Figure 5.

FAC modulates autophagy, lysosome abundance, and cell survival in a MAPK-dependent manner. (a) T80, T80+H-Ras, and T80+K-Ras were treated with 250 μM FAC for the indicated time points (1–18 h). Cell lysates were collected and analyzed by western blotting for the proteins indicated. Results are representative of four independent experiments. (b) Cell lysates collected from T80, T80+H-Ras, and T80+K-Ras followed by western analyses for H-Ras and K-Ras expression. (c) T80 and T80+H-Ras cells were treated with 250 μM FAC for the indicated time points (5 min–3 h). Cell lysates were collected and analyzed by western blotting for the proteins indicated. Results are representative of four independent experiments. (d) Top panel, T80 cells were treated with (1) 10 μM U0126, (2) 250 μM FAC, or (3) 10 μM U0126 and 250 μM FAC for 1–18 h. Cell lysates were harvested and analyzed for the proteins indicated. Results are representative of three independent experiments. Bottom panel, T80 cells were treated with (1) 75 μM LY294002, (2) 250 μM FAC, or (3) 75 μM LY294002 and 250 μM FAC for 1–18 h. Cell lysates were harvested and analyzed for the proteins indicated. Results are representative of three independent experiments. (e) T80 cells were transfected with EGFP–LC3 and treated with (1) 10 μM U0126, (2) 75 μM LY294002, (3) 250 μM FAC, (4) 10 μM U0126 with 250 μM FAC, or (5) 75 μM LY294002 with 250 μM FAC for 18 h. EGFP–LC3 fluorescence was captured using an inverted fluorescence microscope at × 40 magnification. Representative images of experiments conducted in duplicate are shown. (f) results from (e) are shown as the number of cells with punctae quantified by counting 200 cells with >20 punctae. (g) HEY cells were treated with 250 μM FAC (left panel) or in combination with 10 μM U0126 (right panel) for the indicate times followed by western analyses. Results are representative of two independent experiments. (h) HEY cells were seeded onto glass coverslips and exposed to a bolus dose of calcein AM followed by 250 μM FAC treatment in the absence or presence of 10 μM U0126 for 24 h. During the last 1 h of treatment, cells were labeled with Lysotracker red. Cells were imaged using an inverted fluorescent microscope. Results are representative of two independent experiments. (i) HEY cells were treated with 250 μM FAC in the absence or presence of 10 μM U0126 for 24 h. During the last 1 h of treatment, cells were labeled with Lysotracker green. Cells were harvested and analyzed by flow cytometry. Data are expressed as the percentage of Lysotracker green-positive cells. Results are representative of two independent experiments. (j) T80, T80+H-Ras, HEY, TOV112D, and TOV21G cells were treated in combination with 10 μM U0126 and 250 μM FAC compared with either 10 μM U0126 or 250 μM FAC alone. Cell growth was assessed and results shown are representative of two independent experiments. (k) HEY cells were treated with 250 μM FAC in the absence or presence of 10 μM U0126 for 72 h followed by measurement of LDH activity. Results are representative of two independent experiments. * refers to P<0.05