Caveolae

In order to examine correlations between MYOCD/MKL1/MKL2 and caveolae genes in different tissues, we used expression data available from the Genotype-Tissue Expression (GTEx) project (http://www.gtexportal.org/) [50 (link)]. Across-samples normalization for each tissue was performed using the trimmed mean of M-values (TMM) normalization method [51 (link)]. Correlations were calculated with the non-parametric Spearman method using R language for statistical computations [52 ]. Correlation matrices for each tissue were produced by R package "Hmisc".

This study was conducted in a facility approved by the American Association for the Accreditation of Laboratory Animal Care. All procedures were performed in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Our protocol was approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch (UTMB). Primary cortical neuronal cultures were prepared and maintained as described previously^{21 (link)}. Briefly, cortical neurons were isolated from C57BL/6 mice (Jackson Laboratory; 000664) during embryonic days 16–18 using Accutase solution (Sigma; A6964) together with gentle trituration by a fire-polished glass pasture pipet. Dissociated cells were plated at a density of 2 × 10⁵ cells/ml in a 24-well plate containing high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Corning; 10–013-CV) with 2% B-27 Plus supplement (Gibco; A3582801), 10,000 U/ml penicillin, 10,000 μg/ml streptomycin, and 25 μg/ml amphotericin B (Gibco; 15240062). After 2 h, plating medium was removed from cells and replenished with neurobasal medium (Gibco; 12348017) plus 2% B-27 Plus, 0.5 mM GlutaMax (Gibco; 35050-061), 10,000 U/ml penicillin, 10,000 μg/ml streptomycin, and 25 μg/ml amphotericin B supplement. In all, 50% of medium changes were performed every 3–5 days. Cells on days 10–13 in vitro (DIV) were used for all experiments.
For cell treatment, neurons cultured in a 24-well plate were exposed to pharmacological inhibitors for 30 min, being either dynamin inhibitor (Dynasore (Sigma, D7693) or Pitstop2 (Abcam, ab120687)), HSPG inhibitor (Heparin (Sigma, H4784)), or caveolae inhibitor (Nystatin (Sigma, N6261)). AF568- or AF488-tagged TauO exposure were further performed for 1–24 h. Concentrations of inhibitors and TauO and incubation time were mentioned in appropriate methods and figure legends. DMSO (0.02% (v/v)) was the vehicle control in all experiments.

We loaded OVA-FITC into silica nanoparticles and measured the average intracellular fluorescence intensity by FACS analysis to quantitatively detect the uptake of different silica nanoparticles. RAW264.7 cells (3.0 × 10⁵ cells) were precultured in 12-well plates for 12 h and then incubated with free OVA-FITC plus IMQ, OVA-FITC-IMQ-VMSNs/MSNs (25 μg/mL OVA-FITC, 5 μg/mL IMQ) for 15 min, 30 min, 1 h, 2 h, 4 h and 8 h. After that, the cells were washed with PBS for more than 3 times to terminate ingestion and measured average intracellular fluorescence intensity and OVA-FITC positive RAW264.7 cells by FACS analysis. Absorption of VMSNs by cells 4 h after incubation were observed using CLSM under the same conditions.
With the aim of investigating the behavior of internalized silica nanoparticles, we used CLSM to observe the location of silica nanoparticles. RAW264.7 cells were incubated with free OVA-FITC plus IMQ, OVA-FITC-IMQ-VMSNs/MSNs (25 μg/mL OVA-FITC, 5 μg/mL IMQ) for 12 h after well plate growth. To determine the degree of co-localization of OVA and lysosomes, the images were treated with a CLSM after the cell nucleus was labeled by Hoechst and lysosomes stained by Lyso-Tracker Red.
To further investigate the cell uptake mechanism, RAW264.7 cells (3.0 × 10⁵ cells) were incubated in 24-well plates for 12 h. Before adding silica nanoparticles, the cells were incubated with the corresponding mechanism inhibitors for 1h, that is, chlorpromazine (CPZ, 20 μg/mL) was used to inhibit clathrin-mediated endocytosis, genistein (Geni, 2 μg/mL) was employed to suppress the caveolae-mediated endocytosis, and amiloride (Amil, 15 μg/mL) was served as the macropinocytosis inhibitor. The cells were then cultured with the OVA-IMQ-VMSNs/MSNs corresponding inhibitors for 6 h. Results were expressed as percentage uptake in the control group incubated with OVQ-IMQ-VMSNs/MSNs without inhibitors.

TEM was employed to observe the morphology of the EVs and the rat brain microvessel microstructure. Briefly, for EVs samples, 20 μl of EVs suspension was dropped onto the 150 meshes carbon filmed copper grid for 5 min. Thereafter, 2% phosphotungstic acid was dropped on the copper grid to stain for 2 min. The samples were observed under TEM (HT7800, Hitachi) at the 40 k × magnification. For rat microvessel, 1 mm³ rat cortex brain tissues were harvested and fixed in 2.5% glutaraldehyde solution and 1% OsO₄. Afterward, samples were dehydrated by using 30%-95% ethanol, and then subjected to resin penetration and embedding. The embedded samples were cut into 60 nm section by using the ultra-microtome (Leica UC7, Leica). Tissue samples were fished out onto the 150 meshes formvar filmed cuprum grids and stained by 2% uranium acetate saturated alcohol solution for 8 min. After rinsed in 70% ethanol, and ultra-pure water for 3 times, respectively, samples were then stained by 2.6% lead citrate for 8 min, followed by rinsed in ultra-pure water 3 times. Sections were dried overnight and observed under TEM with the magnification of 2 k × or 20 k × . ImageJ software was used to quantify the caveolae structure density.

A549 cells were seeded in six well plates at a density of 2 × 10⁵ cells/well in 3 mL of CCM, supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C. At 24 h post-seeding, the cells were pre-treated with either 10 µg/mL of chlorpromazine (clathrin-coated pit inhibitor), 5 µg/mL of nystatin (caveolae-mediated inhibitor), 2.5 µM of methyl-β-cyclodextrin (MβCD) (caveolae-mediated/cholesterol inhibitor), and 10 µg/mL of wortmannin (macropinocytosis inhibitor) for 1 h. This was followed by treatment with 30 µg/mL of different sizes of AuNPs dispersed in CCM for 3 h. After exposure, the cells were washed three times with PBS, harvested, and transferred to a glass vial. The cells were digested with 3 mL of aqua regia prepared with 37% HCL and 70% HNO₃. Then, 1 mL of hydrogen peroxide was added to the cell suspension. The glass vials were properly closed and heated at 120 °C for 30 min. The samples were diluted to a final acid concentration of 2% HNO₃ and filtered with a 0.22 µM syringe filter (Sigma Aldrich, UK) to ensure the removal of any debris. The total Au concentration in the samples was measured using the same procedure as described in Section 2.5.1.