Journal: Oncogene

Article Title: H19 lncRNA identified as a master regulator of genes that drive uterine leiomyomas

doi: 10.1038/s41388-019-0808-4

Figure Lengend Snippet: TET3 affects DNA methylation and histone modifications of the MED12, TGFBR2, and TSP1 promoters. a UtLM cells were transfected with siCon or siTET3 for 48 h, followed by ChIP-qPCR analysis. Data are presented as mean relative TET3 enrichment over input. n = 3. Red numbers indicate nucleotide positions relative to the transcriptional start sites, with PCR products depicted as red-stripped bars. b Sequences of critical transcription regulatory regions (CTRR) of MED12 , TGFBR2 , and TSP1 . The differentially methylated cytosine residues are marked in red. The red numbers mark the positions of the indicated nucleotides relative to the transcriptional start sites. c UtLM cells were transfected with siCon or siTET3 for 48 h, followed by QMSP analysis. n = 3. d UtLM cells were transfected with siCon or siTET3 for 48 h, followed by ChIP-qPCR analysis. Data are presented as mean relative enrichment over input. n = 3. All data are representative of at least two independent experiments and are presented as mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: Antibodies for TET3 (GeneTex, GTX121453; used at a dilution of 1/500), TGFBR2 (Abcam, ab184948; used at a dilution of 1/1000), TSP1 (Abcam, ab85762; used at a dilution of 1/500), MED12 (Novus Biological, NB100–2357; used at a dilution of 1/500), HMGA2 (Proteintech, 20795–1-AP; used at a dilution of 1/500), GRAF1 (Cell Signaling, 8802; used at a dilution of 1/500), SPARC (Cell Signaling, 8725; used at a dilution of 1/500), COL3A1 (LS-Bio, LS-C159386; used at a dilution of 1/1000), COL4A1 (LS-Bio, LS-C100552; used at a dilution of 1/500), COL5A2 (Origene, TA809611; used at a dilution of 1/500), and GAPDH (Abcam, ab128915; used at a dilution of 1/10000) were purchased.

Techniques: DNA Methylation Assay, Transfection, ChIP-qPCR, Methylation

Journal: Oncogene

Article Title: H19 lncRNA identified as a master regulator of genes that drive uterine leiomyomas

doi: 10.1038/s41388-019-0808-4

Figure Lengend Snippet: H19 and TET3 co-express with fibroid-promoting genes in vivo. a , c RT-qPCR analyses were performed on RNAs extracted from human fibroids and matched myometrium tissues. Spearman’s correlation showed positive correlations between expression of H19 and TET3 ( a , left panel), as well as TET3 and its target genes MED12 , TGFBR2 , and TSP1 ( c ) in a statistically significant manner. No correlation between expression of H19 and HMGA2 at the RNA level was detected ( a , right panel). Spearman’s correlation coefficient, p -values, and sample numbers are presented. b Results of western blotting analysis of HMGA2 in human fibroids and matched myometrium. n = 3. Data are representative of two independent experiments and are presented as mean ± SEM

Article Snippet: Antibodies for TET3 (GeneTex, GTX121453; used at a dilution of 1/500), TGFBR2 (Abcam, ab184948; used at a dilution of 1/1000), TSP1 (Abcam, ab85762; used at a dilution of 1/500), MED12 (Novus Biological, NB100–2357; used at a dilution of 1/500), HMGA2 (Proteintech, 20795–1-AP; used at a dilution of 1/500), GRAF1 (Cell Signaling, 8802; used at a dilution of 1/500), SPARC (Cell Signaling, 8725; used at a dilution of 1/500), COL3A1 (LS-Bio, LS-C159386; used at a dilution of 1/1000), COL4A1 (LS-Bio, LS-C100552; used at a dilution of 1/500), COL5A2 (Origene, TA809611; used at a dilution of 1/500), and GAPDH (Abcam, ab128915; used at a dilution of 1/10000) were purchased.

