Figure 2. Inhibition of PIKfyve causes a rapid loss of β1-integrin from the cell surface and a concomitant accumulation of β1-integrin in internal compartments.

(A–J) HeLa cells were incubated with antibodies to label surface β1-integrin for 1 hr at 4°C. Cells were either fixed (A), fixed after an acid wash (F), or incubated with media containing DMSO or 1 µM apilimod at 37°C for the indicated times. Following incubation, cells were fixed (B–E) or fixed after an acid wash (G–J). Fixed cells were permeabilized and immunostained with Alexa-Fluor-488-conjugated anti-mouse secondary antibodies. Flow diagram (top) outlines the experiment. (K) The surface levels of β1-integrin were inferred from the intensity of β1-integrin within 0.8 µm from the cell border. Surface β1-integrin (for images A–E) is reported as the percentage of the total labeled β1-integrin. (L) Internalized β1-integrin was quantified from cells treated as described in (G–J). β1-Integrin intensity was normalized to the average intensity of cells treated with DMSO for 30 min for each experiment. Data presented mean ± SE. Statistical significance from three independent experiments was analyzed using two-way ANOVA and Sidak’s multiple comparisons tests. (K–L). ****p < 0.001 and ns, not significant. Bar: 10 µm.