Quantstudio 3

Total RNA was extracted from cells using a RNeasy Mini Kit (Qiagen) and treated with RNase‐free DNase (Qiagen). cDNAs were synthesized with random hexamers and Oligo(dT) with Superscript III Reverse Transcriptase (Invitrogen) and stored at −20°C until use. Real‐time PCR was performed using a FastStart Universal SYBR Green Master (Roche) on a QuantStudio™ 3 (Life Technologies). Amplification of β‐actin was also conducted to control the quantity of loaded cDNA in each reaction. Primers sequences are listed in Table S1.

Viral RNA was quantified from serum as well as tissue samples via quantitative reverse transcription-PCR (qRT-PCR) using Applied Biosystems Quant Studio 3 real-time PCR instrument (Life Technologies Corporation, Carlsbad, CA, USA). Serum was inactivated using RNAbee (Tel-Test, Friendswood, TX, USA) and RNA was isolated according to the manufacturer’s instructions. One-step qRT-PCR was performed using RNA UltraSense One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA, USA), and primers and probe specifically designed to detect a region of the EBOV glycoprotein gene [26 (link)]; this One-Step method and primers will detect all RNA corresponding to the Ebola virus GP gene (including antigenome and mRNA).

RNA was isolated by RNeasy Mini Kit (Qiagen). mRNA relative expression for DAB2, DLC1, and PMAIP was determined using SYBR green 1-step iScript (Bio-Rad) with a Life Technologies QuantStudio 3. The primers were: 5′-TTCATTGCCCGTGATGTGACA-3′ (DAB2 forward) and 5′-CCTGTTGCCCGGTTTTTATGG-3′ (DAB2 reverse); 5′-AACCCAAGACTACGGCTATTCA-3′ (DLC1 forward) and 5′-CATAAAGCTGTGCATACTGGGG-3′ (DLC1 reverse); 5′-ACCAAGCCGGATTTGCGATT-3′ (NOXA forward) and 5′-ACTTGCACTTGTTCCTCGTGG-3′ (NOXA reverse); and 5′- CATGTGCAGTACATCCATACGG-3′ (TIMP3 forward) and 5′-CATCATAGACGCGACCTGTCA-3′ (TIMP3 reverse).

Total RNA was extracted from mouse astrocytes using the TRIzol reagent (Invitrogen), and reverse transcription was conducted using an iScript cDNA synthesis kit from Bio-Rad (Hercules, CA, USA), following the manufacturer’s instructions. The qRT-PCR analysis was carried out using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Austin, TX, USA). The PCR cycle parameters were 50 °C for 2 min, followed by 95 °C for 10 min, then 40 cycles at 95 °C for 15 s; the annealing temperature was 56 °C for 1 min. The gene expression levels were determined by QuantStudio 3 from Life Technologies (Carlsbad, CA, USA). The mRNA expression of the target genes was analyzed and normalized to that of 18S, which was used as the housekeeping gene. Data analysis of fold changes in gene expression was performed using the ΔΔCt method and is presented as 2- (average ΔΔCt). The primers used were: CCL2—Fwd 5′-GTTGGCTCAGCCAGATGCA-3′, Rev 5′-AGCCTACTCATTGGGATCATCTTG-3′; IL-6—Fwd 5′-GACTTCCATCGAGTTGCCTTCT-3′, Rev 5′-TTGGGAGTGGTATCCTCTGTGA-3′; and 18S—Fwd 5′-CGGCGACGACCCATTCGAAC-3’, Rev 5′-GAATCGAACCCTGATTCCCCGTC-3′ as a housekeeping gene.

SH-SY5Y cells expressing PrP^C were incubated in Opti-MEM containing GlutaMAX and 20 μm acitretin for 48 h. DMSO only-treated cells were used for comparison with treated cells. Cells were harvested, and RNA was extracted using the RNeasy plus kit (QIAgen) according to the manufacturer's instructions. cDNA was synthesized using 1 μg of prepared RNA using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions. The mRNA expression level of ADAM10 was analyzed by real-time qPCR using the SYBR Green method (Applied Biosystems) with the sense and antisense primers reported previously (66 (link)). Samples were analyzed in triplicate on a Quantstudio 3 (Life Technologies), and relative expression was calculated using ribosomal qPCR as the control.

Following BMP1 knockdown by siRNA (25 nM, 48 h, Ambion), HepG2 cells were harvested and RNA was extracted using the RNeasy Plus Kit (QIAgen) according to the manufacturer’s instructions. cDNA was synthesised according to the manufacturer’s instructions, using 1 µg of prepared RNA using the iScript cDNA Synthesis Kit (BioRad). BMP1 mRNA expression levels were then quantified using real-time qPCR using the SYBR green method (Applied Biosystems) with the following primer sequences: forward primer 5′-ACTACATGGAGCTCTTCGACG-3′ and reverse primer 5′-TCATCCGAGTGGAACTTCACC-3′. Samples were run, in triplicate, using the Quantstudio 3 (Life Technologies) and relative expression was calculated using ribosomal mRNA expression as baseline control.