Journal: The Journal of international medical research

Article Title: MYL9 promotes squamous cervical cancer migration and invasion by enhancing aerobic glycolysis.

doi: 10.1177/03000605231208582

Figure Lengend Snippet: Figure 3. MYL9 promoted the progression of SCC by enhancing aerobic glycolysis. (a) MYL9 knockdown decreased the Lactate production of SCC. (b) MYL9 knockdown inhibited the expression of GLUT1, HK2, and LDHA and (c) MYL9 knockdown inactivated the JAK2/STAT3 pathway. MYL9, myosin light chain 9; SCC, squamous cervical cancer; GLUT1, glucose transporter 1; HK2, hexokinase II; LDHA, lactate dehydrogenase A.

Article Snippet: The membranes were blocked with 5% bovine serum albumin in TBS containing Tween-20 (TBST) for 1 hour at 37 C and incubated with primary antibodies againstMYL9 (mouse, 60233-1-Ig, Proteintech, Rosemont, IL, USA), GLUT1 (rabbit, 21829-1-AP, Proteintech), HK2 (mouse, 66974-1-Ig, Proteintech), LDHA (rabbit, 19987-1-AP, Proteintech), b-tubulin (mouse, Proteintech), and GAPDH (mouse, CW0100M, CWBIO, Beijing, China) overnight at 4 C. Following three washes with TBST, the membrane was incubated with horseradish peroxidase-conjugated goat antirabbit or anti-mouse IgG antibody at 37 C for 1 hour.

Techniques: Knockdown, Expressing

Journal: Journal of translational medicine

Article Title: TIMELESS promotes reprogramming of glucose metabolism in oral squamous cell carcinoma.

doi: 10.1186/s12967-023-04791-3

Figure Lengend Snippet: Fig. 5 TIMELESS augments glycolysis via upregulation of key metabolic Molecules. A Correlation analysis between TIMELESS expression and SIRT1, HIF1A and MYC in HNSC tissues form TCGA database. B Correlation analysis between TIMELESS mRNA levels and SIRT1 mRNA levels in 33 OSCC tissues. C qRT-PCR analysis of the expression levels of TIMELESS and key glycolysis genes in OSCC cells. D Western blot analysis of the expression levels of TIMELESS and key glycolysis genes in OSCC cells. E IHC staining of TIMELESS, SIRT1, HK2, PKM2, LDHA and GLUT1 in nude mice tumors. F The transfection efficiency was examined with western blot. G The level of glucose uptake was examined. H Lactate production was examined. I Cell medium pH. J Oxygen consumption level of cell. Data shown were the mean ± S.E.M. from three independent experiments. *P < 0.05; **P < 0.01

Article Snippet: For the immunohistochemistry assays, we used the Ki67 antibody (AF0198) from Affinity Biosciences, and the PCNA antibody (60097-1-Ig), SIRT1 antibody (13161- 1-AP), HK2 antibody (66974-1-Ig), PKM2 antibody (60268-1-Ig), GLUT1 antibody (66290-1-Ig), LDHA antibody (66287-1-Ig), and TIMELESS antibody (14421-1- AP) from Proteintech Group, Inc.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Transfection

Journal: Investigative Ophthalmology & Visual Science

Article Title: IL-6-Derived Autocrine Lactate Promotes Immune Escape of Uveal Melanoma

doi: 10.1167/iovs.65.3.37

Figure Lengend Snippet: IL-6 increased glycolysis and lactate production in C918 cells. (A) Distribution of differentially expressed transcripts between IL-6/C918 and C918 cells identified in the glycolysis/gluconeogenesis pathway (KEGG pathway), based on transcriptome data; red = upregulated and blue = downregulated. Pathway with blue shading indicated the glycolysis process, and pathway with red shading indicated the pyruvate oxidation process. (B–D) Relative mRNA levels of PFKP (B), PKM (C), and LDHA (D) in IL-6/C918 and C918 cells determined by qPCR. (E) PFKP, PKM, and LDHA protein levels in IL-6/C918 and C918 cells detected by Western blotting. (F) Analysis of TCGA uveal melanoma database using GEPIA2 ( http://gepia2.cancer-pku.cn/#correlation ) showing correlations between IL-6 and PKM or LDHA mRNA levels. (G) Extracellular acidification rate (ECAR) of IL-6/C918 and C918 cells. (H) Lactate concentrations in the culture medium of IL-6/C918 and C918 cells. The results of three independent experiments are presented as mean ± standard deviation.

