Hochest 33342

The treated cells were incubated in medium C for 2 h at 37 °C. We collected the cells and detected the MFI of the cells by FCM (BD Biosciences, Franklin Lakes, NJ, USA).
The treated cells on the confocal petri dish were reincubated in medium C for 2 h at 37 °C. We fixed the cells by 4% paraformaldehyde and stained nuclei with Hochest 33342 (Thermo Fisher Scientific, Waltham, MA, USA). Then, they were imaged with an LSCM (Nikon A1, Tokyo, Japan).

60–70% confluent LX2 cells and PA induced HepG2 cells were washed with PBS twice and incubated with 100 nM MitoTracker Green FM (YeSan, China) at 37 °C for 30 min For fluorescence intensity assay, cells were harvested by using trypsin/EDTA (ThermoFisher Scientific, USA) and resuspended in PBS. The excitation and emission wavelengths were 490 and 516 nm, respectively, and the intensity values corrected for total protein level (mg ml⁻¹). For mitochondrial visualized assay, after incubated with MitoTracker Green FM, the adherent cells were washed with PBS and a single in-focus optical section was acquired with confocal microscopy (Olympus, Japan). The nucleus was stained with Hochest33342 (ThermoFisher Scientific, USA). For every section n = 50 cells, images were analyzed and quantified by ImageJ.

GFP or 3×Flag tagged Cas9 constructs were transfected into HEK293T Standard immunofluorescence staining protocol was followed to label 3×Flag epitopes. Images were acquired and quantitatively analyzed using Operetta High Content Screening system (PerkinElmer) after cells being fixed by 4% (w/v) paraformaldehyde and stained with Hochest 33342 (Thermo Fisher).

Paraffin sections (5 µm) of the lung were used to perform the immunohistochemical assay. After deparaffinization and rehydration, endogenous peroxidase activity and nonspecific staining were blocked with 3% H₂O₂ and 5% sheep serum, respectively. Then the sections were incubated with F4/80 or CD68 primary antibody at 4 °C overnight. Afterward, sections were incubated with poly peroxidase-anti-mouse IgG antibody for 20 min at room temperature and counterstained with hematoxylin for 2 min. The sections were viewed and photographed under a light microscope. The percentage of the immunolabeled area within the total area were measured by using ImageJ software as previous described (Li et al., 2019 (link)).
For immunofluorescence, fixed and permeabilized RAW264.7 were incubated with mouse anti-F4/80 antibody and rabbit anti-iNOS antibody followed by an Alexa Fluor 488-labeled goat anti-mouse IgG antibody (Jackson ImmunoResearch) and by an Alexa Fluor 594-labeled goat anti-rabbit IgG antibody (Jackson ImmunoResearch), respectively. The nuclei were stained by Hochest 33342 (Thermo Fisher). All sections were photographed in five random visual fields. The F4/80⁺ positive cells represented the total macrophages and the F4/80⁺iNOS⁺ represented the M1 phenotype macrophages.

Doxorubicin, 3-(trimethoxysilyl) propyl methacrylate (TMSPM), and magnesium chloride (MgCl₂) solution (1.0 M) were obtained from Sigma Aldrich (Louis, MO). Dulbecco’s phosphate buffered saline (DPBS), SYBR-Safe, Calcein AM, and Hochest 33342 were purchased from Invitrogen (Carlsbad, CA). RPMI-1640 was purchased from ATCC (Manassas, VA). Fetal bovine serum (FBS) and the penicillin/streptomycin solution were purchased from Hyclone (Logan, UT). The CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) kit was from Promega (Madison, WI). Nucleic acid oligonucleotides (Table 1 in supporting information) were synthesized by Integrated DNA Technologies (Coralville, IA). Other chemicals were purchased from Fisher Scientific (Suwanee, GA).

The Colloidal Blue Staining Kit, Hochest 33342 and LipidTOX Red were from Invitrogen. Sodium oleate, sodium palmitate, celecoxib and indomethacin were obtained from Sigma-Aldrich. Western lightning plus-ECL reagent was from PerkinElmer. The synthesized qPCR primers used (Tsingke Technologies Inc., Beijing) are listed in Table S4. Antibodies used in this study are listed in Table S5.

HepG2 cells were grown at low density on glass coverslips. Cells were infected with TFV at an MOI of 10. At 72 h postinfection, cells were fixed for 15 min with 4% formaldehyde in PBS at room temperature. Nonspecific binding was blocked by 30 min incubation in 5% normal goat serum. TFV MCP and caveolin-1 were detected by simultaneously staining cells with specific antibodies diluted in PBS. Cells were then washed, and antibody binding was detected using host-specific Alex Fluor-conjugated secondary IgGs (Invitrogen, Carlsbad, CA, USA) as described previously. The coverslips were then washed several times with phosphate-buffered saline with Tween 20 and incubated with Hochest 33342 (Invitrogen, Carlsbad, CA, USA). The samples were examined under a confocal microscope (Zeiss LSM510, Germany). Mock-infected cells were similarly stained as controls.