Specific primers

Total RNA was purified from cells using RNeasy Mini Kit (20–74104, Qiagen) and reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (4368814, Thermo Fisher Scientific) with RNasin Plus RNase Inhibitor (N2615, Promega). The cDNA from each sample was applied in 20 μl reaction mix using Power SYBR Green PCR Master Mix (AB-4367659, Thermo Fisher Scientific) with specific primers (Sigma-Aldrich). Primer sequences were as follows: human MANF, 5’-GGG CGA CTG CGA AGT TTG TAT-3’, 5’- GTG CTC AGG TCG ATC TGC TT-3’; human PD-L1, 5’- TAT GGT GGT GCC GAC TAC AA-3’, 5’-TGG CTC CCA GAA TTA CCA AG-3’; human SERCA, 5’- CAT GAC AAC CCA CTG AGA AGA GAA-3’, 5’- CGA AGG TCA GAT TGG TCT CATATTT-3’; human GAPDH,5’-TGCACCACCAACTGCTTAGC-3’, 5’-GGCATGGACTGTGGTCATGAG-3’; Murine MANF, 5’- CCACCATATCCCTGTGGAAA-3’, 5’- CGTCCAGGATCTTCTTAGC-3’; Murine PD-L1, 5’-GCA TTA TAT TCA CAG CCT GC-3’, 5’-CCC TTC AAA AGC TGG TCC TT-3’; Murine GAPDH, 5’-TAT GTC GTG GAG TCT ACT GGT-3’, 5’-GAG TTG TCA TAT TTC TCG T-3’.

Total RNA from hemi-hippocampi homogenates was extracted by using the NZY total RNA isolation kit (NZYTech, Lisboa, Portugal) following the company’s datasheet. The total RNA was measured by Nanodrop 1 000 spectrophotometer (Thermo Fisher Scientific, MD, USA), so, 1 µg of RNA was reverse transcribed to obtain cDNA by using oligo(dT) and random primers of the first-strand cDNA synthesis kit (NZYTech, Lisboa, Portugal). All PCRs were performed using supreme NZYTaq DNA polymerase (NZYTech, Lisboa, Portugal) with specific primers (Sigma-Aldrich, Milan, Italy) for tumor necrosis factor-α (TNF-α, forward primer 5′-CAGCCGATGGGTTGTACCTT-3′ and reverse primer 5′-CCGGACTCCGCAAAGTCTAA-3′), interleukin-1β (IL-1β, forward primer 5′-GGACCCCAAAAGATGAAGGGC-3′ and reverse primer 5′-GGAAAAGAAGGTGCTCATGTCC-3′), and IL-10 (forward primer 5′-GCCCTTTGCTATGGTGTCCT-3′ and reverse primer 5′-CTCTGAGCTGCTGCAGGAAT-3′). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, forward primer 5′-GCTACACTGAGGACCAGGTTGTC-3′ and reverse primer 5′-CCATGTAGGCCATGAGGTCCAC-3′) was used as reference gene.

Total RNA of Jurkat cells were isolated with the EZ-RNA II total RNA isolation kit (BI, Biological Industries, Israel) according to manufacturer’s instructions at 24 h after plasma exposure. Quantification of RNA was carried out using a Nanodrop 2000 (Thermofisher Scientific). cDNA was synthesised by reverse transcriptase reaction using SensiFAST cDNA Synthesis Kit (Bioline, UK). Polymerase chain reactions were carried out using SYBR Green Master Mix (ThermoFisher Scientific) and specific primers (Sigma-Aldrich) for P53 (NM_001126114.1), BAD (NM_004322.3), BID (NM_197966.2) and GAPDH (NM_002046.3). The expressions of GAPDH were used as endogenous controls for normalisation. The changes in mRNA expression of P53, BAD and BID were analysed using the threshold cycle 2 ${}^{-\mathrm{\Delta }\mathrm{\Delta }Ct}$ method.