4 6 diamidino 2 phenylindole (dapi)

B cells were isolated from PBMCs using the Pan B Cell Isolation Kit, B Cell Isolation Kit II, or IgG⁺ Memory B Cell Isolation Kit (Miltenyi Biotec). Isolated cells were labeled with anti-human CD19-AF700 (BD), anti-human IgG-APC (BD), DAPI (Thermo Fisher), and the respective HIV-1 Env bait for 30 minutes on ice. BG505_SOSIP.664-GFP or biotinylated YU2_gp140 that was labeled with Streptavidin-PE (BD) were used as HIV-1 Env baits. Env-reactive CD19⁺IgG⁺DAPI^- single cells were sorted into 96-well plates containing 4 μl of lysis buffer (0.5x PBS, 10 mM DTT (Thermo Fisher), 2 U/μl RNasin (Promega), and 1 U/μl RNaseOUT (Thermo Fisher)) per well using a BD FACSAria Fusion. Plates were stored at −80°C until further use.

The murine PMs were cultured overnight on glass coverslips and then treated with lysosome inhibitors for 24 h. Then, the cells were coincubated with labeled αPD-L1 or TEV-bound αPD-L1 for another 2 h. After being washed three times with PBS, the cells were fixed with precooled methyl alcohol for 10 min at −20 °C and then permeabilized with 0.1% Triton X-100 for 10 min at RT. After the cells were blocked with 5% BSA and 3% goat serum in PBS, they were incubated with LAMP1 antibodies (Abcam, Cambridge, UK) overnight at 4 °C in blocking buffer. The following day, after three washes in PBS, the cells were incubated with DyLight 488-labeled secondary antibodies (Multi Sciences Biotech, Hangzhou, China) for 30 min at RT and washed in PBS. Finally, nuclei were stained with DAPI (Thermo Fisher Scientific). Liver and spleen tissues were embedded in Tissue-Tek™ CRYO-O.C.T. (Thermo Fisher Scientific) and processed to obtain 5 μm sections. Then, the tissue sections were stained with mouse F4/80 antibodies (Abcam) at 4 °C overnight followed by staining with DyLight 488-labeled secondary antibodies (Multi Sciences Biotech) for 1 h at 4 °C. The nuclei were stained with DAPI (Thermo Fisher Scientific) for 20 min at RT. The stained sections were imaged using an Olympus IX83-FV3000 confocal microscope (Olympus). Images were analyzed with ImageJ software (NIH).

Samples were fixed with methanol for 10 mins. Cells were washed three times with PBS, 5 mins per wash. After washes, sample were then washed and blocked in 4% Bovine Serum Albumin (BSA) in PBS for 30 mins and incubated with the appropriate primary antibody combinations against GFP (Abcam), E-cadherin (Cell Signaling), and DAPI (ThermoFisher Scientific) or antibody against apical membrane marker Zona Occluden-1 (ZO-1, Thermo Fisher Scientific) and DAPI, overnight at room temperature. Primary antibody was washed away with PBS and samples were washed three times with PBS, 5 mins per wash. Samples were then incubated with monoclonal or polyclonal secondary antibodies (Life Technologies) for 1 hr. Samples were imaged using Nikon A1R Confocal Microscope and NIS elements and Volocity 6.3 software.

Flies were housed at 25°C under an LD cycle on standard media, unless otherwise noted. At each time point ∼10 intestines from female flies <14 days were dissected in PBS (Fisher) and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in PBS and then counterstained with DAPI (Thermo Fisher Scientific, 1:5,000) in PBS-T (PBS + 0.2% Triton X-100, Fisher). Intestines were then blocked in 1% BSA (Bio Basic) + 0.2%Triton X-100 (Fisher) and incubated in the same at room temperature for 2 hr with primary antibodies: mouse anti-Delta (Developmental Studies Hybridoma Bank [DSHB], 1:50), mouse anti-prospero (DSHB, 1:50), mouse anti-histone (Millipore, 1:2000), or rabbit anti-PER (generously provided by Patrick Emery, 1:1,500), then incubated at room temperature for 1 hr in secondary goat anti-mouse/rabbit antibodies (Life Technologies, 1:2000), and counterstained with DAPI (Thermo Fisher Scientific, 1:5,000). Samples were imaged using a slide scanner (Zeiss Axio Scan.Z1) that assembled single images consisting of merged and tiled z stacks of the entire tissue sample in a single plane of focus, or by confocal microscopy (Olympus IX81 FV1000) with a 60× water-immersion lens. Images were analyzed using Zen Blue Edition software (Zeiss) and processed using Photoshop CS5 (Adobe).

Cells treated with various concentrations of CFI-400945 and 0.1% DMSO (control) were seeded on Poly Lysine (Sigma, USA) coated coverslips, washed twice with 1X PBS, fixed in ice cold Methanol (Fisher Chemical, USA) at -20°C for 10 min and then permeabilized with 0.25% Triton-X (Acros Organics, USA) at room temperature (RT) for 10 min. The cells were then blocked in blocking buffer (5% BSA (Jackson Immuno Research Laboratories Inc., USA) in PBST) for 1 hour at RT. The primary antibody (Anti-λ Tubulin 1:500, Sigma, USA) diluted in 1% BSA in PBST was then added and incubated at 4°C overnight. Then, the cells were washed with 1X PBS thrice. Secondary antibody (Anti-Rabbit IgG (H+L) F (ab’) 2, 1:500, Sigma, USA) diluted in 1% BSA in PBST was added and incubated at RT for 2 hours. Three time washes with 1X PBS were performed. Then, 1mL of DAPI (Thermo Fisher Scientific, USA) in PBS (0.5μl of DAPI in 10 ml PBS) was added to the cells and incubated for 10 min at RT. Finally, 3 time washes with 1X PBS were performed and slides were mounted using Mounting Medium (Thermo Fisher Scientific, USA).

F81 cells were seeded in 24-well cell culture plates with pre-placed glass cover slide, and the cell samples were divided into 3 groups when the cells had grown to about 60% confluence. The first group was co-transfected with pEGFP-VP2 and pDsRed-CCT7 (2 μg, the ratio of two plasmids is 1:1), the second group was infected with CPV 12 h after transfection with pDsRed-CCT7 (2 μg) plasmid, and the third group of F81 cells were directly infected with CPV (MOI = 0.1). The supernatant was discarded at 24 h after transfection and/or infection. Cell samples were fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 30 min, then permeabilized with 2% bovine serum albumin (BSA) solution (Sigma-Aldrich, Saint Louis, USA) containing 0.1% Triton X-100 for 10 min at room temperature, and further blocked with 2% BSA for 1 h. After blocking, the transfected cells were washed with PBS, and then DAPI (Thermo Fisher Scientific, MA, USA) was added for nuclear staining. For cells infected with CPV, after washing with PBS, cells were incubated at room temperature for 1 h with a mixture of mouse anti VP2 and rabbit anti CCT7 antibodies, then incubated at room temperature with mixed DyLight™ 488 and TRITC coupled secondary antibody solutions for 1 h, and finally stained with DAPI (Thermo Fisher Scientific, MA, USA). Images were collected on a confocal microscope (LSM900, Carl Zeiss, Germany).