Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Deletion of Lactate Dehydrogenase-A Impairs Oncogene-Induced Mouse Hepatocellular Carcinoma Development

doi: 10.1016/j.jcmgh.2022.06.003

Figure Lengend Snippet: ( A ) qRT-PCR analysis of Ldhb and Ldhd mRNA levels in laser-microdissected tumors (T) and nontumorous surrounding livers (S) from Ldha WT and Ldha KO mice. ( B ) qRT-PCR analysis of Cdkn1a and H2afx mRNA levels in laser-microdissected tumors (T) and surrounding livers (S) from Ldha WT and Ldha KO mice (left); microphotographs showing CDKN1A immunostaining in tumors and surrounding liver of WT and Ldha KO mice (right). ( C ) qRT-PCR analysis of Glut1 , Hk2 , and Gck mRNA levels in microdissected tumors (T) and nontumorous surrounding livers (S) from Ldha WT and KO mice. ( D ) qRT-PCR analysis of Mct4 mRNA levels in microdissected tumors (T) and surrounding livers (S) from Ldha WT and KO mice. ( E ) qRT-PCR analysis of Gls , Glud1 , and Gclc mRNA levels in microdissected tumors (T) and surrounding livers (S) from Ldha WT and KO mice. ( F ) qRT-PCR analysis of the expression of G6pdx and microphotograph showing G6PD immunostaining in Ldha KO tumors (T) compared with peritumoral areas (S) (10×). ( G ) qRT-PCR analysis of Me1 and Pdhx mRNA levels in laser-microdissected tumors (T) and surrounding livers (S) from Ldha WT and KO mice. Gene expression is reported as a fold change of mRNA relative to those from Ldha WT surrounding livers. Relative quantification analysis for each gene was calculated by the 2 ΔΔ Ct method. The histogram represents means ± SD of 6 WT tumors, 3 KO tumors, and 3 WT and KO surrounding livers (1-way analysis of variance). ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001. ns, not significant.

Article Snippet: To investigate by qRT-PCR mRNA expression levels of Glut1 (Mm00441480 _m1), Hk2 (Mm00443385 _m1), Gck (Mm00439129_m1), G6pdx (Mm00656735 _g1), Mct4 (Mm00446102_m1), Glul (Mm00725701_s1), Glud1 (Mm00492353_m1), Gls (Mm01257297 _m1), Gclc (Mm00802655_m1), Nqo1 (Mm01253561_m1), Ldha (Mm001612132_g1), Ldhb (Mm00493146_m1), Ldhc (Mm00496648_m1), Ldhd (Mm00459144_m1), Pdhx (Mm00558275_m1), Me1 (Mm01253561_m1), and LDHA (Hs01378790_g1), total RNA was retro-transcribed to complementary DNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Milan, Italy; Life Technologies, Milan, Italy).

Techniques: Quantitative RT-PCR, Immunostaining, Expressing, Gene Expression, Quantitative Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades

doi: 10.3390/ijms26010274

Figure Lengend Snippet: EGFL8 expression and its relationship with prognosis of patients with ovarian cancer (OC). ( A ) Relative expression levels of EGFL8 mRNA in human OC tissues ( n = 426, red box) and normal ovarian tissues ( n = 88, blue box) analyzed using GEPIA2 platform based on TCGA and GTEx data. ( B ) Representative images show EGFL8 protein expression in ovarian stromal cells (arrows) and vascular endothelial cells (arrowheads) of human OC and normal ovarian tissues, obtained from Human Protein Atlas database, as determined by immunohistochemical staining (magnification, 40×). Bar graphs represent area of immunostained regions in each group. ( C ) Overall survival (OS) and disease-specific survival (DSS) analysis of EGFL8 expression levels in patients with OC. Number of patients at risk (No. at risk) at different time points is displayed on graph. Significant differences between normal and OC groups are indicated by * p < 0.05 and *** p < 0.001. TCGA, The Cancer Genome Atlas; UALCAN, University of Alabama at Birmingham Cancer.

