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: Molecular medicine reports

Article Title: Inhibition of lactate dehydrogenase A by microRNA-34a resensitizes colon cancer cells to 5-fluorouracil.

doi: 10.3892/mmr.2014.2726

Figure Lengend Snippet: Figure 5. Effect of LDHA levels on colon cancer cell resistance to 5‑FU. (A) Expression of LDHA following transient transfection of LDHA into DLD‑1 cells. β‑actin was used as a loading control. (B) After transfection (48 h), vector control cells and cells with overexpression of LDHA were treated with 5‑FU with indicated concentrations for 72 h and cell viability assays were performed. Columns represent the mean of three independent experiments. Bars represent the standard error. *P<0.05 and **P<0.01. LDHA, lactate dehydrogenase A; 5‑FU, 5‑fluoruracil.

Article Snippet: Overexpression vectors containing wild type LDHA (Myc-DDK-tagged) were purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Over Expression

Journal: Disease markers

Article Title: lncRNA-DANCR Promotes Taxol Resistance of Prostate Cancer Cells through Modulating the miR-33b-5p-LDHA Axis.

doi: 10.1155/2022/9516774

Figure Lengend Snippet: Figure 8: Restoration of LDHA rescues the miR-33b-5p-promoted Taxol sensitization. (a) PC3-TXR cells without or with LDHA knockdown were treated with Taxol. Cell survival was evaluated by clonogenic assay and (b) MTT assay. (c) PC3-TXR cells were transfected with control miRNA, miR-33b-5p alone, or plus LDHA overexpression plasmid. Protein expression of LDHA was determined. (d) Glucose uptake and (e) lactate product from the above transfected cells were examined. (f) The above transfected PC3- TXR cells were treated with Taxol at the indicated concentrations. Cell viability was determined by MTT assay. ∗p < 0:05 and ∗∗p < 0:01.

Article Snippet: LDHA overexpression plasmid was purchased from http://Origene.com. miRNA-33b-5p and control miRNA were synthesized by GenePharma (Shanghai, China). siRNA and miRNA were transfected at 25nM for 48 hours.

Techniques: Knockdown, Clonogenic Assay, MTT Assay, Transfection, Control, Over Expression, Plasmid Preparation, Expressing

Journal: Disease markers

Article Title: lncRNA-DANCR Promotes Taxol Resistance of Prostate Cancer Cells through Modulating the miR-33b-5p-LDHA Axis.

doi: 10.1155/2022/9516774

Figure Lengend Snippet: Figure 9: The roles of DANCR-miR-33b-5p-LDHA in Taxol resistance of PCa cells. (a) Control vector, DANCR alone, or plus miR-33b-5p was transfected into PC3-TXR cells. Expressions of miR-33b-5p, (b) LDHA, (c) glucose uptake, and (d) lactate product were detected. (e) The above transfected cells were treated with Taxol at the indicated concentrations. Cell survival was evaluated by MTT assay and (f) Annexin V apoptosis assay. ∗p < 0:05 and ∗∗p < 0:01.

Article Snippet: LDHA overexpression plasmid was purchased from http://Origene.com. miRNA-33b-5p and control miRNA were synthesized by GenePharma (Shanghai, China). siRNA and miRNA were transfected at 25nM for 48 hours.

Techniques: Control, Plasmid Preparation, Transfection, MTT Assay, Apoptosis Assay

Journal: Cell Death & Disease

Article Title: Targeting LINC01711 in FAP + cancer-associated fibroblasts overcomes lactate-mediated immunosuppression and enhances anti-PD-1 efficacy in lung adenocarcinoma

