S6K is required for the LPS-mediated lysosome expansion.

(a) Western blot analysis of protein puromycylation in resting and activated primary macrophages. LPS increases the amount of puromycylated proteins that is blocked by p70S6K inhibitor (LY2584702) or cycloheximide. Lane 1 is control lysates from cells not exposed to puromycin. The band indicated by the arrow is a nonspecific band recognized by the anti-puromycin antibody. p-S6 and β-actin were used to monitor p70S6K activity and as a loading control, respectively. (b) Normalized puromycylation signal (excluding nonspecific band) normalized over β-actin signal. Data are shown as the mean ± standard deviation from 3 independent experiments. Statistical analysis was done with an ANOVA, in which an asterisk indicates conditions that are statistically distinct from control (*p < 0.05). (c) Lysosomes in primary macrophages were pretreated with LY2584702 (LY2) followed by 2 h of LPS where indicated. Images were acquired by live-cell spinning disc confocal microscopy. Scale bar = 5 μm. (d) Lysosomal tubulation was scored for each condition as shown, in which a tubule was defined as longer than 4 μm in length. Tubulation index was determined by normalizing scores to resting cells. (e) Total lysosome volume in primary macrophages treated as indicated. Panels d and e show the mean ± standard error of the mean from 30 to 40 cells per condition per experiment, across 3 independent experiments. (f) Western blot analysis of whole cell lysates from resting and activated primary macrophages with or without LY2584702. (g) Quantification of Western blots showing the levels of LAMP1 and the V-ATPase V₁ subunits H and D, normalized to β-actin. p-S6 and total S6 blots are shown to support effectiveness of LY2584702 treatment. Shown is the mean ± standard deviation of the mean from 5 independent blots. For panels b, c, and e, data were statistically analysed with ANOVA and unpaired post hoc test (*p < 0.05). For each figure with Western blots, see S1 Raw Images for original, unedited Western blots. See S5 Data for original data in Fig 5. CHX, cycloheximide; LAMP1, lysosome-associated membrane protein-1; LPS, lipopolysaccharides; S6K, S6 kinase; V-ATPase, vacuolar H⁺ ATPase pump; LY, Lucifer yellow.