Saponin

C2C12 myoblasts were cultured on 35-mm-diameter confocal dishes and allowed to differentiate for 4–5 days. After treatment with NRG-1β, the myotubes were fixed in 4% (v/v) PFA for 15 min, permeabilized by adding a buffer containing saponin (Beyotime) for 10 min at room temperature, and then incubated in PBS with 3% (w/v) BSA for 1 h at room temperature, followed by incubation with anti-GLUT4 (1:300; Affinity, Suzhou, China) overnight at 4°C. The cells were washed three times with PBS (10 min each time) and then incubated with Alexa Fluor 594-conjugated goat anti-mouse secondary antibody for 2 h at room temperature. Detection was performed using the confocal microscope (TCS SP8 X; Leica Heidelberg, Germany).
Mice were anesthetized with 1% (w/v) pentobarbital sodium and perfused with saline for a few minutes, followed by perfusion with 4% (v/v) PFA in PBS. The gastrocnemius tissues were dissected and soaked overnight at 4°C in 4% (v/v) PFA and then in 30% (w/v) sucrose at 4°C for 48 h. Gastrocnemius slices (20 μm) were prepared and immunofluorescence detected as described above.

In total, 1 × 10⁴ HeLa cells were cultured on cell climbing slices. Following incubation for 6 h with 100 nM BAF, cells were fixed in ice-cold 4% paraformaldehyde and permeabilized in PBS with 0.05% Triton X-100 and 3% BSA. Immunostaining was performed using rabbit anti-CNX antibody (Proteintech, 10427-2-ap) and mouse anti-CD317 antibody (in-house, ref. [26 (link)]) followed by incubation with anti-mouse IgG Alexa Fluor 488 (Beyotime, P0188), anti-rabbit IgG Cy3 (Beyotime, P0183). For monitoring the colocalization of CNX and autophagosomes, cells were transfected with RFP-LC3, fixed in ice-cold 4% paraformaldehyde, and permeabilized with 0.1% saponin (Beyotime, P0095). Then immunostaining for CNX was performed as aforementioned description. Finally, microscopy was performed using a STELLARIS 5 confocal microscope from Leica Microsystem (Mannheim, Germany).

We used a confocal laser scanning microscope to verify the uptake of A-BG-DOX and free DOX by HepG2 cells. Briefly, the cells were seeded on a confocal dish at a density of 1 × 10⁵ cells and incubated overnight. The next day, we replaced the culture media with fresh media (pH 6.5 and 7.4). Next, A-BG-DOX and free DOX were added to the cells and incubated for 24 h. After that, the cells were washed with PBS and fixed with immunol staining fix solution (Beyotime, Shanghai, China) for 10 min. We added immunostaining permeabilization solution with saponin for 10 min (Beyotime, China). After fixing, permeating, and washing, we incubated the samples with monoclonal ANTI-FLAG M2 antibody (Sigma, USA) diluted at a ratio of 1:3000 overnight at 4 °C, and then the cells were washed three times to remove the excess antibodies. Next, we incubated the cells with goat anti-mouse IgG H&L (Alexa Fluor 647) antibody at a dilution of 1:500. Finally, the samples were stained with Dil (Beyotime, China) and DAPI (Sigma, USA) and studied under a Leica TCS SP8 (Leica, Germany) microscope.

The THP-1 cells were obtained from the ATCC cell line, and the virus was a strain HB29 of the SFTSV virus (GenBank No. HM745930, HM745931, HM745932) isolated from diagnosed patients with SFTS by the Chinese Center for Disease Control and Prevention. The antibodies used in this study included Mouse Anti-Human CD14 (FITC marker) (BD) (Franklin Lakes, NJ, USA). Additionally, commonly used reagents and materials included 4% paraformaldehyde (Biosharp) (Hefei, China), Triton X-100 (Beyotime) (Shanghai, China), Saponin (Beyotime), RPMI 1640 (ThermoFisher) (Waltham, MA, USA), EDTA (Gibco) (Grand Island, NY, USA), BSA (Coolaber) (Beijing, China), fetal bovine serum (FBS) (Gibco) (Grand Island, NY, USA), Penicillin/Streptomycin (PS) (Gibco) (Grand Island, NY, USA), phorboll-12-myriate-13-acetate(PMA) (Sigma Aldrich) (St. Louis, MO, USA), DAPI staining solution (Beyotime) (Shanghai, China), 8-well chamber cover glass (ThermoFisher) (Waltham, MA, USA), etc. The detailed procedural steps of the experiment are illustrated in Figure 1A.