Article Snippet: Antibodies against PFKP (sc-514824), PKM (sc-365684), LDHA (sc-137243), PD-L1 (sc-293425), HLA-E (sc-51621), and ubiquitin (sc-8017) were purchased from Santa Cruz Biotechnology (SantaCruz, CA, USA).

Techniques: Western Blot, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: IL-6-Derived Autocrine Lactate Promotes Immune Escape of Uveal Melanoma

doi: 10.1167/iovs.65.3.37

Figure Lengend Snippet: IL-6-induced lactate increased PD-L1 and HLA-E protein levels and immune escape in C918 cells. (A and B) PD-L1 and HLA-E protein and relative mRNA levels in IL-6/C918 cells with PKM or LDHA knocked down examined by Western blotting and qPCR. (C and D) PD-L1 and HLA-E protein and relative mRNA levels in IL-6/C918 cells treated with 50 nM sodium oxamate (SO) examined by Western blotting and qPCR. (E and F) PD-L1 and HLA-E protein and relative mRNA levels examined by Western blotting and qPCR in C918 cells treated with 20 mM lactate. (G) Apoptosis level of IL-6/C918 cells pretreated with 50 nM SO after co-culture with TALL 104 and NK-92MI cells. (H) Apoptosis level of C918 cells pretreated with 20 mM lactate after co-culture with TALL-104 and NK-92MI cells. The results of three independent experiments are presented as mean ± standard deviation.

Article Snippet: Antibodies against PFKP (sc-514824), PKM (sc-365684), LDHA (sc-137243), PD-L1 (sc-293425), HLA-E (sc-51621), and ubiquitin (sc-8017) were purchased from Santa Cruz Biotechnology (SantaCruz, CA, USA).

Techniques: Western Blot, Co-Culture Assay, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: IL-6-Derived Autocrine Lactate Promotes Immune Escape of Uveal Melanoma

doi: 10.1167/iovs.65.3.37

Figure Lengend Snippet: LDHA protein levels were positively correlated with those of PD-L1 in uveal melanoma tissues. (A) Two representative cases of low and high expression of PKM, LDHA, PD-L1, and HLA-E in clinical UM tissues. (B) The relationships of the integrated fluorescence density of PKM, LDHA, PD-L1, and HLA-E.

Article Snippet: Antibodies against PFKP (sc-514824), PKM (sc-365684), LDHA (sc-137243), PD-L1 (sc-293425), HLA-E (sc-51621), and ubiquitin (sc-8017) were purchased from Santa Cruz Biotechnology (SantaCruz, CA, USA).

Techniques: Expressing, Fluorescence

Journal: Molecular Vision

Article Title: The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases

doi:

Figure Lengend Snippet: Confocal images show that pan-LDH and LDH isozyme 5 (LDH-5: liver and muscle type) are expressed in almost all retinal cells and synapses. A : Retina immunolabeled for pan-LDH. B : Retinas double-labeled for pan-LDH and VGLUT1 show colocalization in the OPL and the IPL (aquamarine pixels). C : Retinas double-labeled for pan-LDH and glutamine synthetase (GS) show weak colocalization in the MGC somas and ELM. D : Retina immunolabeled for LDH-5. E : Retinas double-labeled for LDH-5 and VGLUT1 show colocalization in the OPL and the IPL (aquamarine pixels). F : Retinas double-labeled for LDH-5 and COX IV show collocation in the ISs, OPL, and IPL. Inset: Single and double-labeled cone ISs from a high-magnification image revealing colocalization in the ISs. G : High-magnification image of a retina double-labeled for LDH-5 and calbindin shows no colocalization. H : High-magnification image of a retina double-labeled for LDH-5 and CHX10 reveals differential colocalization. I : High-magnification image of a retina double-labeled for LDH-5 and PKCα shows colocalization in the somas but not in the dendrites. COX IV = cytochrome c oxidase subunit IV, ELM = external limiting membrane, IPL = inner plexiform layer, IPL-a = IPL sublamina-a, IPL-b = IPL sublamina-b, ISs = inner segments, LDH = lactate dehydrogenase, MGC = Müller glial cell, ONL = outer nuclear layer, OPL = outer plexiform layer, OSs = outer segments, PKCα-IR = protein kinase C α immunoreactivity, VGLUT1 = vesicular glutamate transporter 1. A–F, scale bar = 40 µm. G–I, scale bar = 20 µm.