Article Snippet: Human predesigned EGFL8 siRNA Set A, which contains three designed small interfering (si) RNA for EGFL8, as well as a scrambled siRNA (negative control), was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Sciences

Article Title: Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades

doi: 10.3390/ijms26010274

Figure Lengend Snippet: EGFL8 protein expression in human OC cells as detected by Western blot analysis. ( A ) EGFL8 protein expression in various human OC cell lines. ( B ) Western blot analysis for EGFL8 siRNA transfection efficiency, comparing non-transfected control (Cont), scrambled negative control siRNA (siNC), and EGFL8 siRNA-transfected A2780 and SKOV3 cells. Data represent mean ± standard deviation (SD) of three independent experiments. * p < 0.05 and *** p < 0.001 vs. respective control.

Article Snippet: Human predesigned EGFL8 siRNA Set A, which contains three designed small interfering (si) RNA for EGFL8, as well as a scrambled siRNA (negative control), was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Western Blot, Transfection, Control, Negative Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades

doi: 10.3390/ijms26010274

Figure Lengend Snippet: Effects of EGFL8 knockdown on cell proliferation. ( A ) Bar graphs showing proliferation of non-transfected control (control), scrambled negative control siEGFL8 (siNC), and siEGFL8-transfected A2780 and SKOV3 cells on days 1, 2, and 3, as assessed by WST-1 assay. Representative phase-contrast microscopic images depict cell morphology in siNC and siEGFL8-transfected A2780 and SKOV3 cells on days 1, 2, and 3. Scale bars = 150 μm. ( B ) Representative fluorescence micrographs of Ki-67 staining (blue: DAPI; green: Ki-67) in siNC and siEGFL8-transfected A2780 and SKOV3 cells at 24 and 48 h. Relative percentage of Ki-67 + cells was calculated by dividing number of cells stained with Ki-67 by total number of cells stained with DAPI. Scale bars = 150 μm. All data are expressed as relative values compared to scrambled control group. Data represent mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. respective scrambled control group.

Article Snippet: Human predesigned EGFL8 siRNA Set A, which contains three designed small interfering (si) RNA for EGFL8, as well as a scrambled siRNA (negative control), was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Knockdown, Transfection, Control, Negative Control, WST-1 Assay, Fluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades

doi: 10.3390/ijms26010274

Figure Lengend Snippet: Effects of EGFL8 knockdown on colony formation and spheroid growth of human OC cells. ( A ) Colony formation assay showing siEGFL8 transfection stimulated colony formation in scrambled negative control siEGFL8 (siNC), and siEGFL8-transfected A2780 and SKOV3 cells on day 7 (magnification, 40×). ( B ) Representative phase-contrast microscopic images of OC cell spheroids generated by 3D cell culture in siNC and siEGFL8-transfected A2780 cells on days 1, 4, 6, 10, 12 and 14, and SKOV3 cells on days 3, 5, and 7. Scale bars = 150 μm. All data are expressed as relative values compared to scrambled control groups. Data represent mean ± SD of three independent experiments. * p < 0.05, ** p < 0.05, and *** p < 0.001 vs. respective scrambled control group.

Article Snippet: Human predesigned EGFL8 siRNA Set A, which contains three designed small interfering (si) RNA for EGFL8, as well as a scrambled siRNA (negative control), was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Knockdown, Colony Assay, Transfection, Negative Control, Generated, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades

doi: 10.3390/ijms26010274

Figure Lengend Snippet: Effects of EGFL8 silencing on the migration and invasion of A2780 and SKOV3 cells. ( A ) Representative phase-contrast microscopy images from wound healing assay showing transfected and non-transfected A2780 and SKOV3 cells migrating into cell-free space. EGFL8 siRNA-transfected cells have significantly increased migratory ability compared with non-transfected control (Cont) and scrambled negative control siRNA (siNC) groups. Scale bars = 650 μm. ( B ) Representative images of transwell invasion assay with A2780 cells. Data represent mean ± SD of three independent experiments. Scale bars = 75 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. respective non-transfected control group. ### p < 0.001 vs. respective scrambled control group.

Article Snippet: Human predesigned EGFL8 siRNA Set A, which contains three designed small interfering (si) RNA for EGFL8, as well as a scrambled siRNA (negative control), was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Migration, Microscopy, Wound Healing Assay, Transfection, Control, Negative Control, Transwell Invasion Assay

Journal: International Journal of Molecular Sciences

Article Title: Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades

doi: 10.3390/ijms26010274

Figure Lengend Snippet: Effects of EGFL8 knockdown on apoptosis in human OC cells. Expression of apoptotic proteins in siEGFL8-transfected and non-transfected A2780 ( A ) and SKOV3 ( B ) cells was detected by Western blot analysis. Bar graphs depict densitometry quantitation of protein expression normalized to β-actin protein. Data represent mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. respective scrambled control group.

