Journal: Science Advances

Article Title: Label-free sensing of exosomal MCT1 and CD147 for tracking metabolic reprogramming and malignant progression in glioma

doi: 10.1126/sciadv.aaz6119

Figure Lengend Snippet: ( A and B ) Baseline phase response of the LSPR biosensor with the functionalized SAM-AuNIs sensing chip with anti-MCT1 AB or anti-CD147 AB after sequential treatment with 11-mercaptoundecanoic acid (MUA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/ N -hydroxysuccinimide (NHS). ( C and D ) Phase response of the LSPR biosensor toward three different concentrations (serial dilution of 1300 μg/ml exosome solution: 1000×, 100×, and 10×) of U251 GM-derived exosomes. Standard curve fitting for phase responses toward anti-MCT1 AB ( R 2 = 0.9871) or anti-CD147 AB ( R 2 = 0.9969). ( E and F ) Representative phase response of the LSPR biosensor toward equal amount of normoxic and hypoxic GM-derived exosomes (50 μg/ml). ( G and H ) Bar graph showing the relative strength of LSPR responses toward exosomal MCT1 (E) and CD147 (F) from normoxic or hypoxic GMs ( n = 3). ( I to K ) Two-dimensional, three-dimensional, and high resolution of three-dimensional AFM topographic images for U251 GM-derived exosomes immobilized on the SAM-AuNIs sensing chip. ( L ) Height profile of single U251 GM-derived exosome by AFM scanning. ( M and N ) Representative separation force responses of the AFM biosensor with the functionalized cantilever sensing tip with anti-MCT1 AB, or anti-CD147 AB toward equal amount (50 μg/ml) of normoxic and hypoxic GM-derived exosomes captured on the SAM-AuNIs sample discs. ( O and P ) Bar graph showing the relative strength of AFM separation force responses toward exosomal MCT1 (M) and CD147 (N) from normoxic or hypoxic GMs ( n = 12). All data were shown as the means ± SD. Significance level: ** P < 0.01, * P < 0.05, hypoxia versus normoxia.

Article Snippet: The ABs used in the experiment were rabbit polyclonal anti–HIF-1α AB (Abcam, catalog no. ab82832), rabbit polyclonal anti-MCT1 AB (Alomone Labs, catalog no. AMT-011), rabbit polyclonal anti-CD147 AB (Abcam, catalog no. ab64616), rabbit polyclonal anti-CD63 AB (Abcam, catalog no. ab68418), rabbit polyclonal anti–β-actin AB (Abcam, catalog no. ab16039), goat anti-rabbit IgG H&L (HRP) secondary AB (Abcam, catalog no. ab6721), and goat anti-rabbit IgG (H + L) Alexa Fluor 488 secondary AB (Thermo Fisher Scientific, catalog no. A11034).

Techniques: Serial Dilution, Derivative Assay, Exosomes, Standard Curve

Journal: Science Advances

Article Title: Label-free sensing of exosomal MCT1 and CD147 for tracking metabolic reprogramming and malignant progression in glioma

doi: 10.1126/sciadv.aaz6119

Figure Lengend Snippet: ( A and B ) Representative phase responses of the LSPR biosensor with the functionalized SAM-AuNIs sensing chip with anti-MCT1 AB or anti-CD147 AB and ( C and D ) separation force responses of the AFM biosensor with the functionalized silicon nitride tip with anti-MCT1 AB or anti-CD147 AB toward equal amount of daughter exosomes (50 μg/ml) from parent U251 GMs with no treatment (control), MCT1 OE, MCT1 KD, CD147 OE, and CD147 KD. ( E to H ) Bar graph showing the relative strength of LSPR responses ( n = 3) or AFM forces ( n = 12) toward exosomal MCT1 [from (A) and (C)] and CD147 [from (B) and (D)]. ( I and J ) Correlation curve between MCT1 or CD147 levels in parent GMs and the strength of LSPR responses toward exosomal MCT1 or CD147, respectively [for MCT1, coefficient of determination ( R 2 ) = 0.9247, and for CD147, R 2 = 0.9654], or the strength of AFM forces toward exosomal MCT1 or CD147, respectively (for MCT1, R 2 = 0.9996, and for CD147, R 2 = 0.9952). The correlation analysis was performed based on the data obtained from (A) to (D). All data were shown as the means ± SD. Significance level: ** P < 0.01, * P < 0.05, MCT1 OE and MCT1 KD group versus control. CD147 OE and CD147 KD group versus control.

Article Snippet: The ABs used in the experiment were rabbit polyclonal anti–HIF-1α AB (Abcam, catalog no. ab82832), rabbit polyclonal anti-MCT1 AB (Alomone Labs, catalog no. AMT-011), rabbit polyclonal anti-CD147 AB (Abcam, catalog no. ab64616), rabbit polyclonal anti-CD63 AB (Abcam, catalog no. ab68418), rabbit polyclonal anti–β-actin AB (Abcam, catalog no. ab16039), goat anti-rabbit IgG H&L (HRP) secondary AB (Abcam, catalog no. ab6721), and goat anti-rabbit IgG (H + L) Alexa Fluor 488 secondary AB (Thermo Fisher Scientific, catalog no. A11034).

Techniques: Silicon, Exosomes, Control, R-Squared, Analysis

Journal: Science Advances

Article Title: Label-free sensing of exosomal MCT1 and CD147 for tracking metabolic reprogramming and malignant progression in glioma

doi: 10.1126/sciadv.aaz6119

Figure Lengend Snippet: ( A to C ) Representative MRI images for the brain of sham-operated mice and U251 and U87 mouse models of glioma. ( D and E ) Representative phase responses of the LSPR biosensor with the functionalized SAM-AuNIs sensing chip with anti-MCT1 AB or anti-CD147 AB and ( F and G ) representative separation force curves of the AFM biosensor with the functionalized silicon nitride cantilever tip with anti-MCT1 AB or anti-CD147 AB toward serum-derived exosomes from sham-operated mice and U251 and U87 mouse models of glioma. ( H to K ) Bar graph summarizing the relative strength of LSPR responses ( n = 3) or AFM forces ( n = 3) toward exosomal MCT1 [e.g., (D) and (E)] and CD147 [e.g., (F) and (G)]. Detailed processes of LSPR and AFM biosensing were described in Materials and Methods. All data were expressed as the means ± SD. Significance level: ** P < 0.01, * P < 0.05, U251 or U87 mouse model of glioma versus sham-operated severe combined immunodeficient mouse. WT, wild type.

Article Snippet: The ABs used in the experiment were rabbit polyclonal anti–HIF-1α AB (Abcam, catalog no. ab82832), rabbit polyclonal anti-MCT1 AB (Alomone Labs, catalog no. AMT-011), rabbit polyclonal anti-CD147 AB (Abcam, catalog no. ab64616), rabbit polyclonal anti-CD63 AB (Abcam, catalog no. ab68418), rabbit polyclonal anti–β-actin AB (Abcam, catalog no. ab16039), goat anti-rabbit IgG H&L (HRP) secondary AB (Abcam, catalog no. ab6721), and goat anti-rabbit IgG (H + L) Alexa Fluor 488 secondary AB (Thermo Fisher Scientific, catalog no. A11034).

Techniques: Silicon, Derivative Assay, Exosomes

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The GPI-anchor biosynthesis pathway is critical for syncytiotrophoblast differentiation and placental development

doi: 10.1007/s00018-024-05284-2

Figure Lengend Snippet: Analysis of placental defects in the Pigl and Pigf Knockout (KO) mice. A Schematic representation of the sequential assembly of the mammalian GPI in the endoplasmic reticulum (ER). The synthesis of the full GPI precursor capable of protein attachment occurs through consecutive enzymatic reactions (1)–(11), leading to the addition of the mannose-4 (Man4) side chain within the ER [step (12)]. The mature GPI assembly is then collectively transferred onto proteins in a single unit [step (13)]. Adapted from . The associated genes corresponding to each reaction step are indicated beneath the respective step numbers. Highlighted in red are the main targets of this study, PIGL which catalyzes the initial step 2 of the pathway and PIGF involved in the regulation of final steps 10 and 11. B Representative hematoxylin and eosin staining of E9.5 placentas from a Pigl and Pigf KO, and littermate wild-type (WT) control. The dashed outline delineates the border between the maternal decidua and the trophoblast compartment. Scale bar = 1 mm. The insets zoom into the developing placental labyrinth zone. Scale bar = 200 µm. Black arrows (WT placenta): fetal blood vessels penetrating into the chorionic ectoderm. Vertical bars: unpatterned appearance of the chorion. Images are representative of ≥ 3 mutants per line. C , D Histological analysis of the placentas at E9.5 shows a strong placental defect in the syncytiotrophoblast layer development of Pigl KO mice ( C ) and Pigf KO mice ( D ). Sections were stained for MCT4 (SynT-II marker) and MCT1 (SynT-I marker). Nuclear counterstain with DAPI. Scale bar = 100 µm. Images are representative of at least 3 independent mutant placentas per line. E , F The bar plots show the mean quantification of MCT4 and MCT1 staining for Pigl KO ( E ) and Pigf KO ( F ) placentas compared to littermate WT controls, measured as CTCF (Corrected total cell fluorescence). Individual data points are shown as dots and error bars show standard deviation. Pairwise comparisons were done by a two-tailed t-test (*p < 0.05, ***p < 0.001)

Article Snippet: Antibodies used were anti-CDH1 (1:100; BD Biosciences 610,181), anti-LAMININ (1:100; Sigma L9393), anti-MCT1 (1:100; Merck Millipore AB1286I), anti-MCT4 (1:100; Merck Millipore AB3314P).

Techniques: Knock-Out, Staining, Marker, Mutagenesis, Fluorescence, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Regulation of oxidative phosphorylation by Nuclear myosin 1 protects cells from metabolic reprogramming and tumorigenesis in mice

doi: 10.1101/2022.06.30.498328

Figure Lengend Snippet: (A) The tumor growth rate over the period of 28 days in Balb/c nude mice injected with WT and NM1 KO cells. (B) Two representative pictures of Balb/c nude mice 28 days after injection of WT and NM1 KO cells under mammary pads tissue. WT and KO mark NM1 cell type injected into a given mouse. (C) Representative tumor tissues isolated from Balb/c nude mice 28 days after injection with NM1 KO cells. (D) Tumor mass measurement of isolated tumors from NM1 KO cell- injected Balb/c nude mice. (E) Immunohistochemistry staining with antibodies against cancer markers Bclxl, Egfr, Ki-67, and Mct1 of control tissue isolated from the place of injection of NM1 WT cells and tumor tissue isolated from mice injected with NM1 KO cells. The scale bar is 250µm. (F, H, E) Staining of sections of vital organs from mice injected with NM1 WT and KO cells. The scale bar is 250µm. Mammary pads tissue isolated from the area of injection of NM1 WT cells was used as a negative control to tumor tissue isolated from NM1 KO-injected mice. The scale bar is 250µm.

Article Snippet: The anti-MCT1 antibody was obtained from Merck (Rockville, MD).

Techniques: Injection, Isolation, Mass Measurement, Immunohistochemistry, Staining, Negative Control

Journal: The Biochemical journal

Article Title: Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

doi: 10.1042/BJ20070374

Figure Lengend Snippet: Primers used for RT–PCR analysis BSG, basigin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Western-blot analysis was carried out as described in [ 22 ] using polyclonal anti-human MCT1 antibody (US Biological, Swampscott, MA, U.S.A.) or monoclonal anti-human vinculin (clone hVIN-1; Sigma), and peroxidase-conjugated rabbit anti-goat IgG (Jackson Immunoresearch Laboratories, West Grove, PA, U.S.A.) or goat anti-mouse IgG (Bio-Rad).

Techniques: Sequencing

Journal: The Biochemical journal

Article Title: Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

doi: 10.1042/BJ20070374

Figure Lengend Snippet: (A) Specific uptake was analysed in BCS-TC2 (black circles) and BCS-TC2.BR2 cells (white circles) after pre-incubation (left panel) up to 4 days in the presence of 4 mM butyrate or (right panel) after 4-day treatment with up to 10 mM butyrate. Results are expressed as a percentage of specific uptake after pre-incubation in the absence of butyrate, and correspond to mean values ± S.D. for three independent experiments with triplicate samples. (B) Representative Western blots corresponding to MCT1 expression under the experimental conditions described in (A). Vinculin (Vin) expression is also shown. (C) Densitometric analysis of MCT1 levels using vinculin expression (a.u., arbitrary units) for normalization. Results correspond to mean values ± S.D. for four independent experiments.

Article Snippet: Western-blot analysis was carried out as described in [ 22 ] using polyclonal anti-human MCT1 antibody (US Biological, Swampscott, MA, U.S.A.) or monoclonal anti-human vinculin (clone hVIN-1; Sigma), and peroxidase-conjugated rabbit anti-goat IgG (Jackson Immunoresearch Laboratories, West Grove, PA, U.S.A.) or goat anti-mouse IgG (Bio-Rad).

Techniques: Incubation, Western Blot, Expressing

Journal: The Biochemical journal

Article Title: Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

doi: 10.1042/BJ20070374

Figure Lengend Snippet: (A) BCS-TC2 cells were transfected with 500 nM scrambled siRNA (S) or with 250 or 500 nM siRNA 1 (si1) or 2 (si2) respectively. At 72 h after transfection, cells were homogenized and analysed by Western blotting using MCT1- or vinculin (Vin)-specific antibodies. Representative blots are shown. (B) Densitometric analysis of MCT1 levels using vinculin expression for normalization. Results correspond to mean values ± S.D. for four independent experiments. (C) Specific transport at 40 μM butyrate was analysed in parallel untransfected and transfected cultures. Results correspond to mean values ± S.D. for three independent experiments using triplicate samples.

Article Snippet: Western-blot analysis was carried out as described in [ 22 ] using polyclonal anti-human MCT1 antibody (US Biological, Swampscott, MA, U.S.A.) or monoclonal anti-human vinculin (clone hVIN-1; Sigma), and peroxidase-conjugated rabbit anti-goat IgG (Jackson Immunoresearch Laboratories, West Grove, PA, U.S.A.) or goat anti-mouse IgG (Bio-Rad).

Techniques: Transfection, Western Blot, Expressing