Hs00174128 m1

Cells were grown in 60 mm² dishes and total RNA was extracted with Trizol reagent (Thermo Fisher), according to the manufacturer’s instructions. One microgram of total RNA was reversely transcribed into cDNA using Superscript III system (Thermo Fisher) and 0.5 μl of cDNA was used per RT-qPCR reaction with Power SYBR Green (Thermo Fisher) master mix. Reactions were read in 7500 StepOne Plus from the Oswaldo Cruz Institute. Primer sequences for Drp1, Fis1, ZO-1, claudin-5, HIF-1α, Mfn2, MFF and TOMM20 are provided in Table S1. For E gene and Spike1 RT-qPCR, we used the protocols described in Corman et al., 2020 (link) and Won et al., 2020 (link), respectively. For the remaining genes, 100 ng of total RNA was used for Taqman reactions using primer probes from Thermo Fisher: Hs00242739_m1 (LTB), Hs00174128_m1 (TNF), Hs00232399_m1 (RELB), Hs00357891_s1 (JUNB), Hs00759776_s1 (FOSL1), Hs00765730_m1 (NFKB1), Hs00174103_m1 (CXCL8), Hs00601975_m1 (CXCL2), Hs00236937_m1 (CXCL1), Hs00173615_m1 (PTX3), Hs00174961_m1 (EDN1), Hs00299953_m1 (SERPINE2) and Hs01028889_g1 (NFKB2). GAPDH (Hs02786624_g1) was used for sample normalization. Gene expression variations were assessed by the 2^ΔΔCt method, with Ct as the cycle number at threshold. Desired PCR result specificity was determined based on melting curve evaluation.

For the analysis of gene expression for IL-1β and TNF-α molecules, the following TaqMan probes were used: Hs01555410_m1 for the human IL1B gene, Hs00174128_m1 for the human TNFA gene, and Hs99999901_s1 for the human 18S rRNA used as an endogenous control) (Thermo Fisher Scientific, Waltham, MA, USA). Screening analysis of the expression profile of 92 genes associated with the function of the immune system was measured using the commercial test kit TaqMan^® Array 96-Well Plate Human Immune Response (Thermo Fisher Scientific, Waltham, MA, USA). Although the provided microplate contains 96 wells, 4 wells are reserved for reference genes (18S rRNA, GAPDH, HPRT1, GUSB). However, we used only 18S rRNA as an internal control, so that results can be compared with those obtained using TaqMan probes in this and our previously published papers. Gene expression measurements were made on the Real-Time PCR–The CFX96™ Touch System (Bio-Rad, Hercules, CA, USA) using a TaqMan Universal Master Mix II, no UNG (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was obtained from the mRNA isolated from the whole blood of patients with ACS and the control group was used for the analysis. All procedures were executed following the manufacturer’s protocols. To calculate the relative expressions of the genes studied, the equation 2^−ΔCt, where ΔCt = Ct_{target gene} − Ct_{18S rRNA}, was used.

The cells were treated for 6 h with ligands with or without LPS. Consecutively, the total RNA was isolated using the RNeasy kit (QIAGEN, Hilden, Germany). Single-stranded cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). The qPCR was performed with a qPCR ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) under usage of TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The following primers were used: (1) Hs00164383_m1, CYB1B1, FAM-MGB (2) Hs00174128_m1, TNFalpha, FAM-MGB (3) Hs00420895_gH, RPLPO, and FAM-MGB (all Thermo Fisher, USA). Fold changes were calculated by the ΔΔct-method relative to the unstimulated control with RPLP0 as the reference [29 (link)].