Cells, Strains, Antibodies, and Reagents
The MBMEC cell line bEND.3 was acquired from ATCC (Manassas, VA, USA) and cultured under conditions of 37°C and 5% carbon dioxide, growing in Dulbecco’s Modified Eagle Medium (DMEM). The mutant strain mpr1Δ of C. neoformans was acquired from ATCC, the C. neoformans grubii wild type (WT) H99 strain originated from our laboratory. Both strains were cultured at 30°C in YPD agar medium (1% yeast extract, 2% peptone, 2% glucose, and 2% agar).
The antibodies and reagents used in this study are as follows. Rabbit polyclonal anti-EGFR antibody, rabbit polyclonal anti-HB-EGF antibody, and rabbit polyclonal anti-p-EGFR antibody were obtained from Abcam (Cambridge, UK). Rabbit polyclonal anti-GAPDH antibody was acquired from Proteintech Group (Rosemont, IL, USA). The inhibitor TAPI-1 was procured from Peptides International (Louisville, KY, USA). CY3-conjugated secondary antibody was procured from Proteintech.
Transcytosis Assays
MBMECs were seeded into Transwell inserts (Corning, NY, USA) and cultured until confluence was achieved. The integrity of the BBB model was assessed by measuring the transendothelial electrical resistance (TEER) using a Millicell-ERS device (Millipore, Inc., Billerica, MA, USA). C. neoformans at a concentration of 4 x 10^5 was added to the upper chamber (blood side) of the insert. After 12 hours, 100 μL of the culture was collected from the lower chamber (brain side) and plated on YPD agar to determine the CFU of C. neoformans that had traversed the MBMECs. An equivalent volume of fresh complete culture medium was added to the lower chamber. Each experiment was conducted at least three times.
siRNA-Based Gene Knockdown
For the purpose of achieving EGFR knockdown, cells were initially seeded at a concentration of 5x10^4 cells per well within 6-well plates and allowed to incubate for a duration of 16 hours in preparation for transfection. Subsequent to a period of 12 hours post-seeding, the medium utilized for transfection was supplanted with a fresh culture medium to support optimal cell conditions. Transfection of mouse EGFR siRNA, procured from Santa Cruz (Dallas, TX, USA), into the cells was executed employing the Lipofectamine RNAiMAX delivery system (Invitrogen), strictly following the guidelines provided by the manufacturer for all siRNA applications.
Real-time PCR Analysis
Total RNA (10-100 ng, depending on the abundance of the target gene) was extracted using TRIzol reagent (Invitrogen, Inc., Waltham, MA, USA) for RT-qPCR, utilizing the 7900 Real-Time PCR System (Applied Biosystems) with a one-step reaction involving the reverse transcription reagent kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and SYBR green PCR master mix (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The primers used for RT-PCR are listed in Table 1. The equations should be inserted in editable format from the equation editor.
Western-Blotting
Cells were lysed using RIPA lysis buffer supplemented with PMSF. Membrane and cytoplasmic proteins were extracted using the Cell Membrane and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Protein concentration was determined using the BCA Protein Assay Kit (Keygen Biotech, Nanjing, China). Density measurement analysis of the protein bands of interest was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
ELISA
Initially, cells were seeded at a concentration of 2 x 10^4 cells per well within a 96-well plate, followed by an overnight incubation period to achieve a confluency level of approximately 70%-80%. Subsequently, centrifugation at 1000g was employed to facilitate the removal of particulate matter. The assay was then conducted using a mouse HB-EGF ELISA kit (Jianglai Biotechnology, Shanghai, China), adhering strictly to the protocol outlined in the accompanying documentation.
Immunofluorescence
Cells seeded on slides were thrice washed with PBS, fixed in 4% paraformaldehyde for 10 min at room temperature. Endogenous peroxidases were inactivated by incubating with 3% H2O2 in PBS for 10 min, followed by three 5-min PBS washes. Non-specific binding sites were blocked with 10% FBS for 15 min at room temperature. The slides were then incubated with an anti-EGFR rabbit antibody at a 1:250 dilution overnight at 4°C. This was followed by three washes with PBS, each for 5 min, facilitated by dispensing 2 mL PBS per wash onto the slide surface with a 1 mL pipette. CY3-conjugated secondary antibody (1:100 dilution) was applied, one drop per slide, and incubated for 30 min at 37°C. After three additional 5 min PBS washes, slides were incubated with Hoechst stain in the dark at room temperature for 15 min before fluorescence microscopy examination.
Statistical analysis
Data are reported as mean ± standard deviation (SD) and were analyzed using IBM SPSS Statistics 25.0 (IBM Corp., Armonk, NY, USA). Differences between treatment and control groups were assessed using analysis of variance (ANOVA) and Student’s t-test, with a P value of <0.05 denoting statistical significance.