Journal: bioRxiv

Article Title: Acinar-ductal metaplasia in the pancreas requires a glycolytic switch and functional mitochondria

doi: 10.1101/2022.06.27.495427

Figure Lengend Snippet: Immunohistochemistry for GLUT1, B, E for HK1 and C, F for LDHA on human tissue microarrays. Scale bar 50 µm. G . Histological scores for GLUT1 IHCs in A and D. H . Histological scores for HK1 IHCs in B and E. I . Histological scores for LDHA IHCs in C and F. Histological scores were given separately for normal tissue, early and late ADM lesions. *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: 2 μm thin slides were then prepared and immunohistochemistry was performed on a Bond Rxm system (Leica) with primary antibodies against Hexokinase 1 (1:500, Cell signaling, 2024), LDHA (1:10.000, Novus Biologicals, NBP1-48336), Glut1 (1:750, abcam, ab115730) or ATP5D (1:100, Sigma Aldrich, HPA002865).

Techniques: Immunohistochemistry

Journal: bioRxiv

Article Title: Acinar-ductal metaplasia in the pancreas requires a glycolytic switch and functional mitochondria

doi: 10.1101/2022.06.27.495427

Figure Lengend Snippet: H&E stainings of pancreata of 10 week old animals. Scale bar = 100 µm. E . Number of ADM lesions per mm 2 acinar tissue of pancreata of 10 week old mice. F, G . Immunohistochemistry for GLUT1 on pancreatic tissue from 10 week old mice. Scale bar = 100 µm H-K . Histological scores for IHCs for GLUT1 ( H ), HK1 ( I ), LDHA ( J ) and ATP5D ( K ) on pancreatic tissue from 10 week-old mice. Histological scores were given separately for normal tissue and ADM lesions. L . Principal component analysis of detected metabolites in tissue samples from 10 week-old animals. M, N . Amounts of glucose ( M ) and succinate ( N ) in the pancreas of 10 week-old animals. Metabolite amounts were plotted against the number of ADM lesions per mm 2 of pancreatic acinar tissue. Results are mean +/-SD, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: 2 μm thin slides were then prepared and immunohistochemistry was performed on a Bond Rxm system (Leica) with primary antibodies against Hexokinase 1 (1:500, Cell signaling, 2024), LDHA (1:10.000, Novus Biologicals, NBP1-48336), Glut1 (1:750, abcam, ab115730) or ATP5D (1:100, Sigma Aldrich, HPA002865).

Techniques: Immunohistochemistry

Journal: bioRxiv

Article Title: Acinar-ductal metaplasia in the pancreas requires a glycolytic switch and functional mitochondria

doi: 10.1101/2022.06.27.495427

Figure Lengend Snippet: Transdifferentiation rates of wild-type acinar cells with or without stimulus and acinar cells harbouring active oncogenes. B-G . Immunofluorescence staining of wild-type and transdifferentiated acinar cells. Cells were stained for GLUT1 (red), KRT19 (green) and DAPI (blue). Scale bar = 50 µm. H . Protein expression of GLUT1 and LDHA in either freshly isolated or transdifferentiated acinar cells. ERK1/2 served as loading control. I . GSVA scores for genesets representing glycolysis and OxPhos in RNAseq data from freshly isolated and cultured acinar cells. J, K . Amounts of glucose ( J ) and lactate ( K ) in supernatants of transdifferentiated acinar cells. Values of unused medium were subtracted from the respective samples. Medium values were normalised to the pellet weight of the acinar cells. L . Isotopologue distribution of hexose-P 2 or 24 h after isolation of acinar cells. M . Amounts of citrate in transdifferentiated acinar cells. Values of freshly isolated acinar cells were subtracted from the respective samples. N, O . Isotopologue distribution of a-ketoglutarate ( N ) and fumarate ( O ) 2 or 24 h after isolation of acinar cells. Results are mean +/-SD, ****p<0.0001.

Article Snippet: 2 μm thin slides were then prepared and immunohistochemistry was performed on a Bond Rxm system (Leica) with primary antibodies against Hexokinase 1 (1:500, Cell signaling, 2024), LDHA (1:10.000, Novus Biologicals, NBP1-48336), Glut1 (1:750, abcam, ab115730) or ATP5D (1:100, Sigma Aldrich, HPA002865).

Techniques: Immunofluorescence, Staining, Expressing, Isolation, Control, Cell Culture

Journal: bioRxiv

Article Title: Acinar-ductal metaplasia in the pancreas requires a glycolytic switch and functional mitochondria

doi: 10.1101/2022.06.27.495427

Figure Lengend Snippet: Extracellular acidification rates of acinar cells 24 h after isolation. Cells were treated with the displayed inhibitors at indicated timepoints. All values were normalized to protein content per well. B . Calculated ATP production from ECAR and OCR rates in acinar cells 24 h after isolation. C . Transdifferentiation rates 48 h after acinar cell isolation. D, E, F . Transdifferentiation rates after treatment with the GLUT1 inhibitor WZB117 ( D ), HK1 inhibitor 2-DG ( E ) or LDHA inhibitor galloflavin ( F ). G . Transdifferentiation rates of human acinar cells after stimulation with TGFa and inhibition with either WZB117, 2-DG or Galloflavin. Results are mean +/-SD.

Article Snippet: 2 μm thin slides were then prepared and immunohistochemistry was performed on a Bond Rxm system (Leica) with primary antibodies against Hexokinase 1 (1:500, Cell signaling, 2024), LDHA (1:10.000, Novus Biologicals, NBP1-48336), Glut1 (1:750, abcam, ab115730) or ATP5D (1:100, Sigma Aldrich, HPA002865).

