Figure S1. MYO18B is essential for... | Rockefeller University Press

Figure S1.

$MYO18B is essential for lysosomal exocytosis.(A) Left: Representative CLSM images of WT HeLa cells stained by Mannitou Ab under unpermeabilized conditions with EGTA and/or Pitstop2 treatment. Bars, 10 µm. Right: Quantitative analysis of cell-surface paucimannose level with EGTA and/or Pitstop2 treatment. The mean ± SD fluorescent intensity of mock treatment was set to unity (n = 10 images; N = 2 biological replicates; One-way ANOVA with Dunnett correction; **P ≤ 0.01; ***P ≤ 0.001). Related to Fig. 1. (B) Representative flow cytometry result of cell-surface LAMP1 level in WT, MYO18B-KO and MYO18B-rescued U2OS cell lines. Related to Fig. 2. (C) Left: Representative flow cytometry results of cell-surface LAMP1 level in MYO18B knock-down U2OS cells. Right: RT-qPCR assessment of MYO18B mRNA level in MYO18B knock-down U2OS cells. Expression of MYO18B was normalized with hypoxanthine-guanine phosphoribosyl transferase (HPRT) (mean ± SD; n = 3, technical replicates; One-way ANOVA with Dunnett correction; ns, P > 0.05; **P ≤ 0.01; ***P ≤ 0.001). Related to Fig. 2. (D) Western blotting detection of total LAMP1 level in WT and MYO18B-KO HeLa and U2OS cells. Related to Fig. 2. (E) Raw fluorescent intensity detected in HEX activity assessment of WT and MYO18B-KO HeLa cells. The fluorescent intensity of intracellular fraction was normalized with protein amount (mean ± SD; n = 3, independently analyzed samples; unpaired two-tailed t test; *P ≤ 0.05; ***P ≤ 0.001). Related to Fig. 2. (F) Left: Representative result of HEX activity assay in WT and MYO18B-KO and MYO18B-rescued U2OS cells (mean ± SD; n = 3, independently analyzed samples; One-way ANOVA with Dunnett correction; *P ≤ 0.05; ****P ≤ 0.0001). Right: Raw fluorescent intensity detected in HEX activity assessment of WT and MYO18B-KO U2OS cells. The fluorescent intensity of intracellular fraction was normalized with protein amount (mean ± SD; n = 3, independently analyzed samples; unpaired two-tailed t test, **P ≤ 0.01, ***P ≤ 0.001). Related to Fig. 2. (G) Representative plasma membrane repairing assay results of WT, MYO18B-KO and MYO18B-rescued U2OS cells. Related to Fig. 2. Source data are available for this figure: SourceData FS1.$

MYO18B is essential for lysosomal exocytosis. (A) Left: Representative CLSM images of WT HeLa cells stained by Mannitou Ab under unpermeabilized conditions with EGTA and/or Pitstop2 treatment. Bars, 10 µm. Right: Quantitative analysis of cell-surface paucimannose level with EGTA and/or Pitstop2 treatment. The mean ± SD fluorescent intensity of mock treatment was set to unity (n = 10 images; N = 2 biological replicates; One-way ANOVA with Dunnett correction; **P ≤ 0.01; ***P ≤ 0.001). Related to Fig. 1. (B) Representative flow cytometry result of cell-surface LAMP1 level in WT, MYO18B-KO and MYO18B-rescued U2OS cell lines. Related to Fig. 2. (C) Left: Representative flow cytometry results of cell-surface LAMP1 level in MYO18B knock-down U2OS cells. Right: RT-qPCR assessment of MYO18B mRNA level in MYO18B knock-down U2OS cells. Expression of MYO18B was normalized with hypoxanthine-guanine phosphoribosyl transferase (HPRT) (mean ± SD; n = 3, technical replicates; One-way ANOVA with Dunnett correction; ns, P > 0.05; **P ≤ 0.01; ***P ≤ 0.001). Related to Fig. 2. (D) Western blotting detection of total LAMP1 level in WT and MYO18B-KO HeLa and U2OS cells. Related to Fig. 2. (E) Raw fluorescent intensity detected in HEX activity assessment of WT and MYO18B-KO HeLa cells. The fluorescent intensity of intracellular fraction was normalized with protein amount (mean ± SD; n = 3, independently analyzed samples; unpaired two-tailed t test; *P ≤ 0.05; ***P ≤ 0.001). Related to Fig. 2. (F) Left: Representative result of HEX activity assay in WT and MYO18B-KO and MYO18B-rescued U2OS cells (mean ± SD; n = 3, independently analyzed samples; One-way ANOVA with Dunnett correction; *P ≤ 0.05; ****P ≤ 0.0001). Right: Raw fluorescent intensity detected in HEX activity assessment of WT and MYO18B-KO U2OS cells. The fluorescent intensity of intracellular fraction was normalized with protein amount (mean ± SD; n = 3, independently analyzed samples; unpaired two-tailed t test, **P ≤ 0.01, ***P ≤ 0.001). Related to Fig. 2. (G) Representative plasma membrane repairing assay results of WT, MYO18B-KO and MYO18B-rescued U2OS cells. Related to Fig. 2. Source data are available for this figure: SourceData FS1.

This Feature Is Available To Subscribers Only