Article Snippet: Human predesigned EGFL8 siRNA Set A, which contains three designed small interfering (si) RNA for EGFL8, as well as a scrambled siRNA (negative control), was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Knockdown, Expressing, Transfection, Western Blot, Quantitation Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades

doi: 10.3390/ijms26010274

Figure Lengend Snippet: Effect of EGFL8 on expression of EMT-related markers and MMPs. ( A ) mRNA levels of key EMT markers in siEGFL8-transfected and non-transfected A2780 and SKOV3 cells as measured by qRT-PCR. ( B ) Confocal microscopy analysis of vimentin expressed in A2780 cells transfected with EGFL8 siRNA or scrambled siRNAs (blue: DAPI; red: vimentin). Scale bars = 30 μm. Bar graph shows quantification of red mean fluorescence intensity (MFI). Relative integrated mean fluorescence intensity (iMFI) of siEGFL8-transfected A2780 cells was normalized to that of scrambled negative control siEGFL8 (siNC). ( C ) Analysis of MMP-2 and MMP-9 expression in siEGFL8-transfected and non-transfected A2780 and SKOV3 cells by qRT-PCR. Bar graphs depict densitometric quantitation of mRNA expression normalized to GAPDH . Data represent mean ± standard deviation (SD) of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. respective scrambled control group. EMT, epithelial–mesenchymal transition; MMP, matrix metallopeptidase.

Article Snippet: Human predesigned EGFL8 siRNA Set A, which contains three designed small interfering (si) RNA for EGFL8, as well as a scrambled siRNA (negative control), was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Confocal Microscopy, Fluorescence, Negative Control, Quantitation Assay, Standard Deviation, Control

Journal: International Journal of Molecular Sciences

Article Title: Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades

doi: 10.3390/ijms26010274

Figure Lengend Snippet: Effects of EGFL8 knockdown on expression of cancer stem cell (CSC) markers in human ovarian cancer (OC) cells. ( A ) Expression levels of CD44 , CD117 , CD133 , Sox2 , Oct4 , Nanog , and KLF4 in siEGFL8-transfected and non-transfected cells were analyzed by qRT-PCR. ( B ) Flow cytometry analysis of ALDH1A1 expression in siEGFL8-transfected and non-transfected A2780 cells. Data represent mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. respective scrambled control group.

Article Snippet: Human predesigned EGFL8 siRNA Set A, which contains three designed small interfering (si) RNA for EGFL8, as well as a scrambled siRNA (negative control), was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Knockdown, Expressing, Transfection, Quantitative RT-PCR, Flow Cytometry, Control

Journal: International Journal of Molecular Sciences

Article Title: Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades

doi: 10.3390/ijms26010274

Figure Lengend Snippet: Effects of EGFL8 knockdown on chemosensitivity of cisplatin, doxorubicin, docetaxel, paclitaxel, curcumin, and rucaparib in A2780 ( A ) and SKOV3 ( B ) cells. Cell viability in scrambled control and siEGFL8-transfected human OC cells 24 h after drug treatment, as shown by WST-1 assay. Data represent mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001, siEGFL8#1 vs. respective scrambled control group. # p < 0.05, ## p < 0.01, and ### p < 0.001, siEGFL8#2 vs. respective scrambled control group.

Article Snippet: Human predesigned EGFL8 siRNA Set A, which contains three designed small interfering (si) RNA for EGFL8, as well as a scrambled siRNA (negative control), was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Knockdown, Control, Transfection, WST-1 Assay

Journal: International Journal of Molecular Sciences

Article Title: Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades

doi: 10.3390/ijms26010274

Figure Lengend Snippet: Effects of EGFL8 knockdown on expression levels of chemoresistance-related genes MDR1 , MRP1 , and Notch1 in scrambled control and EGFL8 siEGFL8-transfected cells as measured by qRT-PCR. Bar graphs show densitometry quantification of mRNA expression normalized to GAPDH . Data represent mean ± SD of three independent experiments. ** p < 0.01 and *** p < 0.001 vs. respective scrambled control group.

