Journal: The Febs Journal

Article Title: Transcriptional regulation of human NMNAT2 : insights from 3D genome sequencing and bioinformatics

doi: 10.1111/febs.70322

Figure Lengend Snippet: Genomic structure of selected interactomes and the impacts of their deletions on NMNAT2 transcription. (A) A cartoon illustrates the location of Region1 (black box; chr1:184664369‐184669585) relative to the regulatory domain of EDEM3 integrated with 4C interactomes output from the w4CSeq pipelines. (B) An illustration of the location of Region2 (black box; chr1:186343515‐186346086) relative to the regulatory domain of TPR and ODR4 integrated with 4C interactomes output from the w4CSeq pipelines. (C) An illustration showing the location of Region3 (black box; chr1:184662408‐184664466) relative to the regulatory domain of EDEM3 integrated with 4C interactomes output from the w4CSeq pipelines. (D–F) RT‐qPCR summary for the impact of deleting Regions 1–3 (D‐F accordingly) on NMNAT2 mRNA levels in undifferentiated and neuron‐like SH‐SY5Y cells. EV, empty vector. Gene expression was normalized to 18S . GAPDH served as a control. n = 3 independent batches of cells per group. Data represent mean ± SEM. Student's t‐test was used to compare statistical significance.

Article Snippet: For comparison of gene expression, RT‐qPCR was carried out using TaqMan Gene Expression Assays for NMNAT2 (Hs00322752_m1), EDEM3 (Hs00981767_m1), TPR (Hs00162918_m1), ODR4 (Hs00215258_m1), 18S (Hs99999901_s1), and GAPDH (Hs02758991_g1) in a 20 μL reaction consisting of 10 μL TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 1 μL TaqMan Gene Expression Assay, 100 ng of cDNA, and water, with the PCR program as follows: 40 cycles of 15 s at 95 °C and 1 min at 60 °C.

Techniques: Quantitative RT-PCR, Plasmid Preparation, Gene Expression, Control

Journal: The Febs Journal

Article Title: Transcriptional regulation of human NMNAT2 : insights from 3D genome sequencing and bioinformatics

doi: 10.1111/febs.70322

Figure Lengend Snippet: Gene expression of NMNAT2 and NMNAT2 ‐associated genes upon knockdown of the target transcription factors (TFs) in undifferentiated and neuron‐like SH‐SY5Y cells. Undifferentiated cells are shown in green and neuron‐like SH‐SY5Y cells in pink. (A) Location of deleted genomic sequences (gray) for TFs knockdown. The initial portions of the gene sequences—covering the promoter area at least partly and exon 1—of the target TFs were targeted to achieve their knockdown. All RNA samples underwent DNase I treatment to eliminate genomic DNA, and all RT‐qPCR primers were designed to span introns, except for SOX11, which lacks introns. RT‐qPCR was performed to examine the knockdown efficiency with CRISPR‐Cas9 deletion on ATF4 , ATF6 , HSF1 , and SOX11 . Fold change was the ratio of mRNA levels after transfection with gRNAs compared to empty vector (EV) for both undifferentiated and neuron‐like SY5Y cells. The incomplete reduction of mRNA expression levels was primarily due to the cells in the knockdown experiments consisting of polyclonal clones, which contain heterozygous and homozygous clones that survived antibiotic selection. n = 3 biological replicates per group for undifferentiated and differentiated SH‐SY5Y cells and two independent batches of differentiation per group. Data represent mean ± SEM. Ordinary one‐way ANOVA with Šídák's multiple comparisons test was conducted, * P = 0.0258, ** P = 0.0012, *** P = 0.0004, **** P < 0.0001. (B) ATF4 knockdown resulted in the upregulation of NMNAT2 and TPR mRNA in SH‐SY5Y cells, as well as NMNAT2 and EDEM3 mRNA in neuron‐like SH‐SY5Y cells. (C) HSF1 knockdown resulted in the upregulation of NMNAT2 , EDEM3 , and TPR mRNA in SH‐SY5Y cells, and NMNAT2 and EDEM3 mRNA in neuron‐like SH‐SY5Y cells. (D) ATF6 knockdown resulted in the upregulation of NMNAT2 mRNA in SH‐SY5Y cells. (E) SOX11 knockdown resulted in the upregulation of NMNAT2 and EDEM3 mRNA in SH‐SY5Y cells. Gene expression was normalized to 18S . GAPDH served as a control (F). KD, TF knockdown. EV, empty vector. n = 3 biological replicates per group for undifferentiated and differentiated SH‐SY5Y cells and two independent batches of differentiation per group. Data represent mean ± SEM. Student's t ‐test was used to compare statistical significance.

Article Snippet: For comparison of gene expression, RT‐qPCR was carried out using TaqMan Gene Expression Assays for NMNAT2 (Hs00322752_m1), EDEM3 (Hs00981767_m1), TPR (Hs00162918_m1), ODR4 (Hs00215258_m1), 18S (Hs99999901_s1), and GAPDH (Hs02758991_g1) in a 20 μL reaction consisting of 10 μL TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 1 μL TaqMan Gene Expression Assay, 100 ng of cDNA, and water, with the PCR program as follows: 40 cycles of 15 s at 95 °C and 1 min at 60 °C.

Techniques: Gene Expression, Knockdown, Genomic Sequencing, Quantitative RT-PCR, CRISPR, Transfection, Plasmid Preparation, Expressing, Clone Assay, Selection, Control