Journal: ACS Biomaterials Science & Engineering
Article Title: Mechanical Regulation of Mitochondrial Dynamics and Function in a 3D-Engineered Liver Tumor Microenvironment
doi: 10.1021/acsbiomaterials.2c01518
Figure Lengend Snippet: Mechanical constraint modulates glycolysis in human liver tumor cells. (A) Alexander and HepG2 cells were cultivated for 7 days in either MC or CS conditions. Mitochondrially encoded cytochrome c oxidase I (MT-CO1) expression was analyzed in whole cell lysates of HepG2 and Alexander cells by immunoblotting; GAPDH—control of equal protein loading. The graphs show the densitometric quantification of MT-CO1 immunoblots. (B) MT-CO1 gene expression level analysis in liver samples from hepatocellular carcinoma (HCC) patients. The mRNA levels of MT-CO1 in 12 HCC tissues and paired adjacent normal tissues around were detected by qPCR. The relative gene expression was normalized to GAPDH expression and calculated using the 2 –ΔΔCT method. (*) P < 0.05 denotes significant ( P = 0.0153) difference determined by the Mann–Whitney test. Assessment of intracellular pyruvate (C) and lactate (D) concentrations in Alexander and HepG2 cells cultivated in either MC or CS. Alexander and HepG2 cells were cultivated for 7 days in either MC or CS conditions, and then cell extracts were collected and deproteinized using <10 kDa MWCO filter spin. Subsequently, levels of pyruvate (C) and lactate (D) were determined using the pyruvate fluorescence assay or lactate fluorescence assay, respectively (both from Sigma Aldrich). n = 4–8 samples out of three independent experiments. (**) P < 0.01 and (***) P < 0.001 denote significant differences. (E) Confocal fluorescence analysis of the cytoskeleton organization of cells under mechanical constraint. Alexander and HepG2 cells were grown for 7 days in either MC or CS conditions. F-actin was stained using the ActinGreen 488 ReadyProbes reagent. F-actin tension was assessed by measuring fluorescence distribution and intensity utilizing confocal microscopy. The fluorescence distribution and intensity of F-actin are shown in the reported pseudo-color scale (0–255). Digital images were processed using the ImageJ software (NIH). F-actin tension was visualized using the look-up table Physics in the ImageJ software (NIH).
Article Snippet: We used the human hepatoblastoma HepG2 cell line (American Type Culture Collection, ATCC, Manassas, VA, USA) and human hepatocellular carcinoma cell line Alexander (PLC/PRF/5, ATCC, Manassas, VA, USA) in this study.
Techniques: Expressing, Western Blot, Control, Gene Expression, MANN-WHITNEY, Fluorescence, Staining, Confocal Microscopy, Software
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