Fig 2: Infection triggers LRRK2‐dependent Rab8A phosphorylation in macrophages A–CRAW264.7 macrophages were left uninfected or infected with (A) Mycobacterium tuberculosis (Mtb), (B) C. albicans (Ca) or (C) L. monocytogenes (Lm) for the indicated time, and Rab8A pT72, Rab10 pT73 and LRRK2 pS935 levels were analysed by Western blot.D–FWT and LRRK2 KO macrophages were left uninfected or infected with (D) Mtb for 24 h, (E) Ca or (F) Lm for 120 min. Rab8A pT72 and LRRK2 pS935 phosphorylation was analysed by Western blot.G–IRAW264.7 macrophages were pre‐treated with 1 μM GSK2578215A (GSK inh) or 0.1 μM MLi‐2 and left uninfected or infected with (G) Mtb for 24 h, (H) Ca or (I) Lm for 120 min. Rab8A pT72 and LRRK2 pS935 phosphorylation was analysed by Western blot and quantified by densitometry. Beta‐actin was used as loading control. Data represent the mean + SEM of three independent biological replicates. One‐way ANOVA followed by Dunnett's test against DMSO control. ns = non‐significant; *P < 0.05; ***P < 0.001 Source data are available online for this figure.

Fig 3: Damaged endolysosomes are targeted to lysophagy in the absence of LRRK2 and Rab8A signalling A, BWT or LRRK2 KO RAW264.7 macrophages, RAW264.7 macrophages pre-treated with 1 µM GSK2578215A (GSK inh) and WT or Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. (A) LC3B and (B) ubiquitin K63-positive vesicle numbers were analysed by immunofluorescence and high-content imaging. Scale bar = 20 µm. Right panels show quantification of number of positive vesicles per cell. Data show the mean ± SEM of three to four biological replicates. ns = non-significant, *P = 0.05, **P = 0.01 by one-way ANOVA followed by Sidak's multiple comparisons test.CRepresentative images of LC3B/LAMP-1 double-positive endolysosomes in control and LLOMe-treated macrophages. Scale bar = 10 µm.DAnalysis of the % of LC3B/LAMP-1 double-positive endolysosomes in LLOMe-treated macrophages. Data show mean ± SEM of three independent biological experiments. *P = 0.05, ***P = 0.001 by Student's t-test.

Fig 4: Effect of Xiaoyaosan on glucose metabolism in hypothalamus of mice injected with 3-MA. (A) Immunofluorescence expression of GLUT4 in hypothalamus of the mice (scale bar = 20 µm). (B) Immunofluorescence intensity of GLUT4 (%) (F (3, 8) = 10.900, p = 0.0034, n = 3). (C) Serum insulin level of the mice (F (3, 16) = 9.543, p = 0.0008, n = 5). (D) The blood glucose of the mice (F (3, 56) = 22.165, p < 0.0001, n = 15). (E) Representative western blotting of GLUT4, Rab8 and Rab10 expressions. (F) Quantitative analysis of protein levels of GLUT4 (F (3, 16) = 4.001, p = 0.0266, n = 5). (G) Quantitative analysis of protein levels of Rab8 (F (3, 16) = 23.664, p < 0.0001, n = 5). (H) Quantitative analysis of protein levels of Rab10 (F (3, 16) = 6.453, p = 0.0045, n = 5). (I) The expression of GLUT4 mRNA (F (3, 12) = 5.221, p = 0.0155, n = 4). (J) The expression of Rab8 mRNA (F (3, 12) = 3.655, p = 0.0443, n = 4). (K) The expression of Rab10 mRNA (F (3, 12) = 6.112, p = 0.0091, n = 4). The values represent the mean ± SEM. * p < 0.05 or ** p < 0.01 compared with the control group. &p < 0.05, &p < 0.01 compared with the 3-MA group. #p < 0.05, ##p < 0.01 compared with the 3-MA group.

Fig 5: RILPL1 is a negative regulator of ciliation.(A) Quantitation of primary cilia in WT and RILPL1-KO A549 cells. (B) Quantitation of cilia length in WT and RILPL1-KO cells. (C) Quantitation of cells with cilia when RILPL1-GFP is expressed in WT or RILP1-KO ad A549 cells. (D) Quantitation of cells with cilia when RILPL1-GFP is expressed in WT hTERT-RPE cells. (E) Quantitation of RILPL1-GFP expression in ciliated versus non-ciliated cells RPE by determining GFP signal intensity per cell in microscopy images. (F) Quantitation of cilia in A549 RILPL1-KO cells that were either un-transfected or transfected with GFP alone, GFP-Rab10 or GFP-Rab8A. (G) Quantitation of cilia in A549 cells lacking Rab8A or Rab10 as indicated, with or without RILPL1-GFP expression.