Alexa fluor 594 conjugate

To assess the lysosomal number and distribution via LAMP1 staining, proinflammatory cytokine-stimulated HaCaT cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature (RT) and subsequently permeabilized with 0.1% Triton X-100 for 15 min at RT. Cells were then blocked with 3% BSA in PBS + 0.1% Tween for 1 h at 4 °C. Immunostaining was performed by overnight incubation with a LAMP1 primary antibody (#9091, Cell Signaling, Leiden, Netherlands; 1:200) in blocking solution at 4 °C. The following day, cells were washed three times with PBS and incubated with a secondary antibody (anti-rabbit IgG (H + L), F(ab′)₂ fragment, Alexa Fluor^® 594 Conjugate; #8889, Cell Signaling, Leiden, Netherlands; 1:1000) in blocking solution for 1 h at 4 °C. After three washes with PBS, coverslips were mounted in SlowFade™ Diamond Antifade Mountant with DAPI (Invitrogen, Thermo Fisher Scientific). Each treatment was analyzed in triplicate in three independent experiments.

Mitotic index and pH2AX foci were quantified by platting 1 × 10⁵ cells onto Falcon^® 8-chamber culture slides (cat. #354118). Cells were allowed to attach overnight and were fixed in 4% paraformaldehyde solution (Electron Microscopy Sciences, cat. #100503-917). Standard immunofluorescence was carried out for α-tubulin (Cell Signaling, cat. #8058) and pH2AX (Cell Signaling, cat. #9718) using secondary anti-rabbit antibody Alexa Fluor 594 conjugate (Cell Signaling cat. #8889), and DAPI staining (Sigma, cat. #D9542) for DNA counterstaining. Slides were photographed using a Zeiss AxioObserver.Z1 fluorescence microscope and at least 5 different fields were captured for each biological replicate. A total of 4 independent biological replicates were photographed per each cell type analyzed. Using image J software, version 1.52a, a total number of cells per field was estimated based on the number of DAPI-positive nuclei. A total number of mitotic cells was estimated based on recognition of mitotic figure visualized by DAPI and α-tubulin staining. A total number of cells positive for pH2AX was estimated, when the number of foci was larger than 20 per nuclei. The percentage of cells in mitosis, cells positive for pH2AX and mitotic cells positive for pH2AX (Tyk-nu cells only) was estimated based on a total number of cells. Data were represented as average ± SE.

Mouse brain was fixed in 4% formaldehyde for 24 h, and then incubated in 30% sucrose until tissues are sink. Fixed brain was flash frozen using pre-cooled isopentane (− 78 °C), sectioned at 30 μm using Microm HM525 Cryostat (Thermo) and picked up on Superfrost Plus slides (VWR, 48311-703). Sections were blocked with 5% normal goat serum and washed in PBS with 1% bovine serum albumin (BSA) and incubated with rabbit- anti-mouse Iba-1 primary antibody (FUJIFILM Wako Pure Chemical Corporation 019-19741, 1:500) or rabbit- anti-mouse Iba-1 primary antibody Alexa Fluor^® 594 conjugate (Cell Signaling, 48934, 1:50) overnight. Sections were washed with phosphate buffer with 1% Tween 20 (PBS-T), and then incubated in goat anti-rabbit IgG (H + L) secondary antibody (Vector laboratory CY-1300, 1:250) at room temperature for 1 h when unconjugated antibody was used. Afterword, sections were washed three times with PBS-T followed by mounting on coverslip using Vectashield DAPI (4′6-diamidino-2-phenylindole 2HCl, Vector Labs, Burlingame, U.S.) mounting media to detect nuclei.