doi: 10.1038/s41419-025-07974-6

Figure Lengend Snippet: A The results of ECAR assays performed in FAP + CAFs transfected with si-LINC01711 or si-NC ( n = 3 biological repeats). The P -value was calculated by two-tailed unpaired t -test. B – D The results of glucose uptake, intracellular lactate production and extracellular lactate production measurement performed in FAP + CAFs transfected with si-LINC01711 or si-NC ( n = 3 biological repeats). The P -value was calculated by two-tailed unpaired t -test. E Flow chart of [U13 C] Glucose stable isotope tracer analysis. F [U13 C] Glucose stable isotope tracer analysis was performed in FAP + CAFs transfected with si-LINC01711 or si-NC. The lactate was shown ( n = 3 biological repeats). The P -value was calculated by two-tailed unpaired t -test. G Silver SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) image revealing proteins immunoprecipitated by LINC01711 and its antisense RNA in FAP + CAFs. H Western blotting validated the interaction between LINC01711 and LDHA. I RNA-pulldown assay was performed using biotin-LINC01711 and recombinant LDHA, followed by western blotting validation. J RIP (RNA immunoprecipitation) assay followed by RT-qPCR analysis confirmed that LINC01711 bound to LDHA, rather than LDHB ( n = 3 biological repeats). The P -value was calculated by two-tailed unpaired t -test. K Dual RNA-FISH (fluorescence in situ hybridization) and immunofluorescence assay showing the colocalization of LINC01711 and LDHA in FAP + CAFs. Scale bars: 10 μm. L RT-qPCR detection of LINC01711 expression in the cytoplasmic and nuclear fractions of FAP + CAFs. M Immunoblot detection of LDHA protein in FAP + CAFs by searching for biotinylated RNA or its antisense sequence of LINC01711 isoform transcribed in vitro. N Molecular docking predicted 3D structure of the LDHA-LINC01711-Δ1 complex. O RIP (RNA immunoprecipitation) assay followed by RT-qPCR analysis confirmed the interaction between LDHA-mutant and LINC01711 ( n = 3 biological repeats). The P -value was calculated by two-tailed unpaired t -test. P Western blotting validated the interaction between the interaction between LDHA-mutant and LINC01711. Q Western blotting confirmed that altering LINC01711 expression would not affect LDHA expression. R RT-qPCR confirmed that altering LINC01711 expression would not affect LDHA expression ( n = 3 biological repeats). The P -value was calculated by two-tailed unpaired t -test. All the results were shown as mean ± S.E.M. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Recombinant human LDHA variants ( P01711 , Solarbio) were mixed with active recombinant His tagged-FGFR1 ( P09665 , Solarbio) in kinase reaction buffers (HER2: 20 mM Tris (pH 7.5), 5 mM MnCl2, 0.5 mM Na3VO4, 1 mM EGTA, 2 mM DTT, 5 mM β-glycerophosphate, 0.01% CHAPS) at 30 ° C for 30 min. Terminate the reaction by soaking in a boiling water bath for 5 min. Quickly freeze the protein in liquid nitrogen and perform Western blot analysis of LDHA-Y10 phosphorylation.

Techniques: Transfection, Two Tailed Test, SDS Page, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Western Blot, Recombinant, Biomarker Discovery, RNA Immunoprecipitation, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Immunofluorescence, Expressing, Sequencing, In Vitro, Mutagenesis

Journal: Cell Death & Disease

Article Title: Targeting LINC01711 in FAP + cancer-associated fibroblasts overcomes lactate-mediated immunosuppression and enhances anti-PD-1 efficacy in lung adenocarcinoma

doi: 10.1038/s41419-025-07974-6

Figure Lengend Snippet: A LDHA activity detection revealed that LINC01711 knockdown inhibited LDHA activity ( n = 5 biological repeats). The P -value was calculated by two-tailed unpaired t- test. B Western blot revealed that phosphorylation level of LDHA significantly decreased when LINC01711 was knockdown. C The results of western blot indicated the level of phosphorylation at the Y10 site of LDHA when FGFR1, Her2 or JAK was knocked down. D Western blot detected the level of phosphorylation at the Y10 site of LDHA in FAP + CAFs treated with PD166866 , oe-LINC01711, and both. E , F RNA-pulldown assay was performed using biotin-LINC01711 and endogenous FGFR1, or biotin-LINC01711 and recombinant LDHA, followed by western blotting validation. G RIP (RNA immunoprecipitation) assay followed by RT-qPCR analysis confirmed that LINC01711 bound to FGFR1 ( n = 3 biological repeats). The P -value was calculated by two-tailed unpaired t -test. H BiFC (Bimolecular fluorescence complementation) experiment revealed LINC01711 could modulates the phosphorylation by FGFR1. I , J Immunoprecipitation experiment revealed that LINC01711 could promotes the interaction between FGFR1 and LDHA. K The in vitro kinase assay was conducted using recombinant His-tagged LDHA and His-tagged FGFR1. The result showed that LINC01711 could significantly promote FGFR1 mediated phosphorylation of LDHA. L Western blot, indicated that overexpression of LINC01711 could upregulated LDHA phosphorylation and the process depends on FGFR1. M Crosslinking followed by western blot revealed that LINC01711 could promote the tetramer formation of LDHA, which was dependent on the participant of FGFR1. N The result of size exclusion chromatography followed by western blot was consistent with crosslinking results. All the results were shown as mean ± S.E.M. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Recombinant human LDHA variants ( P01711 , Solarbio) were mixed with active recombinant His tagged-FGFR1 ( P09665 , Solarbio) in kinase reaction buffers (HER2: 20 mM Tris (pH 7.5), 5 mM MnCl2, 0.5 mM Na3VO4, 1 mM EGTA, 2 mM DTT, 5 mM β-glycerophosphate, 0.01% CHAPS) at 30 ° C for 30 min. Terminate the reaction by soaking in a boiling water bath for 5 min. Quickly freeze the protein in liquid nitrogen and perform Western blot analysis of LDHA-Y10 phosphorylation.