Techniques: In Vivo, Quantitative RT-PCR, Expressing, Western Blot

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 potentiates STAT3 activation via Smad7. A) PRMT5 depletion dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shPRMT5‐1 or shPRMT5‐2 or Control (shCtrl) were harvested and analyzed by using western blotting with indicated antibodies. B) Knockdown of PRMT5 attenuates IL‐6‐induced STAT3 phosphorylation in MCF10A cells. MCF10A cells were transfected with 40 pm siRNA against PRMT5. 36 h later, cells were treated with IL‐6 (10 ng mL −1 ) for the indicated time and harvested for western blotting analysis with appropriate antibodies. C) PRMT5 inhibition attenuates endogenous activation of STAT3 in H358 cells. H358 cells were treated with 20 × 10 −6 m of PRMT5 inhibitors EPZ015666 or GSK591 for the indicated time. Cell lysates were collected and subject to western blotting analysis. SDMA indicates global arginine di‐methylation. D) Smad7 potentiates STAT3 activation in A549 cells. A549 cells stably expressing FLAG‐GFP or FLAG‐Smad7 were harvested and subject to Western blotting analysis using appropriate antibodies. E) Stable knockdown of Smad7 dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shSmad7 or shCtrl were harvested and subject to western blotting analysis using appropriate antibodies. F) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF7 cells. Cells were transfected with siSmad7 (40 pm) and treated with IL‐6 (10 ng mL −1 ) for the indicated time. Cells were harvested and analyzed by western blotting with appropriate antibodies. G) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF10A cells. Cell transfection, treatment, and Western blotting were done as described in Panel F. H) PRMT5 potentiates STAT3 activation dependent of Smad7. MCF10A cells were transduced with lentiviral particles expressing HA‐PRMT5 or HA‐G367A/R368A. After 24 h, cells were transfected with 40 pm siSmad7. 12 h later, cells were stimulated with IL‐6 (2 ng mL −1 ) for the indicated time. Cell lysates were harvested and subject to Western blotting analysis using appropriate antibodies.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Activation Assay, Stable Transfection, Expressing, Control, Western Blot, Knockdown, Phospho-proteomics, Transfection, Inhibition, Methylation, Transduction

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 methylates Smad7 on R57. A) PRMT5 methylates Smad7. HEK293T cells were transfected with expression plasmids carrying MYC‐PRMT5/MEP50 and an SFB‐tagged construct, including gp130, JAK2, STAT3, Smad7, SHP2, and SOCS3. Cell lysates were harvested and precipitated with streptavidin beads. The retrieved complexes and input were analyzed by Western blotting with indicated antibodies. B) PRMT5 methylates the R57 residue on Smad7. HEK293T cells were transfected with MYC‐PRMT5/MEP50 and an SFB‐tagged Smad7 construct, i.e., wildtype Smad7 (WT) or a R‐to‐K substitution of Smad7 as indicated above the blots. Cell lysate was precipitated with streptavidin beads. Arginine di‐methylation of Smad7 was detected by Western blotting analysis. C) Mass spectrum of Smad7 Arg‐57 dimethylated peptide. Mass spectrometry identified Arg‐57 dimethylation of Smad7 in HEK293T cells expressing MYC‐PRMT5/MEP50 and SFB‐Smad7. Mass spectrometry profile of Smad7 sequence covering residue 47–64 is shown, and the dimethylated arginine side chains are indicated. D) PRMT5/MEP50 methylate Smad7, but not the R57K mutant. HEK293T cells were transfected MYC‐PRMT5/MEP50 and SFB‐Smad7 or Smad7 R57K mutant for 36 h. Cell lysates were harvested and immunoprecipitated with SYM10 antibody. The immunocomplexes and inputs were analyzed by Western blotting with indicated antibodies. E) PRMT5 methylates Smad7 in vitro. MYC‐PRMT5 or MYC‐G367A/R368A together with MYC‐MEP50 were immunopurified using anti‐MYC antibody from transfected HEK293T cells. Purified recombinant GST‐Smad7, GST‐Smad7 R57K mutant, and GST‐Smad4 were produced in E. coli . GST proteins and MYC‐PRMT5/MEP50 proteins were incubated in the presence of S‐adenosyl‐methionine to allow methylation reaction. Dimethylated Smad7 on R57 was detected by using Western blotting analysis.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Transfection, Expressing, Construct, Western Blot, Residue, Methylation, Mass Spectrometry, Sequencing, Mutagenesis, Immunoprecipitation, In Vitro, Purification, Recombinant, Produced, Incubation