Article Snippet: Pan-lactate dehydrogenase , pan-LDH , Human LDH , Rabbit , Santa Cruz , 1:500 , .

Techniques: Immunolabeling, Labeling, Membrane

Journal: Molecular Vision

Article Title: The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases

doi:

Figure Lengend Snippet: LDH histochemistry of the light-adapted mouse retina. Different concentrations of sodium DL-lactate showed selective activity of lactate dehydrogenase (LDH) in the ISs. A : Retinas incubated in the absence of the sodium DL-lactate. No LDH reactivity is observed. B : Retinas incubated with 1 mM sodium DL-lactate show LDH activity in the IS. C : Retinas incubated in 5 mM sodium DL-lactate show high LDH activity in the ISs and low LDH activity in the OPL. D : Retinas incubated in 10 mM sodium DL-lactate show intense LDH activity in the IS and strong LDH activity in the OPL, INL, and GCL. E : Retinas incubated in the absence of sodium DL-lactate and 1 mM sodium pyruvate. F and G : Retinas incubated with equimolar concentrations of sodium DL-lactate and sodium pyruvate show selective LDH activity in the ISs. H : Retinas incubated with 10 mM sodium DL-lactate and 5 mM sodium pyruvate show reduced activity of LDH in most retinal layers except the ISs. I : LDH activity in different retinal layers presented as mean ± SEM of relative optical density. The LDH activity in ISs was significantly greater at each concentration of lactate relative to the other retinal layers as indicated by asterisks (p<0.05). Bars for each retinal layer that share the same letter (a, b, c, or d) exhibit significant concentration-dependent increases in LDH activity with increasing concentration of lactate (p<0.05). GCL = ganglion cell layer, INL = inner nuclear layer, IPL = inner plexiform layer, ISs = inner segments, NFL = nerve fiber layer, ONL = outer nuclear layer, OPL = outer plexiform layer. Scale bar = 20 μm.

Article Snippet: Pan-lactate dehydrogenase , pan-LDH , Human LDH , Rabbit , Santa Cruz , 1:500 , .

Techniques: Activity Assay, Incubation, Concentration Assay

Journal: Molecular Vision

Article Title: The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases

doi:

Figure Lengend Snippet: Confocal images reveal a unique metabolic signature of Müller cell mitochondria. A : Retinas immunolabeled for glutamate dehydrogenase 1 (GDH1) reveal intense expression in rod and cone ISs and synaptic terminals, and a strong to intense in the ELM, IPL, GCL, and MGC end-feet. The expression in the ONL is punctate and strong to intense. B : Strong to intense colocalization of GDH1-IR and glial high-affinity glutamate-aspartate transporter (GLAST-IR) is seen throughout the MGCs. C : Retinas immunolabeled for GABA-transaminase (GABA-T) reveal an intense expression in rod and cone ISs and synaptic terminals, and a strong to intense in the ELM, IPL, GCL, and MGC end-feet. The expression in the ONL is punctate and strong to intense. D : Strong to intense colocalization of GABA-T-IR and GLAST-IR is seen throughout the MGCs. E : Retinas immunolabeled for the lipoamide subunit of the 2-oxoglutarate (α-ketoglutarate) dehydrogenase complex (OGDH) reveal intense expression in the rod and cone IS and synaptic terminals and a strong to intense in the ELM, IPL, GCL, and MGC end-feet. The expression in the ONL is punctate and strong to intense. F : OGDH-IR and GLAST colocalization is strong in the ELM and end-feet, and moderate distal and proximal processes, OPL, and somas. G : Retinas immunolabeled for succinate thiokinase (STK) reveal intense expression in the rod and cone ISs and synaptic terminals and strong to intense expression in the ELM, IPL, GCL, and MGC end-feet. The expression in the ONL is moderate and more like that of COX IV-IR ( , , and ). H : STKH-IR and GLAST colocalization is strong in the ELM, proximal processes, and end-feet and moderate in the distal processes, OPL, and somas. COX IV-IR = cytochrome oxidase IV immunoreactivity, ELM = external limiting membrane, GCL = ganglion cell layer, INL = inner nuclear layer, IPL = inner plexiform layer, ISs = inner segments, MGC = Müller glial cell, NFL = nerve fiber layer, ONL = outer nuclear layer. A–H, scale bars = 40 µm.

