POLR3 gene and protein expression dynamics in 4H 203 6 (D9542, Sigma-Aldrich®) was added in a dilution of 1:1000 in washing buffer and incubated for 5 min at RT. The cells were then washed a final time with PBS for 10 minutes. After washing, the cells were imaged with the CellInsight™CX7 LED Pro High-Content Screening Platform (Thermo Scientific™). Example images are displayed in Supplementary Figure 2. After imaging cell segmentation was performed with ImageJ (plugin: Trackmate). Using the region of interest (ROIs) data, quantification was performed. The measured integrated intensity was divided by the measured area (25-40 µm) and multiplied by the mean of 5 background measurements. qPCR Total RNA was extracted from iPSCs, NES and neurons using Trizol Reagent (Thermo Fisher Scientific) according to manufacturer's protocol. cDNA synthesis was performed using SuperScript™ IV First-Strand Synthesis System (Invitrogen, 18091050) with 1 μg of RNA and random hexamer primers, according to the manufacturer’s protocol. qPCR was carried out on a QuantStudio Real-Time PCR System (Thermo Fisher Scientific) and Sensi Fast Sybr HiROX-kit (Bioline, BIO-92005). Each reaction consisted of 2 µl 1:12 diluted cDNA, 10 μM of forward and reverse primers, and 5 μL of the Sensi mix mix, with a final reaction volume of 10 μL. The following cycling conditions were used: initial denaturation at 95 °C for 10 minutes, followed by 40 cycles of denaturation at 95 °C for 20 seconds, annealing and extension at 60 °C for 30 seconds. A melt curve analysis was performed at the end of the amplification cycles to confirm the specificity of the PCR products. Gene expression levels were normalized to SDHA reference gene, and relative expression levels were calculated using the 2^−ΔΔCt method. All samples were run in technical duplicates, and data from seven controls and five 4H individuals were used for analysis. Primer sequences for the target genes and reference genes are provided in Supplementary Table II. Statistics Linear mixed-effect models All statistical analyses were performed using R (version 4.4.1). The Shapiro-Wilk test (S-W test) for normality was performed to determine whether the underlying distribution of POLR3 gene expression, Pol III protein expression and localization as well as the expression of Pol III transcripts, are Gaussian (Normal). If this test failed on the response scale of these expressions/transcripts/subunits, a log-transformation to the data was applied, and the S-W test was carried out again to confirm normality. A linear mixedeffect model (LMEM) was applied to the log-transformed POLR3 gene expression, Pol III protein expression, and Poll III transcripts. For each LMEM, the fixed effects of cell type (iPSC,
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