Tcs sp8 mp

The encapsulation of MF into liposomes
was verified by spectrofluorometric measurements (spectrofluorometer
Chronos DFD, ISS) and confocal microscopy (Leica TCS SP8MP, Leica
Microsystems, Germany) based on autofluorescence properties of MF
(Figures S5 and S6). The excitation and
emission spectra of MF in 96% ethanol were measured using Chronos
DFD, ISS, and Vinci software. Transition of MF 2 μM in water
into the model of lipid membrane (EPC, 38 μM) was assessed directly
(t = 0) after mixing both aqueous dispersions by
spectrofluorometric measurements. The cellular uptake of MF liposomal
(1 μM) by THP-1-XBlue-MD2-CD14 cells was observed at 24 h, and
the uptake of PEG-VIII (10 μM) with incorporated
18:1 Liss Rhod PE (0.1 mol %) by BV-2 cells was observed immediately
(t = 0), 60, 90, 120, and 240 min after treatment
using super-resolution live cell imaging (Leica TCS SP8MP, Leica Microsystems,
Germany).

Three fluorescence systems were used in the experiment: two‐photon laser confocal microscope (Leica TCS SP8‐MP, WZ, German) for presentation of subcellular structure, High‐Content Screening System (PerkinElmer Operetta, MA, USA) for providing long period live cell imaging and an inverted laser confocal microscope (Zeiss LSM 880 with Airyscan, Oberkochen, Germany) for high quality confocal image. In living cell fluorescence imaging, all miRNAs (for cell incubation or transfection) were used at 50 nm. The concentrations and incubation time of MitoTracker Green FM, LysoTracker Green DND‐26, ER‐Tracker Green, Hoechst 33342, cytochalasin B, rottlerin, dynasore, nocodazole, EIPA, Dibucaine, and Sphingosine were listed in Extended data, Table S2 of the Supporting Information. Cell transfection with small RNAs was according to manufacturer's protocol. To observe cells on upright microscope, cells were prepared on glass‐bottom dish. All cells were living when images were taken. Fluorescence images taken by Operetta High‐Content Screening System were analyzed by Columbus 2.4.1, a cellular imaging and analysis software provided by PerkinElmer. Fluorescence intensity of images taken by Leica TCS SP8‐MP was analyzed by Adobe Photoshop. The fluorescence intensity data were shown in Figure 1a, 1b, 3e, 3f, 4d, and 6a.

Gelatin-coated coverslips placed in MW6 plates were used to grow HUVECs until subconfluence. Cells were pretreated with HT-C6 for 30 min and then induced with 20 ng/mL TNF-α for 15 min in presence of the compound. After fixation in formalin solution (Sigma-Aldrich/Merck, Darmstadt, Germany), cells were permeabilized in Triton X-100 0.5%-PBS and blocked in 5% BSA-PBS. RelA/p65 mouse mAb (sc-8008; Santa Cruz Biotechnology, Dallas, TX, USA) was used for incubation during 1 h at room temperature in a wet chamber. Coverslips were washed in PBS prior to incubation with Alexa Fluor 568-linked anti-mouse IgG antibody (A11004, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Then, 3 washes in PBS were performed and 1 mg/mL Hoechst 33342 was used for 5 min incubation. Coverslips were mounted in glass slides using 1:10 (vol-vol) glycerol-water solution. Fluorescence-confocal microscope Leica TCS SP8 MP and Leica Application Suite X software version 5.1.0 (Leica Microsystems, Wetzlar, Germany) were used for sample visualization and imaging capture. Fiji software (https://imagej.net/contribute/citing, accessed on 29 June 2023) [21 (link)] was used for quantification of nuclear/cytosolic signals in at least 5 images of each experimental condition. Three independent replicates were performed for this assay.