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: Arg methylation enhances Smad7 binding to gp130. A) Smad7 methylation increases its association with gp130. HEK293T cells were transfected with SFB‐Smad7 or Smad7 R57K mutant and HA‐gp130, together with MYC‐PRMT5/MEP50. Cell lysates were harvested and immunoprecipitated with Streptavidin beads. Western blotting analysis was done with appropriate antibodies. B) PRMT5 depletion blocks Smad7 methylation and its interaction with endogenous gp130. A549 tet‐on cells expressing SFB‐Smad7 were cultured with or without 1 µg mL −1 Dox for 3 d and then transfected with 40 × 10 −12 m siPRMT5. Cell lysates were harvested and immunoprecipitated with streptavidin beads. Endogenous gp130 was detected from the immunoprecipitates by using Western blotting analysis. C) Methylated Smad7 binds more tightly to gp130. HEK293T cells were transfected with indicated expression plasmids for MYC‐PRMT5, MYC‐G367A/R368A, and MYC‐MEP50 as well as SFB‐Smad7 or SFB‐R57K. Dimethylated Smad7 was immunopurified using SYM10 antibody, while total Smad7 was retrieved using an‐FLAG antibody. Bacterially expressed GST‐gp130‐ICD was purified using glutathione‐sepharose and eluted with elution buffer (10 × 10 −3 m glutathione, pH 8.0). In the in vitro binding experiments for evaluating the Smad7‐gp130 interaction, recombinant GST‐gp130‐ICD was added to the immunopurified Smad7. gp130‐ICD binding to immobilized Smad7 was analyzed by using Western blotting. D) Unmethylatable Smad7 R57K mutant loses its ability to potentiate STAT3 activation. MCF10A tet‐on cells stably expressing SFB‐Smad7 or Smad7 R57K were induced with 10 ng mL −1 Dox for 3 d, and treated with indicated concentrations of IL‐6. Cell lysates were collected and subject to Western blotting analysis.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Methylation, Binding Assay, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Cell Culture, Purification, In Vitro, Recombinant, Activation Assay, Stable Transfection

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 promotes STAT3 transcriptional and growth‐promoting responses. A) PRMT5 inhibition attenuates CDC25C expression in A549 cells. EPZ015666 or GSK591 (20 × 10 −6 m ) were added to A549 cells for 48 h. Cell lysates were harvested and analyzed by using qRT‐PCR to examine CDC25C mRNA levels. Data are shown as mean ± SD; n = 3. *** P < 0.001. B) PRMT5 inhibition attenuates CCNB1 expression in A549 cells. Cell treatment, harvest, and qRT‐PCR analysis were done as described in Panel A. Data are shown as mean ± SD; n = 3. *** P < 0.001. C) PRMT5 deficiency disables IL‐6/STAT3 responsiveness. GSEA showed that downregulated genes in PRMT5‐depleted A549 cells (shPRMT5‐2) were highly enriched in the IL‐6/STAT3 signaling gene set. Red, upregulated genes; blue, downregulated genes. NES = ‐1.73, FDR q value = 0.002. D) Heatmap showing expression levels (log 2 FPKM; left) and relative expression changes (log 2 (shPRMT5‐2/shCtrl); right) of the IL‐6/STAT3 signaling genes. E) Depletion of PRMT5 reduces DNA synthesis. A549 cells stably expressing shPRMT5‐1 or shPRMT5‐2 or Control (shCtrl) were subject to EdU staining to determine DNA incorporating rate (RiboBio),20x. F) Statistic analysis of the result in panel E. Data are shown as mean ± SD; n = 3. 0.01 < * P < 0.05. G) Inhibition of PRMT5 attenuates invasiveness in A549 cells. A549 cells were treated with PRMT5 inhibitors EPZ015666 or GSK591 (20 × 10 −6 m ) for 2 d, starved overnight in FBS‐free medium. 1 × 10 5 cells were plated in a transwell chamber and stained with crystal violet after 12 h. Purple color indicates crystal violet staining of the invaded cell population. H) PRMT5 depletion blocks colony formation. A549 stable cells were subject to crystal violet staining and photography.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, DNA Synthesis, Stable Transfection, Control, Staining

