Epoch biotek spectrophotometer

Total RNA was isolated using the High Pure RNA Isolation Kit (Roche Diagnostics GmbH, Mannheim, Germany). RNA concentration and purity were assessed using a Take-3 system on an Epoch BioTek spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA), and the data were analyzed in Gen5 software (BioTek Instruments, Inc., Winooski, VT, USA). RNA integrity number (RIN) was determined to evaluate the integrity and level of degradation of RNA using an Agilent 2100 Bioanalyzer and Agilent RNA 6000 Pico Kit (Agilent Technologies, Palo Alto, CA, USA). RIN values were equal to or higher than 9.

Frozen spinal cords were suspended in RLT buffer and then homogenized using an IkaT10 basic Ultra-Turrax homogenizer. RNA was extracted using the RNeasy Plus Mini Kit (Qiagen Science, Hilden, Germany) following manufacturer’s protocol and RNA quantity was assessed through spectrophotometric analysis using an Epoch Biotek spectrophotometer (Biotek Instruments, Winooski, VT, United States). Utilizing the RT² First Strand Kit (Qiagen Sciences, MD, United States), 1 μg RNA/sample was reverse transcribed into cDNA. A 1-μg cDNA sample was then used as a template for RT-qPCR employing TaqMan^® gene expression assays (Applied Biosystems, Foster City, CA, United States) (Supplementary Table 1) in the 7500 Fast Real-Time PCR System. Samples were ran in duplicate for target genes and were normalized using HPRT1 as an endogenous control. Relative quantification of transcript expression was performed using the 2^–ΔΔCt method where C_t represents the threshold cycle.

RNA was extracted from NSps using TRIzol and pipet homogenization. Glycogen was added to the TRIzol sample after homogenization to aid in RNA recovery. RNA quantification was assessed using Epoch Biotek spectrophotometer (Biotek Instruments, Winooski, VT, USA). Utilizing the RT2 First Strand Kit (QIAGEN, Germantown, MD, USA), a 0.5 µg RNA sample was reverse-transcribed into cDNA. A 25 ng cDNA sample was then used as a template for RT-qPCR, employing TaqManR gene expression assays (Applied Biosystems, Foster City, CA, USA) (Supplementary Table S1) in the Applied Biosystems StepOnePlus Real-Time PCR System. Samples were run in duplicate for target genes and were normalized using HPRT1 as an endogenous control. Relative quantification of transcript expression was performed using the 2^−ΔΔCt method, where Ct represents the threshold cycle.

At E15, E17, and E20, comparable size spinal cords from vehicle, control, and MMC fetuses were dissected from the lumbar region and snap-frozen and stored at −80 °C until used for gene expression analysis. In MMC fetuses, only the spinal cord from the open area were dissected. Frozen spinal cords were homogenized using an IkaT10 basic Ultra-Turrax homogenizer in RLT buffer and then RNA was extruded using the RNeasy Plus Mini Kit (Qiagen Science, Hilden, Germany) following the manufacturer’s protocol. RNA quantity was assessed through spectrophotometric analysis using an Epoch Biotek spectrophotometer (Biotek Instruments, Winooski, VT, USA).

To obtain nuclear and cytoplasmic fractions, NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, Waltham, MA, USA) were used. Protein concentrations in the nuclear and cytoplasmic fractions were measured using a QuantiPro BCA Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) and Epoch BioTek spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). RelB and p52 NF-κB were detected in both fractions by Western blot analysis using primary anti-RelB (Abcam, Cambridge, MA, USA) and anti-NF-κB2 p100/p52 antibodies (Cell Signaling Technology, Danvers, MA, USA) and secondary goat anti-rabbit-IgG-HRP pAbs (Cell Signaling Technology, Danvers, MA, USA). Anti-GAPDH rabbit mAbs (Cell Signaling Technology, Danvers, MA, USA) were used as a cytoplasmic loading control, and anti-poly(ADP ribose) polymerase (PARP) (46D11) rabbit mAbs (Cell Signaling Technology, Danvers, MA, USA) were used as a nuclear loading control.

RNA was isolated from neuron cells using the Qiagen RNAeasy Mini Kit (Qiagen, Germantown, MD, USA), as recommended by the manufacturer. The RNA concentration was measured using the Take-3 system on an Epoch BioTek spectrophotometer and quantified using Gen5 software (BioTek Instruments, Inc, Winooski, VT, USA). Real-time PCR was performed using 96-well Mitochondria RT² profiler PCR array plates (Qiagen Germantown MD, USA) and ABI 7500 thermocycler (Life Technologies, Carlsbad, CA, USA) at 95 °C for 10 min, 40 cycles of 95 °C for 15 sec, and 60 °C for 1 min. Expression values were collected using the SDS Software system (Applied Biosystems). Assays were performed in three independent replicates. Gene expression was normalized to the expression values of reference genes (GAPDH, b2m, and Hsp90ab1) and evaluated against a negative control, which were uninfected cells. Each PCR array plate contained lyophilized primers for 84 genes associated with the mitochondrial functions into six categories: mitochondrial membrane potential and polarity, mitochondrial transport, transport of small molecules, targeting of proteins to mitochondria, import proteins, outer-membrane translocation, inner-membrane translocation, fusion and fission, mitochondrial localization, and apoptosis.