Oligo d t 16

Human brain astrocytes were incubated with 1 μM β-lapachone for 4 hrs under co-treatment of 125 μM CoCl₂ or hypoxia (3% O₂). RT-PCR was performed and analysed as previously [12 (link)]. Total RNA was obtained from cells using the TRI Reagent® (Molecular Research Center, Cincinnati, OH, USA), according to the manufacturer*s instructions. 5 μg of total RNA was converted to first-stranded cDNA using moloney murine leukaemia virus reverse transcriptase and oligo-(dT)¹⁶ (Invitrogen). Equal amounts of cDNA were subsequently amplified by PCR in a 20-μL reaction volume containing 1× PCR buffer, 300 μM dNTPs, 10 μM specific primer for VEGF (5′-GAGAATTCGGCCTCCGAAACCATGAACTTTCTGT-3′ and 5′-GAGCATGCCCTCCTGCCCGGCTCACCGC-3′), HIF-1α (5′-AGTCGGACAGCCTCAC-3′ and 5′-TGCTGCCTGTATAGGA-3′) and GAPDH (5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′) and 1.25 U Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA). PCR products were separated on 1% agarose gels and visualized using SYBR® Safe DNA gel stain (Invitrogen) under UV transillumination.

Total RNA from PBMCs for RNASeq was used to synthesize cDNA with the SuperScript IV Reverse Transcriptase (18090010; Invitrogen) and oligo-dT₁₆ (N8080128; Invitrogen; for RT-PCR) or random hexamers (N8080127; Invitrogen; for RT-qPCR). Nested RT-PCR was performed with a single forward primer (5′-GCT​TCT​GTC​CGC​CTG​CA-3′) and two reverse primers (5′-AGC​CCC​GAT​GAA​CCC​CTA-3′ and 5′-TCT​TGT​CAC​GCT​CAG​CCC-3′). For RT-qPCR, two TaqMan probes targeting different exon junctions of the CD274 mRNA (Hs01125296_m1 and Hs01125301_m1 for the exon 1–2 and 6–7 junctions, respectively) were used. GUSB was used as an endogenous control (4310888E; Applied Biosystems). Gene expression was quantified by the ΔΔCT method, as previously described (Ogishi et al., 2021 (link)).

RAW264.7 cells were either stimulated with 100 ng/mL LPS or infected with L. donovani (MOI: 50) for 24 h as described above. RNA was extracted using a TRIzol reagent (Invitrogen). The concentration of total RNA was measured using a DU730 Life Science UV/vis spectrophotometer (Beckman Coulter, Chaska, MN, USA), and 4 µg of total RNA was used as the template for the synthesis of cDNA. A tube containing 500 ng of oligo (dT)16 and 10 nmol of dNTPs (Fisher Scientific, Loughborough, UK) with template RNA was incubated for 5 min at 65 °C. Then, 5× first-strand buffer, 200 nmol of DTT (Thermo, Waltham, MA, USA), and 200 U of M-MLV (Thermo) were added, and the tube was incubated at 37 °C for 50 min. The reaction was stopped by incubation for 15 min at 70 °C. The synthesized cDNA was used for the expression analyses of Sirpa and Actb. The designed primers are listed in Table 1 [15 (link)]. A real-time polymerase chain reaction (PCR) assay was conducted using 1 µL of reverse transcription PCR product as the template and 10 µL of SYBR Select Master Mix (Thermo) with the Applied Biosystems QuantStudio 5 Real-Time PCR System (Thermo). Data were analyzed with 2−ΔΔCt methods through normalization with Actb. The thermal cycling conditions were 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min.