Article Snippet: Pan-lactate dehydrogenase , pan-LDH , Human LDH , Rabbit , Santa Cruz , 1:500 , .

Techniques: Immunolabeling, Expressing, Membrane

Journal: Molecular Vision

Article Title: The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases

doi:

Figure Lengend Snippet: Integrated summary of relative Ab intensity levels obtained from the immunoreactivity of bioenergetic regulating and buffering enzymes in the neuronal and glial compartments of the adult light-adapted C57BL/6N mouse retina. The numbers on the x-axis (from 1 to 22) indicate the specific retinal compartments (see key). The values on the y-axis represent the integrated mean relative intensity values of immunoreactivity levels for enzymes that regulate glycolysis (HK-1, HK-2, PFK-L1, PK-M1, and PK-M2), aerobic glycolysis (pan-LDH and LDH-5), the tricarboxylic acid (TCA) cycle (OGDH and STK), oxidative phosphorylation (OXPHOS; cytochrome c oxidase subunit IV, COX IV), and the ~P transferring kinases (NDPK, AK1, AK2, CK-B, CK-M, and mi-CK) obtained from Appendix 1. Overall, the findings reveal highly compartmentalized and graded expression levels of the different enzymes in the outer compared to the inner retina as well as in the Müller glial cells (MGCs) compared to the neurons. Glycolytic enzymes (solid black squares and line) are higher in the outer retina than in the inner retina, except in the rod and cone outer segments (ROSs; COSs). Specifically, the highest levels (i.e., ≥3 on intensity scale) are in the rod and cone inner segments (RISs; CISs), rod spherules, and cone pedicles where most of the retinal mitochondria are located [ , ]. Enzyme levels are slightly lower (i.e., 2.5–3.0) in the cone somas, the inner plexiform layer (IPL), the IPL sublamina-a, and sublamina-b (IPL-a; IPL-b) and still lower (i.e., 2.0–2.5) in the rod somas, inner nuclear layer (INL) somas, ganglion cell layer (GCL), and MGC end-feet/nerve fiber layer (NFL), and lowest (i.e., 1.0–1.75) in the ROS and COS, bipolar cell (BC) dendrites, horizontal cell (HC) somas and processes, and MGC somas. No glycolytic enzyme immunoreactivity was detected in the MGC external limiting membrane (ELM) or processes. Similar to glycolysis, aerobic glycolysis is higher in the outer retina than in the inner retina, except again for the ROSs and COSs. Compared to the RIS, aerobic glycolysis is just slightly lower in the CISs, rod and cone somas, and spherules and pedicles, as seen with the lactate dehydrogenase (LDH) activity . The BC dendrites, HC somas, and HC process have no apparent aerobic glycolytic capacity. Except amacrine cells in the proximal INL and the GCL, aerobic glycolysis is low (i.e., 1.0–2.0) in the inner retina. Aerobic glycolysis is absent to low (i.e., ≤1.0) throughout the MGCs. OXPHOS capacity is present and relatively high in all outer and inner retinal compartments, except in the ROS and COS (no COX IV or COX activity detected) and the INL somas (i.e., ≤2.0). As the IS showed high expression of GDH1 and the inner retinal neurons had the highest GLS expression levels , the high OXPHOS capacity in these compartments may be supported by non-glucose derivatives. Except the MGC end-feet/NFL, OXPHOS capacity was not detected in any MGC compartments, consistent with the COX activity and . In the outer retina, the TCA cycle expression profile essentially mirrors that of OXPHOS. In the inner retina, except in the INL somas, the TCA cycle expression is moderately high (i.e., 2.5 to 3.0) and mirrors that of OXPHOS. The TCA cycle expression profile in MGCs is characterized by moderate to high expression (i.e., 2.5 to 3.0) in the ELM, proximal processes, and end-feet/NFL and lower expression (i.e., 2.0) in the distal process and somas. This is in marked contrast to their expression of enzymes related to glycolysis, aerobic glycolysis, and OXPHOS. MGCs also have a capacity for glutamate and GABA catabolism, as well as GTP production . Together, this suggests that MGCs support their bioenergetics demands using non-glucose derivatives to generate GTP. The ~P transferring kinases were differentially distributed among all compartments of the inner and outer retinal neurons. The ~P transferring kinases were more highly expressed in all cone compartments (CIS, somas, and pedicles, i.e., 3.5) compared to similar rod compartments (i.e., 2.0–2.5). The ~P transferring kinases were also moderately to highly expressed in the BC dendrites, HC somas, and HC processes, compartments that had low glycolytic and/or aerobic glycolytic capacity. In the MGCs, the ~P transferring kinases were moderately expressed in the ELM and distal processes and had lower expression (i.e., ≥2.0) the somas, proximal processes, and end-feet/NFL. As the MGCs lack OXPHOS capacity, except in the end-feet/NFL, the ~P transferring kinases likely convert the GTP produced in the TCA cycle to ATP, as well as buffer the ATP concentration.