Article Snippet: Human predesigned EGFL8 siRNA Set A, which contains three designed small interfering (si) RNA for EGFL8, as well as a scrambled siRNA (negative control), was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Knockdown, Expressing, Control, Transfection, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades

doi: 10.3390/ijms26010274

Figure Lengend Snippet: Effects of EGFL8 knockdown on ERK/MAPK pathway in human OC cells. Western blot analysis of ERK1/2 and phospho-ERK1/2 (pERK1/2) protein expression in scrambled control and siEGFL8-transfected A2780 and SKOV3 cells. Bar graphs display densitometric quantification of protein expression normalized to β-actin. Data presented as mean ± SD of three independent experiments. ** p < 0.01 and *** p < 0.001 compared to respective scrambled control group. ### p < 0.001 compared to U0126-untreated siEGFL8-transfected A2780 and SKOV3 cells.

Article Snippet: Human predesigned EGFL8 siRNA Set A, which contains three designed small interfering (si) RNA for EGFL8, as well as a scrambled siRNA (negative control), was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Knockdown, Western Blot, Expressing, Control, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades

doi: 10.3390/ijms26010274

Figure Lengend Snippet: EGFL8 knockdown promotes human ovarian cancer (OC) cell proliferation by activating ERK/MAPK signaling pathway. ( A ) Results of WST-1 assay showing proliferation rates and representative phase-contrast microscopic images depicting cell morphology in scramble- and siEGFL8-transfected A2780 and SKOV3 cells at 24 h post-transfection. Scale bars = 150 μm. ( B ) Representative fluorescence micrographs of Ki-67 staining (blue: DAPI; green: Ki-67) in scramble- and siEGFL8-transfected A2780 and SKOV3 cells at 24 h. Scale bars = 150 μm. All data expressed as relative values compared to scrambled control group. Data represent mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. respective scrambled control group. ### p < 0.001 compared to U0126-untreated siEGFL8-transfected A2780 and SKOV3 cells.

Article Snippet: Human predesigned EGFL8 siRNA Set A, which contains three designed small interfering (si) RNA for EGFL8, as well as a scrambled siRNA (negative control), was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Knockdown, WST-1 Assay, Transfection, Fluorescence, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades

doi: 10.3390/ijms26010274

Figure Lengend Snippet: EGFL8 knockdown-mediated upregulation of EMT-regulating transcription factors, metastasis-promoting molecules, and stemness-regulating molecules in human OC cells acts via ERK/MAPK signaling pathway. Expression levels of key genes associated with malignancy in scramble- and siEGFL8-transfected A2780 ( A ) and SKOV3 ( B ) cells 24 h post-transfection measured using qRT-PCR. Bar graphs display relative mRNA expression levels of EMT-regulating transcription factors ( Twist1 , Zeb1 , and Zeb2 ), ( B ) metastasis-promoting molecules ( MMP-2 , MMP-9 , and Notch1 ), and stemness-regulating molecules ( Oct4 , CD44 , and CD117 ) in A2780 and SKOV3 cells. GAPDH used as housekeeping gene for normalization. Data presented as relative values compared to respective scrambled control group and represent mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. respective scrambled control group. # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. U0126-untreated siEGFL8-transfected A2780 and SKOV3 cells.

Article Snippet: Human predesigned EGFL8 siRNA Set A, which contains three designed small interfering (si) RNA for EGFL8, as well as a scrambled siRNA (negative control), was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Knockdown, Expressing, Transfection, Quantitative RT-PCR, Control

Journal: Biomimetics

Article Title: Identification of Individual Target Molecules Using Antibody-Decorated DeepTip TM Atomic-Force Microscopy Probes

doi: 10.3390/biomimetics9040192

Figure Lengend Snippet: Florescence images of DeepTip TM probes: ( a ) Probes decorated with an anti-LDH antibody and incubated with a fluorescent anti-anti-LDH antibody. ( b ) Control probes not decorated with an anti-LDH antibody, but incubated with a fluorescent anti-anti-LDH antibody. The broken lines correspond to the silhouette of the cantilevers ( c ) Bar chart comparing the mean fluorescent intensity of the decorated and non-decorated samples. Statistical significance: p < 0.01 (**).

