Figure 3. MAMs/GEMs are Responsible for Mitochondrial Ca²⁺ Overload and Apoptosis in GM1-Accumulating Cells.

(A) Levels of mitochondrial Ca²⁺ in β-gal^−/−, β-gal^+/+ loaded with GM1 (treated or not treated with MBCD) and β-gal^−/−/CyP-D^−/− MEFs were measured ratiometrically using mitochondria-targeted pericam and compared to those of wild-type cells. Mitochondrial Ca²⁺ measurements are shown at baseline and at peak values in response to a pulse of histamine. Values are expressed as mean ± standard deviation of twenty individual mitochondria in an average of 15 cells. Groups were compared by the Student t-test for unpaired samples. (*P<0.05, **P<0.01)

(B) Inmmunoblots of MAMs and MBCD-extracted MAMs probed with HRP-conjugated β-subunit of Cholera toxin (GM1).

(C and D) β-gal^−/− and β-gal^+/+ MEFs were transfected with MFN2 siRNA. Untreated cells, mock transfected cells and cells transfected with a scrambled siRNA were used as controls. Cell lysates were analyzed on immunoblots probed with anti MFN2 and anti actin antibodies.

(E and F) β-gal^−/− and β-gal^+/+ MEFs were transfected with IP3R-1 siRNA. Untreated cells, mock transfected cells and cells transfected with a scrambled siRNA were used as controls. Cell lysates were analyzed on immunoblots probed with anti IP3R-1 and anti actin antibodies.

(G) Flow cytometry analyses of : Annexin V⁺ β-gal^−/− and β-gal^+/+ MEFs untreated or treated with increasing concentration of MBCD (50, 100, 150 μM); β-gal^−/− and β-gal^+/+ MEFs transfected with MFN2 or IP3R-1 siRNAs; mock transfected or scrambled siRNA transfected cells were used as controls.