Gfp expressing huvecs

The human Müller cell line MIO‐M1 was a gift from Prof. S. Yoshida (University of Kurume, Kurume, Fukuoka, Japan), and was cultured in DMEM‐low glucose (Sigma) supplemented with 10% FBS (Gibco; Waltham, MA, USA) and 1% penicillin‐streptomycin (Gibco). GFP‐expressing HUVECs (Angio‐Proteomie; Boston, MA, USA) were cultured in EGM medium (Lonza; Basel, Switzerland). HBVPs (Sciencell; Carlsbad, CA, USA) were cultured in pericyte media (Sciencell). All cell lines were cultured as recommended by the manufacturer and incubated at 37 °C with 5% CO₂. For knockdown experiments, siRNAs were transfected using G‐fectin (Genolution; Seoul, Republic of Korea) as instructed by the supplier. siRNAs targeting TRAP1, HIF1α, calpain‐1, calpain‐2, and CypD were synthesized by Genolution as follows:

Species	Name	Sequence (5′→3′)
Mouse	siTRAP1‐#1	AAACATGAGTTCCAGGCAGAG
Mouse	siTRAP1‐#2	GCCCGTTCTCTGTACTCAGAA
Mouse	siHIF1α‐#1	GGGTTATGAGCCGGAAGAACT
Mouse	siHIF1α‐#2	GATGGAAGCACTAGACAAAGT
Human	siTRAP1‐#1	AAACATGAGTTCCAGGCCGAG
Human	siTRAP1‐#2	CCCGGTCCCTGTACTCAGAAA
Human	siCalpain‐1	GGAACAACGTGGACCCATA
Human	siCalpain‐2	CTATTGGCTTCGCGGTCTA
Human	siCypD‐#1	GGACTCTAATACCTGTTTA
Human	siCypD‐#2	GGCAGATGTCGTCCCAAAG
Human	siANG2	GTGACTGCCACGGTGAATAAT
Human	siHIF1α	GGCCACATTCACGTATATGAT

John Wiley & Sons, Ltd.

HUVECs (Lonza, Switzerland) were fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS and subsequently permeabilized with 0.3% Triton-X (Sigma-Aldrich) in PBS. Next, 15 µL/mL of both ActinRed (Thermo Fisher Scientific) and NucBlue (Thermo Fisher Scientific) was added to the Triton-X solution to visualize the F-actin filaments and nuclei, respectively. Images were captured using a Leica DMI 6000 m microscope (Leica Microsystems, Germany) with a pE300^ultra LED illumination system (CoolLED, United Kingdom) and an RGB filter cube.
Live-cell images of the GFP-expressing HUVECs (Angio-Proteomie, USA) were taken with an EVOS FL cell imaging system using the GFP filter cube. The brightness and contrast of all images were adjusted using ImageJ.

In preparation for cell seeding, HUVECs (Lonza, Switzerland) or GFP-expressing HUVECs (Angio-Proteomie, USA) were cultured in collagen I-coated T75 flasks (CELLCOAT®, Greiner Bio-One) until reaching approximately 80% confluency. These cells were then trypsinized, centrifuged, and resuspended in endothelial growth medium (EGM) (Cell Applications, Inc., CA, USA) containing 25 mM hydroxyethyl piperazine-ethanesulfonic acid (HEPES). The cell suspension was filtered through a 40-µm pore-size filter (BD Falcon^™) and then seeded in the previously prepared mLSI MFBB.