Cells were seeded in 8-well chamber slides (µ-Slide 8 well, ibidi GmbH, Gräfelfing, Germany) at a density of 1 × 10⁴ and cultivated for 24 h. They were treated with 10 µM of α-mangostin or 10 µg/mL of extract for 72 h. After treatment, cells were fixed with 4% formaldehyde for 30 min at room temperature and then blocked with 5% BSA containing 0.1% Triton-X100 (Merck, Rahway, NJ, USA) for 1 h at room temperature. Incubation with the PD-L1 antibody at 1:200 dilution was performed at 4 °C overnight. Cells were washed three times with PBS and incubated with fluorescence secondary antibody Alexa Fluor^® 594 Conjugate (#8889, Cell Signaling Technology, MA, USA) at 1:500 dilution for 2 h at room temperature in the dark. Cells were counterstained with Hoechst for visualization of nuclei and Cell Mask™ (1:500) for visualization of cell membranes. Images were captured using an Olympus fluorescence microscope (IX83, Evident Life Science, Waltham, MA, USA) equipped with CellSens imaging software, Olympus CellSens dimension 3.1.1 (Build 21264). The maximum emission and excitation wavelengths of Alexa Fluor^® 594, Hoechst, and Cell Mask^TM were 590–617, 361–497, and 522–535 nm, respectively. The fluorescence intensities of images were quantified by ImageJ software (version 1.54d) [43 (link)].

Mouse Trim59, Trim27, WASH, and control siRNAs were purchased from Ribo, Shenzheng, China. Anti- Trim59 (ab69639, Abcam), anti-Trim27 (A6405, Abclonal), anti-WASH (SAB-4200552, Sigma), anti-Oct4 (ab18976, Abcam), anti-FLAG (M20008, Abmart), anti-HA (#T501-1, Signalway Antibody), anti-Ub (YT4793, Immunoway), anti-K48 (#4289, Cell Signaling Technology), anti-K63 (BML-PW0600, Enzo), anti-ACTR2 (ab134082, Abcam), anti-MYH9 (ab138498, Abcam), anti-CAPZA1 (ab166892, Abcam), anti-CAPZB (ab175212, Abcam), anti-β-actin (SC-81178, Santa), Phalloidin-iFluor 488 (ab176753, Abcam), DAPI (#4083, Cell Signaling Technology), Alexa Fluor 488 Conjugate (#4416, #4408, Cell Signaling Technology), and Alexa Fluor 594 Conjugate (#8890, #8889, Cell Signaling Technology) antibodies were purchased.
Murine FL Trim59 clone was obtained from the ATCC. Trim59 mutants were constructed by performing PCR with four primers according to a previous method^{33 (link)}. All primers used in this study are listed in supplementary Table S2b. YFP–WASH and YFP-WASH K220R mutant were from Patrick Ryan Potts, UT Southwestern Dallas, TX 75390, USA; plasmids encoding hemagglutinin (HA)-tagged ubiquitin (HA-Ub), HA-K48-Ub, and HA-K63-Ub were obtained from Y. Xiong (University of North Carolina, Chapel Hill).

Mouse brain was fixed in 4% formaldehyde for 24 hours, and then incubated in 30% sucrose until tissues are sink. Fixed brain was flash frozen using pre-cooled isopentane (−78 °C), sectioned at 30 μm using Microm HM525 Cryostat (Thermo) and picked up on Superfrost Plus slides (VWR, 48311–703). Sections were blocked with 5% normal goat serum and washed in PBS with 1% bovine serum albumin (BSA) and incubated with rabbit- anti-mouse Iba-1 primary antibody (FUJIFILM Wako Pure Chemical Corporation 019–19741, 1:500) or rabbit- anti-mouse Iba-1 primary antibody Alexa Fluor^® 594 conjugate (Cell Signaling, 48934, 1:50) overnight. Sections were washed with phosphate buffer with 1% Tween 20 (PBS-T), and then incubated in goat anti-rabbit IgG (H+L) secondary antibody (Vector laboratory CY-1300, 1:250) at room temperature for 1 hour when unconjugated antibody was used. Afterword, sections were washed three times with PBS-T followed by mounting on coverslip using Vectashield DAPI (4′6-diamidino-2-phenylindole 2HCl, Vector Labs, Burlingame, U.S.) mounting media to detect nuclei.