Rnase free distilled water

10⁶ THP-1 cells were seeded into 6cm sterile dishes with 100nM phorbol 12-myristate 13-acetate (PMA, Sigma) for 48hrs at 37°C. Cells were stimulated with 0.5mg/mL lipopolysaccharide (LPS, E. coli O55:B5, Sigma) and incubated for the indicated time-points up to 8hrs at 37°C. Cells were washed in PBS (Invitrogen) and lysed in 500μL RLT Buffer (Qiagen) containing 1% beta-mercaptoethanol (Sigma). Total RNA from 10⁶ THP-1 cells was extracted using the RNeasy Mini Kit (Qiagen), eluted in 50μL RNase-free distilled water (Invitrogen), and treated with DNase for 1hr at 37°C using the Turbo DNA-free kit (Invitrogen). RNA concentration was measured using a NanoDrop spectrometer, and the RNA was then stored at -80°C.

Expressed proteins were confirmed by RT-PCR. Cells were extracted from the hydrogels using a glass tissue grinder and lysed using 1 mL of Trizol (Invitrogen, Carlsbad, CA, USA). Subsequently, 0.2 mL of chloroform was added to the lysed cells, and the cell debris was centrifuged at 12,000× g for 15 min at 4 °C to collect RNA from the supernatant fraction. The collected RNA was precipitated with isopropanol and spun down as described above. The RNA pellet was washed with ethanol with the same method as the above centrifugation procedure. An RNA solution was then prepared by dissolving the RNA in 40 µL of RNase-free distilled water (Invitrogen, Carlsbad, CA, USA). After determining the concentration of the resulting RNA solution, the required amount of RNA was added to a One-Step RT-PCR Kit (Enzynomics, Daejeon, Korea) tube along with expression markers such as β-actin, IL1B, Col I, Col II, MITF, NPR-A, Rhodopsin, and RPE65. Afterward, RT-PCR was performed with a TAKARA PCR Thermal Cycler Dice (TaKaRa, Kyoto, Japan). Finally, electrophoresis was performed to analyze the PCR products on a 1% agarose gel by means of Mupid-One (TaKaRa, Kyoto, Japan) for 30 min at 100 V. The electrophoresed PCR products were visualized by a UV transilluminator (MultiImage Light Cabinet, Alpha Innotech, San Leandro, CA, USA).

qPCR was performed to detect the localization of known genes (list of used primers is attached in Supplementary Table S1). PCR primers were designed using Primer3 (Untergasser et al., 2012 (link)). The expected length of qPCR products was 80–120 bp and the annealing temperature was 60°C. Geneious prime (version 2021.2) was used to increase the specificity of designed primers and to avoid targeting RNA isoforms.
qPCR reaction mix with a total volume of 7 μl contained 2 μl of cDNA, 0.29 μl of forward and reverse primers mix (1:1, 10 μM each), 3.5 μl of 2x TATAA SYBR^® GrandMaster^® Mix (TATAA Biocenter, Sweden) and 1.21 μl of RNase-free distilled water (ThermoFisher, USA). qPCR was performed using CFX384 Real-Time System (Bio-Rad, USA) as follows: the initial denaturation for 3 min at 95°C, 45 cycles of denaturation for 15 s at 95°C, annealing at 60°C for 20 s and extension at 72°C for 20 s. qPCR melting curves were analyzed to test the reaction specificity. qPCR data were analyzed using workflow published in Sindelka et al., 2008 (link).