A1933

Cells were plated at a density of 1 × 10⁵ cells/well in 24-well plates and then grown on glass coverslips for 12 h and infected with S. oralis, followed by staining with 5 mM SYTO® Green-Fluorescent Nucleic Acid Stains (Thermo Fisher, USA) for 30 min. After gentle washing for three times with PBS, the cells were fixed for 10 min in 4% paraformaldehyde (wt/vol). The cells were pre-blocked with 5% BSA (A1933; Sigma–Aldrich), labeled with TRITC Phalloidin (Yeasen, 1 μg/mL), and incubated with anti-rabbit Alexa Fluor 594 (Invitrogen) secondary antibodies (1:300) for 1 h at room temperature. The cell nuclei were stained with by DAPI dihydrochloride staining solution. Images were collected using a confocal microscope (SP2-AOBS Leica Microsystems).

The HL-1 cardiomyocytes were rinsed with phosphate-buffered saline (PBS, 806552, Sigma-Aldrich) and fixed in 4% paraformaldehyde (P6148, Sigma-Aldrich) at room temperature for 30 min. After washing three times with prechilled PBS, the HL-1 cardiomyocytes were permeabilized with 0.15% Triton X-100 (T8787, Sigma–Aldrich) in PBS for 15 min and blocked with 1% bovine serum albumin (BSA, A1933, Sigma–Aldrich) for 30 min at room temperature. For cytoplasmic labeling, HL-1 cardiomyocytes were incubated with a primary antibody against α-actin (A3853, Sigma-Aldrich) at a dilution of 1:1000 at 4 °C overnight and washed with PBS three times to remove the remaining antibodies. Then, the cardiomyocytes were incubated with Cy3-labeled secondary goat anti-mouse IgG antibodies (AP130C, Sigma-Aldrich) for 2 h at room temperature without light. The cardiomyocytes were washed with PBS three times again to remove the unbound fluorescent probes. For nuclear labeling, the cardiomyocytes were incubated with 4′,6-diamidino-2-phenylindole (DAPI, D9542, Sigma-Aldrich) for 10 min at room temperature and washed with PBS to remove the unbound DAPI. Fluorescence images were captured by a laser confocal fluorescence microscope (Nikon A1HD25, Japan).

All operations in this section were performed at low temperature. To evaluate PMN and MN populations, processed CSF cells or PBLs were stained with 1 μg/ml Hoechst 33342 for 5 min, then fixed in PBS containing 1% paraformaldehyde, and checked through an Influx flow cytometer (BD). For cell immunostaining, processed CSF cells or PBLs were blocked for 15 min in PBS containing 0.5% bovine serum albumin (Sigma-Aldrich, A1933) and 3% FBS and then incubated for 30 min with anti-VSIG4 (4 μg/ml, Santa Cruz, sc-53977), anti-CD1C (2 μg/ml, NOVUS, NBP2-62220), or equal concentrations of isotype-matched control antibodies. After washing three times with PBS containing 3% FBS, the suspension was incubated with 2 μg/ml Alexafluor 488-conjugated goat anti-mouse antibody (4408S, Cell Signaling) for 30 min in PBS containing 0.5% bovine serum albumin and 10% goat serum (B900780, Proteintech) and then incubated for another 5 min with 1 μg/ml Hoechst 33342. The cells were washed three times and fixed in PBS containing 1% paraformaldehyde. Cellular fluorescence was measured using an Influx flow cytometer, and data were analyzed using FlowJo software.