Article Snippet: Pan-lactate dehydrogenase , pan-LDH , Human LDH , Rabbit , Santa Cruz , 1:500 , .

Techniques: Phospho-proteomics, Transferring, Expressing, Membrane, Activity Assay, Produced, Concentration Assay

Journal: Cell communication and signaling : CCS

Article Title: Blockade of glycolysis-dependent contraction by oroxylin a via inhibition of lactate dehydrogenase-a in hepatic stellate cells.

doi: 10.1186/s12964-019-0324-8

Figure Lengend Snippet: Fig. 5 Oroxylin A inhibition of LDH-A led to blockade of aerobic glycolysis in HSCs. LX2 cells were treated with oroxylin A and/or galloflavin at indicated concentrations, or transfected with LDH-A siRNA for 24 h. a Measurements of glucose uptake. b Measurements of glucose consumption indicated by GOD activity. c Measurements of intracellular lactate levels. d Real-time PCR analyses of mRNA expression of HK2, PFK1 and PKM2. e Western blot analyses of protein expression of HK2, PFK1 and PKM2 with quantification. f Measurements of intracellular HK2 enzyme activity. g Measurements of intracellular PFK1 enzyme activity. h Measurements of intracellular PKM2 enzyme activity. i Real-time PCR analyses of mRNA expression of LDH-A. j Western blot analyses of protein expression of LDH-A with quantification. k Real-time PCR analyses of mRNA expression of HK2, PFK1 and PKM2. For statistical significance of panels a-h: *p < 0.05 vs. control, **p < 0.01 vs. control, ***p < 0.001 vs. control. For statistical significance of panels i-k: *p < 0.05 vs. control siRNA, **p < 0.01 vs. control siRNA, ***p < 0.001 vs. control siRNA

Article Snippet: LDH-A siRNA (sc-43,893) and control siRNA (sc-37,007) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Transfection, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control

Journal: Cell communication and signaling : CCS

Article Title: Blockade of glycolysis-dependent contraction by oroxylin a via inhibition of lactate dehydrogenase-a in hepatic stellate cells.

doi: 10.1186/s12964-019-0324-8

Figure Lengend Snippet: Fig. 6 Oroxylin A inhibition of LDH-A led to reduction of HSC contraction. LX2 cells were treated with oroxylin A and/or galloflavin at indicated concentrations, or transfected with LDH-A siRNA for 24 h. a Collagen gel contraction assays with quantification. b Cytoskeleton fluorescence staining, scale bar: 20 μm. c Western blot analyses of MLC2 phosphorylation with quantification. d Immunofluorescence analyses of MLC2 phosphorylation, scale bar: 20 μm. e Cytoskeleton fluorescence staining, scale bar: 20 μm. f Western blot analyses of MLC2 phosphorylation with quantification. For statistical significance of panels a-d: *p < 0.05 vs. control, **p < 0.01 vs. control. For statistical significance of panels e-f: **p < 0.01 vs. control siRNA

Article Snippet: LDH-A siRNA (sc-43,893) and control siRNA (sc-37,007) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Transfection, Fluorescence, Staining, Western Blot, Phospho-proteomics, Immunofluorescence, Control