Fig. 2.

Enzymatic activity of MTMR6, MTMR6/R9, MTMR8, and MTMR8/R9 toward PtdIns(3)P (A) or PtdIns(3,5)P₂ (B). (C) Measurement of cellular PtdIns(5)P levels. HeLa cells were transfected with vector (control), cotransfected with either MTMR6 plus MTMR9 or MTMR8 plus MTMR9, and PtdIns(5)P levels were measured as previously described (45). Results are presented as relative mass compared with that in vector-transfected cells. (D) Overexpression of MTMR8 plus MTMR9 reduces levels of PtdIns(3)P in COS-7 cells. Cells were stained with anti-PI(3)P antibodies followed by anti-mouse Alexa568-conjugated antibodies. Nuclei are visualized with DAPI. Cotransfection efficiency was determined in a separate set of plates, because of limited available fluorescence channels, and was found to be greater that 95% in all cases. (Magnification: 63×.) (E) Quantification of immunofluorescence shown in D, expressed as an average number of PI(3)P spots per cell normalized to the number seen in MTMR8/R9 expressing cells. (F) Overexpression of MTMR8/R9 has no effect on apoptosis. HeLa cells were treated with the indicated constructs for 36 h, then with 100 µM etoposide to induce apoptosis. After 8 h, cells were treated with APOPercentage dye to selectively stain apoptotic cells with dark spots. Percentage of apoptotic cells are counted as the number of darker cells out of every 100 cells (*P < 0.01, t test).