Transit lt1

HepG2-hNTCP-C4 cells were transfected with a reporter plasmid carrying a tandem repeat of the peroxisome proliferator-activated receptor (PPAR) response elements (PPREs) upstream of the firefly luciferase gene, together with expression plasmids encoding PPARγ and retinoid X receptor (RXR) α, using TransIT-LT1 (Takara Bio Inc.) according to the manufacturer's protocol. The cells were treated with the indicated compounds for 24 h and the luciferase activity was measured as described (Watashi et al., 2013 (link)) to assess the transcriptional activity of PPARγ.

B16 mouse melanoma cells [35] (link) were transfected with the pcDNA3-mHEL plasmid by lipofection using Trans IT-LT1 (Takara), and cultured with G418 (2 mg/ml, Wako). Drug-resistant stable clones (B16-mHEL) were subsequently selected. B16 cells were retrovirally transduced with the pMX-mHEL-IRES-GFP, and then cloned by limiting dilution method to establish B16-mHEL-GFP cells. 40 LB, Balb/c 3T3 fibroblasts expressing exogenous CD40-ligand and BAFF, have been described previously [17] (link). 40 LB cells were transduced with the pMX-mHEL-IRES-GFP vector, and a single clone expressing mHEL and eGFP, termed 40 LB-mHEL, was selected by limiting dilution. To express FasL, 40 LB cells were first transduced with the pSIREN-RetroQ-shFas vector. The resultant Fas-knocked-down cells (40 LB-Fas⁻) were then transduced with the pMX-FasL-IRES-hCD8 vector and a single clone expressing FasL and hCD8 (40 LB-FasL cells) was selected by limiting dilution. Finally, the 40 LB-FasL cells were transduced with the pMX-mHEL-IRES-GFP vector to obtain a single clone expressing mHEL and eGFP (40 LB-mHEL-FasL). B16 and 40 LB cells, and their derivatives, were maintained in D-MEM medium (high glucose; Wako) supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (GIBCO) in a humidified atmosphere at 37°C with 5% CO₂.

A pseudovirus assay was performed as previously described (29 (link)
– (link)
32 (link), 46 (link), 49 (link)
– (link)
57 (link)). Briefly, HIV-1-based, luciferase-expressing reporter viruses were pseudotyped with the S proteins of B236, B52, and their derivatives. HEK293T cells (3,000,000 cells) were cotransfected with 4 µg psPAX2-IN/HiBiT (58 (link)), 4 µg pWPI-Luc2 (58 (link)), and 2 µg plasmids expressing parental S or its derivatives using PEI Max (Polysciences, Cat# 24765-1) according to the manufacturer’s protocol. Two days post transfection, the culture supernatants were harvested, and the pseudoviruses were stored at −80°C until use. For pseudovirus infection, the amount of input virus was normalized to the HiBiT value measured by the Nano Glo HiBiT lytic detection system (Promega, Cat# N3040), which indicates the amount of p24 HIV-1 antigen. For target cells, the HOS-TMPRSS2 cells stably expressing a variety of Rhinolophus bat ACE2 (Fig. 1D) and the HEK293 cells transfected with the plasmids expressing R. pusillus and R. macrotis ACE2 and their derivatives with TransIT-LT1 (Takara, Cat# MIR2300) (Fig. 3B and D) were used. Two days post infection, the infected cells were lysed with a Bright-Glo Luciferase Assay System (Promega, Cat# E2620), and the luminescent signal was measured using a GloMax Explorer Multimode Microplate Reader (Promega).

Transient transfection of HEK 293 cells was performed with TransIT-LT1 (Takara). For transfection, 14 μg of expression plasmid (3MST/pCI)9 (link) was mixed with 45 μL of TransIT-LT1 in 1.5 mL of Opti-MEM (Invitrogen) and then added to HEK 293 cells at 60–70% confluency in a 9 cm dish. Cells were harvested at 48 h post-transfection and washed with ice-cold PBS. After precipitation by centrifugation, cell pellets were resuspended in the ice-cold buffer and sonicated.