Trackit software

The protocol was modified from Kuo’s report [16 (link)]. In brief, 293T (5 × 10⁵) cells were seeded in 3.5 cm glass bottom dish. After 48 h, cells were transfected with RFP-Rab37 or GFP-PD-1 for 18 h. TIRF microscopy system was built on an inverted microscopy Olympus IX81 (Olympus, Tokyo, Japan) equipped with a high sensitivity EMCCD Camera (iXOn3897, Andor technology, New York, United States) and a UPON 100X oil objective lens (NA = 1.49, Olympus) to capture 100–200 nm images below the plasma membrane interface. We defined each green fluorescence spot as a cargo-containing vesicle and trafficking by Rab37 in cells, and then tracked each vesicle trafficking distance with trackIT software (Olympus). The cutoff length of moving vesicle was 3 μm. The vesicle trafficking event was measured as the ratio of moving vesicles to the total vesicles in cells. A total of at least 20 moving vesicles per cell were tracked and 6 cells were scored.

For confocal analysis, cells (1 × 10⁵) were fixed and then incubated with primary antibodies against Rab37 and TIMP1. After incubation with secondary fluorescent antibodies, the images were obtained using Olympus FV1000 confocal microscope. For immuno-EM analysis, primary antibodies and the conjugated 10 or 15 nm gold particles were listed in Supplementary Table 2. For TIRF analysis, cells expressing WT-Rab37 were transiently transfected with GFP-TIMP1 for 24 h before image analysis. To capture the Rab37 trafficking events, we tracked each vesicle trafficking distance with trackIT software (Olympus, Tokyo, Japan).

TIRF analysis was modified from Tsai's report 24 (link). RAW264.7 macrophage cells were transfected with GFP-CHI3L1 and RFP-Rab37 for 16 h and 1 × 10⁴ cells were re-seeded in 3.5 cm glass bottom dish. TIRF microscopy system (Olympus IX81) equipped with a high sensitivity EMCCD Camera (iXOn3897, Andor technology) and a UPON 100X oil objective lens (NA = 1.49, Olympus) was used to capture 100-200 nm images below the plasma membrane. We defined each green fluorescence spot as a CHI3L1-containing vesicle, and then tracked each vesicle trafficking distance with trackIT software (Olympus).