Mounting medium with dapi

Primary cilia and nuclei of ASCs were stained as previously described [42] (link) using antibodies against acetylated tubulin (Sigma-Aldrich), Alexa Fluor 568 goat anti-mouse IgG (Invitrogen), and Mounting Medium with DAPI (Abcam). The cells were visualized using fluorescent microscope Olympus BX61 with UPLan SApo 40 × objective.

Wortmannin, dynasore, SU6655, NH₄Cl, SP600125, IPA-3, MβCD and chlorpromazine were obtained from Sigma (Sigma, MO, United States), Akti-1/2 and bafilomycin A1 were obtained from Abcam (Abcam, Cambridge, United Kingdom), nystatin and EIPA was obtained from Solarbio (Solarbio, Beijing, China).
The rabbit anti-dynamin-2, anti-clathrin heavy chain, anti-Rab5, anti-Rab7, anti-PI3K p85 (phos pho Y458) + PI3 Kinase p55 (phospho Y199), anti-Akt, anti-Src, anti-Src (phospho Y416), anti-phospho-Akt (Ser473) and anti-GAPDH monoclonal antibodies were obtained from Abcam (Abcam, Cambridge, United Kingdom). The rabbit anti-JNK and anti-JNK (Thr183/Tyr185) monoclonal antibodies were obtained from Cell Signaling Technology (Cell Signaling Technology, Danvers, United States). The rabbit anti-PI3 Kinase p85 alpha polyclonal antibody, the mouse anti-Flag monoclonal antibody, goat anti-rabbit and goat anti-mouse HRP-labeled secondary antibody, Alexa fuor-488-conjugated anti-mouse, Cy™3-conjugated anti-rabbit IgG (H + L) and mounting medium with DAPI was purchased from Abcam (Abcam, Cambridge, United Kingdom). The rabbit anti-caveolin-1 monoclonal antibody was obtained from Beyotime (Beyotime Biotechnology, Shanghai, China). The mouse anti-BRSV G protein monoclonal antibody was provided by China Animal Health and Epidemiology Center.

The specific probe of chi_circ_0006511 conjugated with Cy3 was designed and synthesized by RiboBio CO., LTD (Guangzhou, China). The cellular localization of chi_circ_0006511 was detected by FISH kit (RiboBio, Guangzhou, China). Briefly, the GIMAs cultured with slides in 24 wells were fixed with 4% paraformaldehyde, permeabilized with pre-cooled PBS containing 0.5% Triton X-100 for 10 min, added with prehybridization solution and incubated at 37 °C for 30 min, and then 50 μmol of probe was added to the pre-warmed hybridization solution and incubated with cells at 37 °C overnight. In dark conditions, the slides were washed with 4× (0.1% Tween-20), 2× and 1 × SSC solutions at 42 °C, respectively, and then the slides were fixed on the slides with Mounting Medium with DAPI (abcam, Cambridge, UK). Pictures were taken with a confocal microscope (Zeiss, Oberkochen, Germany).

Cells were grown in a poly-L lysine-coated 4 well Millicell EZ slides (EMD Millipore) for 48 h and pre-extracted with 0.2% Triton-X on ice 90 s before fixation with 4% paraformaldehyde in PBS for 20 min at room temperature (RT) and then permeabilized by incubation with ice-cold methanol. After permeabilization, cells were blocked with 5% BSA in PBS for 1 h at RT, followed by incubation with 5% BSA in TBS-T containing primary antibodies at a ratio of 1:200 overnight at 4 °C. Cells were washed and incubated with secondary fluorescent antibodies for 1 h at RT. Alexa Fluor 488 anti-rabbit and Alexa Fluor 488 anti–mouse antibodies were purchased from Invitrogen. After washing, the nuclear content was stained with Mounting Medium with DAPI (Abcam) overnight at 4 °C. Images were obtained with fluorescence microscope BX53 (Olympus). Images were quantitatively assessed using ImageJ software with ‘Find maxima’ function. More than 100 cells were analyzed per condition in each experiment in a blinded manner. Antibodies used in immunofluorescence analysis are listed in Supplementary Data 7.

According to the instructions of the reagent instructions of the PKH26 Red Fluorescent Cell Linker Mini Kit (MINI26-1KT, #MKCM1863, Sigma-Aldrich), SCDEs were incubated with excess dye at room temperature for 4 h and then removed by ultracentrifugation at 100,000 × g for 1 h. After being co-cultured with PKH26-labeled exosomes for 16 h, BMDMs were fixed, penetrated, blocked, and incubated with CD11b antibody (1:400; Abcam, #1211 MA, USA) overnight at 4 °C, and then incubated with secondary antibody. After being washed, the slides were removed from the 24-well plate and mounted with Mounting Medium with DAPI (Abcam, #104139, MA, USA). PKH26-labeled SCDEs being uptaken by primary bone marrow-derived macrophages was observed by the ultra-high resolution laser confocal microscope (ZEISS LSM 900, Germany).

First, 3 × 10⁴ HEK293 cells/well were plated in 8-well chamber slides for immunofluorescence (Ibidi, Gräfelfing, Germany). Transient transfection was performed as described above, and cells were washed and fixed in Methanol (Sigma-Aldrich) for 5 min at −20 °C and blocked in blocking solution (Phosphate Buffered Saline (PBS, Sigma-Aldrich) containing 3% Bovine Serum Albumin (BSA, Sigma-Aldrich). Rabbit anti-B7-H4 primary antibody (1/200 in blocking solution) was incubated overnight at 4 ℃ in a wet chamber. Subsequently, cells were washed three times with PBS-BSA for 10 min prior to incubation with anti-rabbit FITC secondary antibody (1/100) for 1 h in a wet chamber and darkness at room temperature. Cells were washed and mounted in a Mounting Medium with DAPI (Abcam, Cambridge, UK) and visualized in a confocal microscope (ZEISS LSM880 Airyscan, Jena, Germany). For quantitation of B7-H4 subcellular distribution, at least 50 positive cells were scored. Cells were rated as membrane staining (M) or membrane/cytoplasm (M/C). Nuclei were identified by DAPI staining.