Techniques: Isolation, Cell Isolation, Inhibition

Journal: bioRxiv

Article Title: Lactate metabolic coupling between the endplates and nucleus pulposus via MCT1 is essential for intervertebral disc health

doi: 10.1101/2025.02.24.640004

Figure Lengend Snippet: a) Representative images of COL X, EMCN, and TNAP in the endplate region of 12-month-old Mct1 CTR and Mct1 cKO mice. White arrowheads indicate hypertrophic chondrocytes, yellow arrowheads show blood vessels. Scale bar: columns 1, 3 = 200 μm, columns 2, 4, = 50 μm. b) Representative images showing GLUT1, MCT4, LDHA, and LDHB in the AF and NP compartments, scale bar = 50 μm. n = 4 mice/genotype (2 males, 2 females), 3 discs/animal assessed. Dotted lines indicate boundaries between different tissue compartments within the disc.

Article Snippet: Tissue sections were then blocked with 10% serum (Thermo Fisher Scientific, 10,000C) in PBS-T or M.O.M.™Immunodetection Kit (Vector Laboratories, BMK-2202), and then incubated with primary antibodies against LDHA (1:100; Novus; NBP2-19320), LDHB (1:50; Santa Cruz; sc100775), pan-Kla (1:100, PTM Bio, PTM-1401-RM), and H3K18la (1:100, PTM Bio, PTM-1406RM).

Techniques:

Journal: Aging (Albany NY)

Article Title: Metformin suppresses Nrf2-mediated chemoresistance in hepatocellular carcinoma cells by increasing glycolysis

doi: 10.18632/aging.103777

Figure Lengend Snippet: Metformin increased glucose uptake and glycolysis in HepG2/DDP cells. ( A ) Metformin increased glucose uptake. Indicated cells were treated with or without metformin (1mM) for 24 hours and glucose uptake assay were conducted with Glucose Uptake-Glo. Cell Titer-Glo was also carried out to measure the relative viability, which was used to normalize the data in glucose uptake assay. Data from 3 independent biological samples of 3 replicates were statistically analyzed by student’s t-test (*** P<0.0001). ( B ) Metformin increased the expression of glucose transporter Glut1 and Glut4. HepG2/DDP cells were treated with or without metformin (1mM) for 24 hours and total cell lysates were separated by SDS-PAGE. Glut1 and Glut4 protein levels were detected by Western blot using specific antibodies to Glut1 and Glut4. Tubulin was used as internal control. Representative images of were shown. Quantification of N= 2 biological repeats were shown in bar graph. ( C ) Metformin increased intracellular glucose concentration in HepG2/DDP cells. HepG2/DDP cells were treated with or without metformin (1mM) for 24 hours, washed extensively and intracellular glucose concentration was measured by using Glucose-Glo kit. Data from 2 independent biological samples of 3 replicates were plotted and statistically analyzed by student’s t-test (** P<0.001). ( D ) Metformin increased the protein levels of glycolytic enzymes HK2 and LDHA. Experiment was conducted as in (B) except HK2 and LDHA antibodies were used. Representative images of were shown. Quantification of N= 3 biological repeats were shown in bar graph. ( E ) Metformin increased intracellular lactate production. Indicated cells were treated with or without metformin (1mM) for 24 hours, washed extensively then intracellular lactate concentration was measured by using lactate-Glo kit. Data from 2 independent biological samples of 3 replicates were plotted and statistically analyzed by student’s t-test (** P<0.001, *** P<0.0001). ( F ) Metformin increased intracellular NAD/NADH production. HepG2/DDP cells were treated with or without metformin (1mM) for 24 hours and lactate concentration was measured by using NAD/NADH -Glo kit. Data from 2 independent biological samples of 3 replicates plotted and statistically analyzed by student’s t-test (* P<0.05, **P<0.001). ( G ) Metformin increased intracellular NADP/NADPH production. HepG2/DDP cells were treated with or without metformin (1mM) for 24 hours and lactate concentration was measured by using NADP/NADPH -Glo kit. Data from 2 independent biological samples of 3 replicates were plotted and statistically analyzed by student’s t-test (*** P<0.0001).

Article Snippet: Membranes were blocked in 5% non-fat milk and probed with primary antibodies in 5% non-fat milk at the following concentration: Nrf2 (Promab Biotechnologies, #30597) at 2000X, Tubulin (Promab Biotechnologies, #20374) 0.2 ug/ml, Glut1 (R&D Systems, MAB14181) 2 ug/ml, Glut4 (Abcam, ab654) at 2500X dilution, HK2 (R&D Systems, MAB8179) at 0.2 ug/ml, LDHA (R&D Systems, AF7304).

Techniques: Expressing, SDS Page, Western Blot, Control, Concentration Assay

Journal: eLife

Article Title: Insights into metabolic heterogeneity of colorectal cancer gained from fluorescence lifetime imaging

doi: 10.7554/eLife.94438

Figure Lengend Snippet: ( A ) Representative immunohistochemical images of GLUT3 expression. Scale bar = 50 μm (magnification x200) and 20 μm (magnification x630). ( B ) Representative immunohistochemical images of LDHA expression. Scale bar = 50 μm (magnification x200) and 20 μm (magnification x630). ( С ) Semi-quantitative evaluation of the expression level by staining intensity.

Article Snippet: Next, slides were incubated with primary polyclonal antibodies to Glucose Transporter 3 GLUT3 (E-AB-31557, Elabscience, China) or to Lactate dehydrogenase A LDHA (E-AB-19947, Elabscience, China) for 15 min. For the antibodies detection «Bond polymer refine detection system» (Leica Biosystems, UK) was used.

Techniques: Immunohistochemical staining, Expressing, Staining