SARS-CoV-2 Transcriptional Regulation Assay

Cells were grown in 60 mm² dishes, and total RNA was extracted with Trizol reagent (ThermoFisher, Carlsbad, CA, USA), according to the manufacturer’s instructions. One microgram of total RNA was reversely transcribed into cDNA via the SuperScript III system (ThermoFisher, Carlsbad, CA, USA), and 0.5 µL of cDNA was used per RT-qPCR reaction with Power SYBR Green (ThermoFisher, Carlsbad, CA, USA) 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 the E gene and Spike1 RT-qPCR, we used the protocols described in [45 (link)] and [46 (link)], respectively. For the remaining genes, 100 ng of total RNA was used for Taqman reactions using primer probes from ThermoFisher (Carlsbad, CA, USA): 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 the threshold. Desired PCR result specificity was determined based on melting curve evaluation.