Article Snippet: The following materials were used in this study: sulfo-LC-SPDP (sulfosuccinimidyl 6-(3′-(2-pyridyldithio)propionamido)hexanoate; ThermoScientific, Waltham, MA, USA, 21650), TCEP-HC (Tris(2-carboxyethyl)phosphine hydrochloride Thermo Scientific, Waltham, MA, USA, 20490), EDTA (ethylenediaminetetraacetic acid; Sigma Aldrich, Burlington, MA, USA, E9884), MicroDeck TM -G-150 surfaces (Bioactive Surfaces, Galapagar, Madrid, Spain), lactate dehydrogenase (LDH; Sigma Aldrich, Burlington, MA, USA, L1006-12.5KU), biotinylated anti-lactate dehydrogenase (anti-LDH) antibodies (IgG polyclonal antibody; Origene, Rockville, MD, USA, AP21329BT-N), poly(ethylene glycol)-(N-hydroxysuccinimide 5-pentanoate) ether 2-(biotinylamino)ethane (NHS–PEG–biotin; Sigma Aldrich, Burlington, MA, USA, 757799-100μγ), streptavidin (Sigma Aldrich, Burlington, MA, USA, S4762-5 mg), DeepTip TM SiN R11 atomic-force microscopy probes (Bioactive Surfaces, Galapagar, Madrid, Spain), Goat Anti-Rabbit IgG H&L Alexa Fluor ® 488 (Abcam, Cambridge, UK, ab150077), 5-iodoacetamidofluorescein (5-IAF; Thermo Scientific, Waltham, MA, USA, 62246), sodium pyruvate (P2256 Sigma-Aldrich, Burlington, MA, USA,), β-nicotinamide adenine dinucleotide (NADH; N8129-500 mg), and glutaraldehyde (50 wt.

Techniques: Incubation, Control

Journal: Biomimetics

Article Title: Identification of Individual Target Molecules Using Antibody-Decorated DeepTip TM Atomic-Force Microscopy Probes

doi: 10.3390/biomimetics9040192

Figure Lengend Snippet: Representative force–displacement (F–z) curves obtained from the interaction between anti-LDH antibodies and LDH. In this figure, six types are shown: ( i ) no−interaction curve; ( ii ) elastomeric curve; ( iii ) non−elastomeric curve; ( iv ) elastomeric curve—two peaks; ( v ) double-peak curve—combined; ( vi ) three- or four-peak curve. An additional 7% of the curves were discarded due to them exhibiting an abnormal shape. Compression forces are positive and positive displacement corresponds to the retraction of the AFM probe from the substrate.

Article Snippet: The following materials were used in this study: sulfo-LC-SPDP (sulfosuccinimidyl 6-(3′-(2-pyridyldithio)propionamido)hexanoate; ThermoScientific, Waltham, MA, USA, 21650), TCEP-HC (Tris(2-carboxyethyl)phosphine hydrochloride Thermo Scientific, Waltham, MA, USA, 20490), EDTA (ethylenediaminetetraacetic acid; Sigma Aldrich, Burlington, MA, USA, E9884), MicroDeck TM -G-150 surfaces (Bioactive Surfaces, Galapagar, Madrid, Spain), lactate dehydrogenase (LDH; Sigma Aldrich, Burlington, MA, USA, L1006-12.5KU), biotinylated anti-lactate dehydrogenase (anti-LDH) antibodies (IgG polyclonal antibody; Origene, Rockville, MD, USA, AP21329BT-N), poly(ethylene glycol)-(N-hydroxysuccinimide 5-pentanoate) ether 2-(biotinylamino)ethane (NHS–PEG–biotin; Sigma Aldrich, Burlington, MA, USA, 757799-100μγ), streptavidin (Sigma Aldrich, Burlington, MA, USA, S4762-5 mg), DeepTip TM SiN R11 atomic-force microscopy probes (Bioactive Surfaces, Galapagar, Madrid, Spain), Goat Anti-Rabbit IgG H&L Alexa Fluor ® 488 (Abcam, Cambridge, UK, ab150077), 5-iodoacetamidofluorescein (5-IAF; Thermo Scientific, Waltham, MA, USA, 62246), sodium pyruvate (P2256 Sigma-Aldrich, Burlington, MA, USA,), β-nicotinamide adenine dinucleotide (NADH; N8129-500 mg), and glutaraldehyde (50 wt.

Techniques: