Journal: Scientific Reports

Article Title: GM2-GM3 gangliosides ratio is dependent on GRP94 through down-regulation of GM2-AP cofactor in brain metastasis cells

doi: 10.1038/s41598-019-50761-5

Figure Lengend Snippet: Study of the GM2 hydrolytic process in BRV5CA1 and GRP94-silenced clones. ( A ) HexA, HexB and total activities in the cellular models. HexA activity is calculated by the difference of total activity and HexB activity by thermal denaturation. The percentages of HexA activity respect to the total activity are specified. ( B ) HexA activity measured using the specific substrate for HexA. The percentages of HexA of silenced clones are written down; ( C ) Western Blot for HexA and GM2-AP and relative quantification; Hela cells are used as positive control of HexA, JAR cells are used as a positive control of GM2-AP; the bands of interest have been cropped from the original gel (available as Fig. ); ( D ) Immunofluorescence assay for GM2 and GM3 and relative quantification. Nuclei were stained in blue and GM2 and GM3 in red, ***p < 0.005; ( E ) Immunofluorescence assay of GM2 and co-localization with Lysotracker, in control and shGRP94-2 cells. Scale bars = 20 µm; ( F ) Immunofluorescence assay of LC3B and GM2, in control and shGRP94-2 cells. Scale bars = 20 µm; ( G ) q-RT-PCR of GM2A gene in the cell clones, ***p < 0.005.

Article Snippet: Quantitative real-time PCR was performed using a 7900HT Fast Real-Time PCR System (Applied Biosystems) with validated Taqman Gene Expression Assays (Hs00942655_m1, for HEXA and Hs00166197_m1 for GM2A, Applied Biosystems).

Techniques: Clone Assay, Activity Assay, Western Blot, Quantitative Proteomics, Positive Control, Immunofluorescence, Staining, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: GM2-GM3 gangliosides ratio is dependent on GRP94 through down-regulation of GM2-AP cofactor in brain metastasis cells

doi: 10.1038/s41598-019-50761-5

Figure Lengend Snippet: Representation of the hypothesis formulated based on the results of this work. ( A ) Normal expression of GRP94 enables the correct folding of GM2-AP, which, once in the lysosome, catalyses the hydrolysis of the endocyted GM2 to GM3. ( B ) Hypothesis of cell biology in GRP94-silenced cells. The deficiency of GRP94 leads to unfolded/misfolded GM2-AP, which impairs its hydrolytic activity in the lysosome. As a consequence, the endocyted GM2 accumulated in the lysosome and, on one hand, it difficults the autophagosome fusion to the lysosome, and on the other hand, limits the production of GM3, which alters the balance of GM3-GM2 in the plasma membrane, related to the cellular metastatic potential.

Article Snippet: Quantitative real-time PCR was performed using a 7900HT Fast Real-Time PCR System (Applied Biosystems) with validated Taqman Gene Expression Assays (Hs00942655_m1, for HEXA and Hs00166197_m1 for GM2A, Applied Biosystems).

Techniques: Expressing, Activity Assay, Clinical Proteomics, Membrane

Journal: Molecular neurodegeneration

Article Title: Elevated ganglioside GM2 activator (GM2A) in human brain tissue reduces neurite integrity and spontaneous neuronal activity.

doi: 10.1186/s13024-022-00558-4

Figure Lengend Snippet: Fig. 8 A system coupling proteomic profiling and functional assays identified GM2A as a neurotoxic factor to human brain

Article Snippet: Nitrocellulose membranes were incubated with Intercept blocking buffer (LI-COR) for 1 hr. at room temperature, followed by overnight incubation with rabbit anti-GM2A antibody (1:1000, Proteintech), mouse anti-GAPDH (1:10,000, Proteintech), rabbit anti-HOMER1 (Synaptic Systems, 1:1000), or mouse anti-Vglut2 (Abcam, 1:1000).

Techniques: Functional Assay

Journal: Cancer science

Article Title: Identification of ganglioside GM2 activator playing a role in cancer cell migration through proteomic analysis of breast cancer secretomes.

doi: 10.1111/cas.12935

Figure Lengend Snippet: Fig. 2. mRNA expression of ganglioside GM2 activator (GM2A). (a) Gene expression levels obtained from the Oncomine database were com- pared between breast cancer (BC; n = 53) and normal breast tissues (n = 6).(20) (b) GM2A expression levels in BC tissues were compared between estrogen receptor (ER)-negative (n = 440) and ER-positive samples (n = 1508).(21) (c) Gene expression level of GM2A and GM2A-correlated genes (correlation coefficient > 0.9) in MAP2K- and RAF1-transfected MCF-7 breast cancer cell lines is represented as a heat-map. (d) Interaction net- works between GM2A and GM2A-correlated genes by Ingenuity Pathway Analysis. The asterisks (*) indicate multiple identifiers in the dataset file map to a single gene in the molecular network.

Article Snippet: The primary antibodies used in this study were directed against the following proteins: GM2A, ATP6AP2 (Atlas Antibodies, Stockholm, Sweden), FBLN1, and IGFBP5 (Abnova, Taiwan, China).

Techniques: Expressing, Gene Expression, Transfection

Journal: Cancer science

Article Title: Identification of ganglioside GM2 activator playing a role in cancer cell migration through proteomic analysis of breast cancer secretomes.

doi: 10.1111/cas.12935

Figure Lengend Snippet: Fig. 3. Effect of ganglioside GM2 activator (GM2A) alteration in breast cancer cells on cell motility ⁄ migration. (a) mRNA expression of GM2A in siRNA-treated breast cancer (BC) cells was measured by quantitative real-time PCR. Error bars represent SD of quadruplicated measurements. (b) Protein level of GM2A in siRNA-treated BC cells was measured by Western blot. (c) Cell migration was analyzed for siRNA-treated BC cells (tripli- cates). (d) Immunoblot analysis of GM2A overexpression in BC cells. (e) Cell migration was analyzed in GM2A-overexpressing (Over) BC cells (trip- licates). *P < 0.01; **P < 0.001.

Article Snippet: The primary antibodies used in this study were directed against the following proteins: GM2A, ATP6AP2 (Atlas Antibodies, Stockholm, Sweden), FBLN1, and IGFBP5 (Abnova, Taiwan, China).

Techniques: Migration, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Over Expression

Journal: Cancer science

Article Title: Identification of ganglioside GM2 activator playing a role in cancer cell migration through proteomic analysis of breast cancer secretomes.

doi: 10.1111/cas.12935

Figure Lengend Snippet: Fig. 4. Ganglioside GM2 activator (GM2A) levels in plasma samples of breast cancer (BC) patients. (a) Immunoblot of GM2A in plasma samples (10 lg) from healthy controls and BC patients. Black circles indicate estrogen receptor (ER)-negative samples. (b) ELISA analysis of GM2A in plasma samples of BC patients and healthy controls. (c) Receiver– operating characteristic (ROC) curve of GM2A for discriminating BC from healthy controls. (d) ROC curve of GM2A for discriminating ER-negative BC from healthy controls. AUC, area under the ROC curve.

Article Snippet: The primary antibodies used in this study were directed against the following proteins: GM2A, ATP6AP2 (Atlas Antibodies, Stockholm, Sweden), FBLN1, and IGFBP5 (Abnova, Taiwan, China).

Techniques: Clinical Proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cancer Science

Article Title: Identification of ganglioside GM 2 activator playing a role in cancer cell migration through proteomic analysis of breast cancer secretomes

doi: 10.1111/cas.12935

Figure Lengend Snippet: Twelve proteins selected as marker candidates for breast cancer

Article Snippet: To overexpress GM2A in cell lines, 4 μg GM2A human cDNA (Origene, Rockville, MD, USA) and porcine cytomegalovirus as a control were transfected into the cells using X‐tremeGENE HP DNA transfection reagent (Roche, Mannheim, Germany).

Techniques: Marker