Journal: The Journal of Pathology

Article Title: Hypoxia‐inducible factor 1‐alpha does not regulate osteoclastogenesis but enhances bone resorption activity via prolyl‐4‐hydroxylase 2

doi: 10.1002/path.4906

Figure Lengend Snippet: HIF induction during osteoclast differentiation. (A) Western blot analysis of HIF‐1α protein expression in CD14+ monocytes treated with 25 ng/ml M‐CSF for 2–24 h versus the untreated normoxic control (Nx). Hx = hypoxia (2% O 2 , 24 h). (B) RT‐qPCR comparing HIF1A , HIF2A, LDHA , and SLC2A1 ( GLUT1 ) mRNA on days 3, 5, 7 and 9 of osteoclast differentiation. * p < 0.05; ** p < 0.01. (C) Western blot analysis of HIF‐1α, GLUT1, and LDHA protein on days 3, 5, and 9 of osteoclast differentiation. (D) Normalized HRE‐driven luciferase reporter luminescence on days 3, 5, 7, and 9 of osteoclast differentiation. * p < 0.05; ** p < 0.01.

Article Snippet: Primary antibodies were against HIF‐1α (clone 54, 1:1000; BD Biosciences, Oxford, UK), GLUT1 (ab14683, 1:2500; Abcam, Cambridge, UK), LDHA (NBP1‐48336, 1:2000; Novus Biologicals, Cambridge, UK), and β‐tubulin (clone TUB2.1, 1:2500; Sigma‐Aldrich, Dorset, UK).

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, Luciferase

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