Techniques: Activity Assay, Knockdown, Two Tailed Test, Western Blot, Phospho-proteomics, Recombinant, Biomarker Discovery, RNA Immunoprecipitation, Quantitative RT-PCR, Fluorescence, Immunoprecipitation, In Vitro, Kinase Assay, Over Expression, Size-exclusion Chromatography

Journal: Cell Death & Disease

Article Title: Targeting LINC01711 in FAP + cancer-associated fibroblasts overcomes lactate-mediated immunosuppression and enhances anti-PD-1 efficacy in lung adenocarcinoma

doi: 10.1038/s41419-025-07974-6

Figure Lengend Snippet: A – E ECAR assay, glucose uptake assay, lactate production assay and LDHA activity assay were performed in FAP+ CAFs transfected with si-LINC01711 or si-NC, treated with LDH inhibitor or not ( n = 3 biological repeats). The P -value was calculated by two-tailed unpaired t -test. F – J . ECAR assay, glucose uptake assay, lactate production assay and LDHA activity assay were performed in FAP + CAFs transfected with si-LINC01711 or si-NC, co-transfected with LDHA WT or LDHA mutant ( n = 3 biological repeats). The P -value was calculated by two-tailed unpaired t -test. K – O ECAR assay, glucose uptake assay, lactate production assay and LDHA activity assay were performed in FAP + CAFs transfected with si-LINC01711 or si-NC, co-transfected with si-FGFR1 or si-NC ( n = 3 biological repeats). The P -value was calculated by two-tailed unpaired t -test. P Flow chart of in vitro co-culture model. Q Flow cytometry analysis on CD8-positive T cells infiltration, GZMB + CD8-positive T cells infiltration and CD69 + CD8-positive T cells infiltration in in vitro co-culture model. R Flow chart of in vivo model. S Representative image of subcutaneous tumors. n = 5 biological repeats. T Tumor volume of subcutaneous tumors ( n = 5 biological repeats, the P -value was determined by two-way ANOVA with Tukey’s multiple comparison test). U Tumor weight of subcutaneous tumors ( n = 5 biological repeats, the P -value was calculated by two-tailed unpaired t -test). All the results were shown as mean ± S.E.M. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Recombinant human LDHA variants ( P01711 , Solarbio) were mixed with active recombinant His tagged-FGFR1 ( P09665 , Solarbio) in kinase reaction buffers (HER2: 20 mM Tris (pH 7.5), 5 mM MnCl2, 0.5 mM Na3VO4, 1 mM EGTA, 2 mM DTT, 5 mM β-glycerophosphate, 0.01% CHAPS) at 30 ° C for 30 min. Terminate the reaction by soaking in a boiling water bath for 5 min. Quickly freeze the protein in liquid nitrogen and perform Western blot analysis of LDHA-Y10 phosphorylation.

Techniques: ECAR Assay, Activity Assay, Transfection, Two Tailed Test, Mutagenesis, In Vitro, Co-Culture Assay, Flow Cytometry, In Vivo, Comparison

Journal: bioRxiv

Article Title: Mitochondrial Dysfunction and Senescence Accompany Glioblastoma Cell Death Triggered by a Putative Metabolic Inhibitor

doi: 10.1101/2025.09.08.674975

Figure Lengend Snippet: (A) Immunoblot showing overexpression of LDHA at 5 and 10 mg concentration of the LDHA plasmid in U87 cells, represented with LDHA and Myc tag LDHA. Quantification of the percentage of overexpression observed. (B) Cell viability of control, empty vector, and LDHA plasmid overexpression. Cell viability graphs of STP, TMZ, and GSK without overexpression of LDHA and with overexpression of LDHA. (C) Immunoblot representing LDHA knockdown by siLDHA. Quantification of the percentage decrease in LDHA expression. (D) Cell viability analysis of STP, TMZ, and GSK upon knockdown of LDHA. (E) Kinetic evaluation of LDHA enzyme activity assay with STP, and sodium oxamate. (F) Cell viability of SKNSH cells upon treatment with STP and Diazepam. n=3, *p<0.033, **p<0.002, ***p<0.001, one-way ANOVA Tukey test.