Cells growing on different substrates were fixed with 4% paraformaldehyde for 10 min at room temperature and incubated with 0.1% Triton X‐100 in PBS for 3–5 min. After blocking with blocking solution containing 3% bovine serum albumin (BSA; A1933; Sigma) for 45 min, cells were incubated with antibodies (YAP1, H00010413‐M01, Novus, Littleton, CO, USA; pFAK, #8556; Myo‐II, #3403; Plakoglobin, #2930; β‐Tubulin, #2128; and CD44, #3570; all from Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Then, cells were incubated with secondary antibody (A32740 or A32742; Invitrogen) or phalloidin (O7466; Invitrogen) dissolved in PBS containing 1% BSA for 2 h. After washing three times with PBS for 5 min, cells were incubated with Hoechst 33342 (62249; Thermo Scientific) for 15 min at room temperature, washed again three times with PBS for 5 min, and imaged using a fluorescence microscope (Axio Observer; Zeiss) or confocal microscope (Ti‐E; Nikon, Tokyo, Japan).
For cross‐linked collagen substrates, cells were blocked with 5% BSA for 1 h, co‐incubated with primary and secondary antibodies against collagen I (ab6308; Abcam, Cambridge, UK), and analyzed using fluorescent immunoassays with the Axio Observer instrument.

Sphere formation assay was carried out as reported previously [24 (link)]. Concisely, transfected MDA-MB-231 or MCF-7 cells were inoculated in ultra-low attachment 6-well plates (Corning, NY, USA; 5 × 10³ cells per well) for 10–14 days. Cells were maintained in DMEM/F12 serum-free medium (PM150312A, Procell) added with 5 μg/mL insulin (Sigma-Aldrich), 0.4% bovine serum albumin (BSA; A1933, Sigma-Aldrich), 2% B27 (MAB1285, Sigma-Aldrich), 20 ng/mL basic fibroblast growth factor (bFGF; PHG0369, Thermo Fisher Scientific), and 20 ng/mL EGF (Peprotech, Rocky Hill, NJ, USA). Generated spheres were photographed and then counted using a light microscope (Zeiss, Oberkochen, Germany). Sphere formation efficiency is calculated as (number of spheres/number of inoculated cells) × 100%, using MShot Image Analysis System. Only spheres with diameters greater than 75 μm were counted.

Immunostaining of the spermatozoa was performed as described previously (Sha, Xu, et al., 2017). Briefly, the prepared spermatozoa were smeared onto poly‐L‐lysine coated slides, allowed to air‐dry, washed in phosphate‐buffered saline (PBS), fixed in 4% PFA (F8775, Sigma, United States) for 10 min at RT, and washed twice in PBS, followed by permeabilization with 0.2% Triton X‐100 (93443, Sigma, USA). Then, the samples were blocked with PBS containing 1% bovine serum albumin (A1933, Sigma, USA) and 2% normal goat serum (NS02L, Millipore, USA) for 30 min at RT. Slides were incubated with rabbit anti‐EIF4G1 (15704‐1‐AP, Proteintech, USA), rabbit anti‐COXIV (11242‐1‐AP, Proteintech, USA), or rabbit anti‐ATP6 (55313‐1‐AP, Proteintech, USA) primary antibodies and costained with anti‐acetylated tubulin (66200‐1‐Ig, Proteintech, USA) primary antibodies 1 hr at RT, followed by incubation with Alexa Fluor^® 594‐conjugated goat anti‐rabbit IgG (ZF‐0516, zsbio, China) and Alexa Fluor^® 488‐conjugated goat anti‐mouse IgG (ZF‐0512, zsbio, China) secondary antibodies for 45 min at RT. Slides were subsequently washed three times in PBS, mounted with Vectashield containing DAPI (Vector Laboratories) and examined under a laser scanning confocal immunofluorescence microscope (LSM510 Exiter, Carl Zeiss, Germany). The specific antibodies used in this assay are listed in Table S2.

A total of 10³ CD133⁺ cells were isolated from NSCLC tissues and the lung cancer cell line A549 and cultured at 37°C with 5% CO₂ in serum-free DMEM/F12 containing 100 U/mL penicillin and 100 μg/mL streptomycin, 20 ng/mL EGF (PHG0311, Gibco), 10 ng/mL bFGF (13256-029, Gibco), 5 μg/mL insulin (HY-P1156, MedChemExpress, Monmouth Junction, NJ), 0.4% bovine serum albumin (BSA) (A1933, Sigma-Aldrich Chemical Company, St. Louis, MO), and 2% B27 (17504044, Gibco). After 10 days of culturing, the sphere number was counted under an inverted microscope (Olympus, Tokyo, Japan, IX73).