A1 confocal system

The toxicities of selected enhancers were evaluated on two widely used non-malignant cell lines: HaCaT spontaneously immortalized human keratinocytes and 3T3 Swiss albino mouse embryonic fibroblasts. The cell lines were cultivated as described previously^{13 (link),14 (link)}. For toxicity studies and confocal microscopy, cells were seeded at a density of 10,000 cells per well into 96-well plates and 100,000 cells per well into 4-well cell-imaging slides. Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake assay after 48 h incubation with the enhancer^{13 (link),14 (link)}, see Supporting Information for details.
Morphological changes were evaluated using laser scanning confocal microscopy using a Nikon A1 + confocal system after 48 h incubation of the cells with enhancers using concentrations of compounds corresponding to their TC₁₅ and TC₈₅ as described before^{13 (link),14 (link)}. Staining for actin and tubulin cytoskeleton was performed for 90 min using 5 U/mL Alexa Fluor 555 phalloidin and 2 µg/mL α-tubulin antibody and Alexa Fluor 488 conjugate, respectively (Supporting Information).

Digital images of fluorescent labeling were collected using Nikon A1 confocal system (Japan) equipped with Nikon 90i upright fluorescence microscope and BioRad Laser Sharp 2000 imaging program (Digital BioRad Center, Pleasanton, CA, USA). Alexa Fluor 568 labeling was viewed through 561 excitation laser line with 10 nm resolution of spectra. Alexa Fluor 488 and ByLight 405 were viewed through 488 and 405 excitation laser line, also through 10 nm resolution spectra. Confocal images were captured with ×20 and ×40 objective at iris of 2.0–2.5 in box size of 1,024×1,024. All Z-scan was set up at 1 μm layer of laser scan step and saved as “avi” video files and the clearest double or triple labeling image was selected and converted to Photoshop 7.0.1 (Adobe, CA, USA) through Bio-Rad Plug-In software and stored as “tiff” file at 1,024×1,024 pixels. The electron microscopy microphotographs presented here were taken with FEI Tecnai G² Spirit electron microscope.

Immunofluorescence staining was performed as previously described (Sun et al., 2017 (link)). Briefly, anesthetized mice were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Lumbar spinal cords (L3–L5) and DRGs were quickly removed and post-fixed in PFA overnight at 4°C. Then the tissues were transferred to 0.1 M phosphate buffer containing 30% sucrose at 4°C. The 40-μm-thick sections prepared by cryostat were washed 3 times and then blocked in 5% goat serum containing 0.3% Triton X-100 for 1 h at room temperature (RT). After incubation in the primary antibody overnight at 4°C, sections were incubated with the secondary antibodies at RT for 2 h and then were counterstained with DAPI. For primary antibodies, we used rabbit anti-CGRP (1:500, ImmunoStar, United States), Isolectin IB₄ Conjugate (1:500, Invitrogen, United States), mouse anti-PKC-γ (1:200, Santa Cruz Biotechnology, United States), mouse mouse anti-PV (1:2000, Sigma-Aldrich, United States), rabbit anti-VGAT (1:500, Millipore, United States); For secondary antibodies, we used Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse (1:500, Abways Technology, China). The Fluorescence images were captured using a laser scanning confocal microscope (Nikon A1 Confocal System, Japan).

Macrophages were infected as described above with Brucella–GFP (MOI of 100:1) and the bacteria number was assessed in cells infected for 24 h, as previously described [47 (link)]. Cells were washed twice with PBS and fixed in 4% paraformaldehyde (pH 7.4) at room temperature for 30 min. After fixation, coverslips were washed three times with PBS. Coverslips were mounted in slides using ProLong Gold with DAPI mounting medium (Invitrogen, Carlsbad, California, USA). Confocal microscopy analysis was performed in a Nikon A1 confocal system. Three coverslips were analyzed per sample and images were taken using a 10× objective for six random areas of each coverslip. Images presented here are representative.

MDA-MB-157 and BT-549 cells were stained with 2 μM Calcein AM before harvesting the cells for spheroid formation. An ATPS technology was used to form spheroids [21 (link)]. Spheroids were suspended in an ice-cold 4 mg/ml solution of type I rat tail collagen (Corning) and then incubated at 37 °C for 30 min to allow collagen gelation. Invasion of the spheroids in collagen matrix was captured using fluorescent confocal microscopy (Nikon A1 confocal system) at a 10X magnification. FITC filter was used to capture images with a z-spacing of 20 μm. NIS Elements software was used for image acquisition. Z-projected images were reconstructed by collapsing the stacks using ImageJ (NIH) and total area of pixels was found using color thresholding of the resulting images.