Article Snippet: U87 cell line was purchased from ATCC (USA), fetal bovine serum (FBS) (Atlanta Bio, USA), Dulbecco’s modified Eagle medium (DMEM), PSA, and Trypsin (Corning, USA), stiripentol and temozolomide (Fisher, USA), GSK2837807 sodium oxamate (Sigma, USA), siLDHA and Lipofectamine RNAimax (Life technologies, USA), LDHA plasmid (Origene, USA), Transit LT-1 (Mirrus, USA), Opti-MEM (Gibco, USA), BCA (Thermoscientific, USA), Direct-Zol RNA extraction kit (Zymo research, USA), CMH 2 DCFDA and CMXROS (Invitrogen, USA), primary antibodies β-actin, ALDHA13, AOX1, CPA4, TXNIP, KYNU, (Proteintech, USA), LC3 (Sigma) secondary antibodies anti-mouse, anti-rabbit, and senescence kit (Cell signaling, USA).

Techniques: Western Blot, Over Expression, Concentration Assay, Plasmid Preparation, Control, Knockdown, Expressing, Enzyme Activity Assay

Journal: Drug Design, Development and Therapy

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism

doi: 10.2147/DDDT.S90530

Figure Lengend Snippet: Overexpression of miR-125b in chondrosarcoma cells downregulates glucose metabolism. Notes: ( A ) Glucose uptake and ( B ) lactate product were measured in miR-125b overexpressing JJ012 (left) and CH-2879 (right) cells compared with negative control. ( C ) Western blotting experiments showed that the expressions of HK II, PDK1, and LDHA were downregulated in miR-125b overexpressing JJ012 (left) and CH-2879 (right) cells compared with negative control. β-actin was used as a loading control. Columns, mean of three independent experiments; bars, SE. * P <0.05.

Article Snippet: Overexpression vectors containing wild-type ErbB2 (RC212583) and LDHA (RC228293) were purchased from www.origene.com .

Techniques: Over Expression, Negative Control, Western Blot, Control

Journal: Drug Design, Development and Therapy

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism

doi: 10.2147/DDDT.S90530

Figure Lengend Snippet: Restoration of ErbB2 in miR-125b overexpressing cells recovers the glucose metabolism and doxorubicin sensitivity. Notes: ( A ) JJ012 cells were transfected with 100 nM pre-miR-negative control and pre-miR-125b for 48 hours, followed by transfection with vector control and overexpression vector containing wild-type ErbB2 for 24 hours, and then cells were collected and prepared for Western blotting with antibody against ErbB2, HK II, PDK1, and LDHA. β-actin was used as a loading control. ( B ) JJ012 cells were transfected with pre-miR-125b and ErbB2 as described in ( A ), then the glucose uptake (left) and lactate product (right) were checked. ( C ) JJ012 cells were transfected with pre-miR-125b and ErbB2 as described in ( A ); cells were collected and replaced in 48-well plates for overnight, followed by doxorubicin treatments at the indicated concentrations for 48 hours. Cells were analyzed by cell viability assays. ( D ) The JJ012 cells were transfected with control siRNA or siErbB2 for 48 hours, and then cells were collected for Western blot analysis (middle) and the measurements of glucose uptake and lactate product. Columns, mean of three independent experiments; bars, SE. * P <0.05; ** P <0.01.

Article Snippet: Overexpression vectors containing wild-type ErbB2 (RC212583) and LDHA (RC228293) were purchased from www.origene.com .

Techniques: Transfection, Negative Control, Plasmid Preparation, Control, Over Expression, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism

doi: 10.2147/DDDT.S90530

Figure Lengend Snippet: Overexpression of miR-125b resensitizes doxorubicin resistant chondrosarcoma cells through the inhibition of glucose metabolism. Notes: ( A ) Overexpression of miR-125b resensitized doxorubicin resistant cells. Cells were transfected with pre-miR-125b for 48 hours and then were treated with doxorubicin at indicated concentrations for 48 hours, followed by the measurement of cell viability. ( B ) Western blotting experiments showed the overexpression of miR-125b in JJ012 doxorubicin resistant cells decreased the expressions of ErbB2, HK II, PDK1, and LDHA to the same levels as those of JJ012 parental cells. ( C ) Glucose uptake (left) and lactate product (right) results showed the overexpression of miR-125b in JJ012 doxorubicin resistant cells decreased the glucose metabolism to the same levels as those of JJ012 parental cells. ( D ) Exogenous overexpression of LDHA into miR-125b pretransfected JJ012 parental cells (left) showed resistant to doxorubicin treatments at the indicated concentrations for 48 hours (right). Columns, mean of three independent experiments; bars, SE. * P <0.05; ** P <0.01. Abbreviation: Doxo R, Doxo resistant.

Article Snippet: Overexpression vectors containing wild-type ErbB2 (RC212583) and LDHA (RC228293) were purchased from www.origene.com .

Techniques: Over Expression, Inhibition, Transfection, Western Blot