pkm2 elisa kits  (Elabscience Biotechnology)

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: Discover oncology

Article Title: NCAPD3 is involved in papillary thyroid carcinoma proliferation, metastasis, and aerobic glycolytic pathway.

doi: 10.1007/s12672-025-02767-x

Figure Lengend Snippet: Fig. 6 Effect of NCAPD3 silencing on the levels of LDHA, PKM2, and lactate in K1 and TPC-1 cells. K1 and TPC-1 cells were transfected with NC siRNA and two siRNA of NCAPD3 (siRNA1 or siRNA2). After transfection, levels of LDHA and PKM2 in cell lysates and lactate level in cul- ture medium supernatants were measured. * P < 0.05, siRNA1 vs. NC; # P < 0.05, siRNA2 vs. NC

Article Snippet: The levels of LDHA and PKM2 in the cell lysates were detected using LDHA ELISA kits (KL-PKM2-Hu, Kanglang Biology, Shanghai, China) and PKM2 ELISA kits (E-EL-H0556, Elabscience, China), respectively.

Techniques: Transfection

Journal: Discover oncology

Article Title: Blocking lncRNA NOP14-AS1 overcomes 5-Fu resistance of colon cancer cells by modulating miR-30a-5p-LDHA-glucose metabolism pathway.

doi: 10.1007/s12672-025-02156-4

Figure Lengend Snippet: Fig. 6 Restoring LDHA in miR-30a-5p-overexpressing CRC cells recovers glucose metabolism. A Protein expressions of LDHA were shown after transfecting HT-29 5-Fu resistant cells with control miRNA, miR-30a-5p alone, or miR-30a-5p plus LDHA overexpression plasmid. B Glu- cose uptake and C lactate production were examined in the above cells. *, p < 0.05

Article Snippet: LDHA overexpression plasmid (#RC209378) was obtained from Origen. com.

Techniques: Control, Over Expression, Plasmid Preparation

Journal: Discover oncology

Article Title: Blocking lncRNA NOP14-AS1 overcomes 5-Fu resistance of colon cancer cells by modulating miR-30a-5p-LDHA-glucose metabolism pathway.

doi: 10.1007/s12672-025-02156-4

Figure Lengend Snippet: Fig. 5 LDHA is directly tar- geted by miR-30a-5p in CRC cells. A Targetscan.org pre- dicted binding of miR-30a-5p on LDHA 3’UTR. B Pearson’s correlation analysis showed negative correlation between LDHA and miR-30a-5p expres- sions in colon cancer tissues. C LDHA expressions were meas- ured by qRT-PCR in colon can- cer tissues (n = 40) and normal colon tissues (n = 40). D LDHA expressions were compared in colon cancer cell lines and normal colon epithelial cells. E Protein expressions of LDHA were shown after transfecting colon cancer cells with control miRNA or miR-30a-5p. F Lucif- erase assays were performed in HT-29 and G DLD-1 cells that were co-transfected with control miRNA or miR-30a-5p along with WT-LDHA 3’UTR or Mut-LDHA 3’UTR. Luciferase activities were examined. **, p < 0.01; ***. p < 0.001

Article Snippet: LDHA overexpression plasmid (#RC209378) was obtained from Origen. com.

Techniques: Binding Assay, Quantitative RT-PCR, Control, Transfection, Luciferase

Journal: Discover oncology

Article Title: Blocking lncRNA NOP14-AS1 overcomes 5-Fu resistance of colon cancer cells by modulating miR-30a-5p-LDHA-glucose metabolism pathway.

doi: 10.1007/s12672-025-02156-4

Figure Lengend Snippet: Fig. 8 Roles of the NOP14-AS1-miR-30a-5p-LDHA axis in 5-Fu resistant colon cancer cells. A Pearson’s correlation analysis showed posi- tive correlation between NOP14-AS1 and LDHA expressions in colon cancer tissues. B HT-29 5-Fu R cells were transfected with control, NOP14-AS1 alone, or NOP14-AS1 plus miR-30a-5p. Expressions of miR-30a-5p and C LDHA were examined by qRT-PCR and Western blot. D The above cells were treated with 5-Fu. Cell responses to 5-Fu were determined by cell viability assay and E Annexin V apoptosis assay. *, p < 0.05; **, p < 0.01

Article Snippet: LDHA overexpression plasmid (#RC209378) was obtained from Origen. com.

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Viability Assay, Apoptosis Assay

Journal: Discover oncology

Article Title: Blocking lncRNA NOP14-AS1 overcomes 5-Fu resistance of colon cancer cells by modulating miR-30a-5p-LDHA-glucose metabolism pathway.

doi: 10.1007/s12672-025-02156-4

Figure Lengend Snippet: Fig. 7 Restoring LDHA in miR-30a-5p-overexpressing CRC cells recovers 5-Fu resistance. A HT-29 5-Fu resistant cells were treated with con- trol, Oxamate alone, 5-Fu alone, or 5-Fu plus Oxamate. Cell viability was examined. B Control miRNA, miR-30a-5p alone, or miR-30a-5p plus LDHA overexpression plasmid was transfected into HT-29 5-Fu R cells, followed by treatment with control or 5-Fu. Cell responses to 5-Fu were determined by cell viability assay and C apoptosis assay. *, p < 0.05; **, p < 0.01

Article Snippet: LDHA overexpression plasmid (#RC209378) was obtained from Origen. com.

Techniques: Control, Over Expression, Plasmid Preparation, Transfection, Viability Assay, Apoptosis Assay