 and L9 mice were examined using the indicated antibodies. The right panels show the relative densitometric analysis of the western blots. The results are presented as the means ± SD. Significance was assessed with one-way ANOVA followed by a post hoc Tukey test (*p < 0.05, **p < 0.01). b Quantitative RT-PCR analysis of Hk2 mRNA in the hearts of 3-month old male L9 mice. The results are presented as the means ± SD with the unpaired t-test applied to determine significance (n = 4 ~ 5; *p < 0.05 vs. NTG). c Western analysis of HK2 in the hearts of 3-month old male L9 and L9/HK2 mice. Whole cell extracts were examined with an anti-HK2 antibody. The white and black arrowheads respectively indicate the transgenic human HK2 and the endogenous mouse HK2. d Quantitative RT-PCR analysis of Hk2 mRNA in the hearts of 3-month old male of L9/HK2 mice. Primers with common binding sites for human and mouse Hk2 genes were used. The results are presented as the means ± SD with the unpaired t-test applied to determine significance (n = 4; **p < 0.01 vs. NTG). e Kaplan-Meier survival analysis of L9 and L9/HK2 mice. Data were analyzed using log-rank test. f Hexokinase assay of the hearts of 3-month old male L9 and L9/HK2 mice. The results are presented as the means ± SD. Significance was assessed with one-way ANOVA followed by a post hoc Tukey test (n = 3; **p < 0.01 vs. NTG, ††p < 0.01 vs. L9). g ATP content assay in the hearts of 4-week old male L9 mice. The results are presented as the means ± SD with the unpaired t-test applied to determine significance (n = 4 ~ 5). Experiments 1 and 2 were performed independently. a–d, f Similar results were obtained in at least two independent experiments
Fig 2: HK3 interactome reveals interaction with BH3-only protein BIM.A DESeq2 normalized counts and differential expression analysis of BIM transcript expression in HK3-null cells and control ±ATRA. Data represent the mean of three biological replicates. B Confocal imaging of proximity ligation assay (PLA) using either endogenously HiBiT-tagged HK2 or HK3 HL60 cell lines. Fluorescent foci indicate the colocalization of proteins. Primary antibodies used were mouse anti-HiBiT, combined with rabbit anti-VDAC or rabbit anti-BIM. Control: secondary antibodies only. C PLA quantification. Negative controls: First column, secondary antibodies only, second column HL60 not expressing a HiBiT-tag (n = 2, 2 technical replicates each, mean ± SEM). Anti-VDAC was used as a positive control for HK2 interactions. D Co-IP of HK3 and BIM. IP was performed in HL60 HK2-null cells treated with ATRA for 2 days using an anti-BIM antibody immobilized on Protein A/G magnetic beads. HK3, BCL-2, and BIM immunoblotting of flowthrough (FT) and pulled-down (IP) proteins are shown. BCL-2, a known BIM interacting protein, was used as a positive control. E NOXA western blotting of HL60 control, HK2- and HK3-null cell lines treated for 4 days with DMSO or ATRA. Quantification of NOXA protein expression of two independent experiments is shown below. NOXA expression was normalized to total protein and Cas9 control cell expression.
Fig 3: CircHIPK3 knockdown repressed cell proliferation, migration, and glycolysis while facilitated apoptosis in lung cancer cells. Lung cancer H1975 and A549 cells were introduced with sh-circHIPK3 or sh-NC. (a) CCK-8 assay for the cell viability of the treated H1975 and A549 cells. (b) Flow cytometry for the apoptosis of the transfected H1975 and A549 cells. (c) Transwell assay for the migration ability of the treated lung cancer cells. (d–f) Assessment of the glucose consumption, lactate production, and the enzyme activity of HK2. (g) Western blot analysis for the protein levels of Ki-67, MMP-9, and HK2 in transfected cells. *P < 0.05.
Fig 4: Schematic depiction of the signaling cascade in aMHC/miR-143/145TG mice. A hypothetical signaling model for a reductive state in aMHC/miR-143/145TG mice is shown. Our data indicate that the overexpression of miR-143 plays a pivotal role in the pathogenesis of the aMHC/miR-143/145TG phenotype, but the key targets for miR-143 triggering this process have not been identified and are shown with a question mark. *: Although the expression of HK2 was suppressed in transgenic hearts, in vitro HK activity was comparable in the L9 and NTG mouse hearts. Since the expression levels of GR, ?-GCSC, p62 and G6PD are controlled by Nrf2, these four molecules are surrounded by a red box. A detailed explanation is given in the Discussion section
Fig 5: TRAP1 regulates Warburg metabolism through modulation of PFK1 activity/expression. (A) Representative fluorescence images showing proximity ligation assay signals (red), detected in HCT116 cells stained with TRAP1 and PFK1 (left panel). The right panel represents the negative control stained with not related antibodies. Nuclei are DAPI-labeled (blue). Scale bar, 10 µm. (B, C) HKII, PFK1, and TRAP1 immunoblot analysis (B) and relative PFK1 and HKII activity (C) in transient TRAP1-silenced HCT116 cells. (D, E) PFK1 and TRAP1 immunoblot analysis (D), and relative PFK1 activity and lactate production (E) in transient TRAP1-silenced SA54 CRC spheres. (F, G) PFK1 and TRAP1 immunoblot analysis (F), and relative lactate production and PFK1 activity (G) in PKF1-silenced HCT116 cells transfected with TRAP1 cDNA. (H, I) PFK1 and TRAP1 immunoblot analysis (H) and relative lactate production and PFK1 activity (I) in shTRAP1 HCT116 cells transfected with PFK1 cDNA. Graphs represent mean ± SD of three experiments. The Mann–Whitney test was used to establish the statistical significance between two groups (P < 0.05). (G) ANOVA test: P = 0.0001; Bonferroni post hoc test: siNEG pmock vs siPFK1, lactate production: P < 0.01, PFK1 activity: P < 0.01, siNEG pmock vs siNEG pTRAP1, lactate production: P < 0.001, PFK1 activity P < 0.001; siNEG pTRAP1 vs siPFK1 pTRAP1, lactate production P < 0.05, PFK1 activity: P < 0.001. (I) ANOVA test: P = 0.004; Bonferroni post hoc test: scramble pmock vs shTRAP1 pmock, lactate production: P < 0.01, PFK1 activity: P < 0.01; scramble pPFK1 vs shTRAP1 pPFK1 lactate production: P < 0.05, PFK1 activity: P < 0.01.
Supplier Page from Abcam for Hexokinase Activity Assay Kit (Colorimetric)