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 promotes lung tumorigenesis. A) Depletion of PRMT5 attenuates tumorigenesis. LLC cells stably expressing shControl or mouse sh‐mPRMT5‐1 or sh‐mPRMT5‐2 were subcutaneously injected into female nude mice. Ten days after implantation, tumors were dissected and photographed. B) Measurement of tumor weight in Panel A. Data are shown as mean ± SD; n = 5 for each group. 0.01 < * P < 0.05. C) PRMT5 depletion impairs STAT3 signaling in tumors. Tumor samples were analyzed by Western blotting to examine phosphorylated STAT3 (p‐STAT3) and STAT3 target gene products such as c‐Myc and Survivin. D) PRMT5 is highly expressed in nonsmall cell lung cancer tissues (NSCLC). NSCLC tissue microarray (Alenabio) was subject to immunohistochemistry (Servicebio) using PRMT5 antibody. E) Statistic analysis of IHC score in Panel D. Statistical analysis was performed using a two‐tailed Student's t ‐test. Data are shown as mean ± SD. Lung cancer samples = 45. Normal lung tissue samples = 55. *** P < 0.001. F) A working model for PRMT5‐mediated STAT3 activation.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Stable Transfection, Expressing, Injection, Western Blot, Microarray, Immunohistochemistry, Two Tailed Test, Activation Assay

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: Density of UG nucleotide sequences 100bp upstream and downstream of m6A modifications identified by cross-linking induced mutation sites (CIMS; A ) or cross-linking induced truncation sites (CITS; B ) in relation to random sequences (red line). Grey shading represents 95% confidence regions. ( C ) Schematic of HaloTag immunoprecipitation and dot blot procedure. ( D ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of HaloTag-labeled proteins in HEK293T cells overexpressing HaloTag, TDP43-HaloTag or YTHDF2-HaloTag from 3 biological replicates. ( E ) Diagram illustrating insertion of the HaloTag open reading frame into the endogenous TARDBP locus immediately 5’ to the TDP43 start codon, resulting in a fusion of HaloTag to the N-terminus of TDP43. ( F ) Halo-TDP43 HEK293T cells labeled live with JF646 Halo dye (red), then fixed, permeabilized, and immunostained with anti-TDP43 antibody (green) prior to imaging. DAPI (blue) marks the nucleus of each cell. Scale bar = 10µm. ( G ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of endogenous HaloTag-TDP43 or exogenous HaloTag. Additional replicates shown in Sup. Fig. 1.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Mutagenesis, Immunoprecipitation, Dot Blot, Modification, Isolation, Immunoaffinity Purification, Labeling, Imaging

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) HaloTag-TDP43 immunoprecipitation was followed by DART-seq to delineate m6A sites within TDP43 target RNAs. HaloTag-TDP43 HEK293T cells were transfected with APOBEC1-YTH or APOBEC1-YTHmut and crosslinked before immunoaffinity purification of HaloTag-labeled proteins. Immunoprecipitated RNAs were then sequenced and C-T transitions were identified in the context of DRACH motifs (red shaded box, D=A/G/T, R=A/G, H=A/C/T). Absolute counts ( B ) and relative frequency ( C ) of base pair transitions observed by RNA-seq in each condition. Shaded boxes represent transition types expected from APOBEC1 activity. ( D ) Example m6A sites identified by DART-seq in RPL10A . C-T transitions are highlighted in red, and DRACH motifs in pink. Green arrow, transcription start site; red hexagon, transcription stop site; thick blue bars, coding exons; thin blue bars, untranslated region. ( E ) Absolute count and relative distribution ( F ) of DART-seq reads in cells expressing APOBEC1-YTH and APOBEC1-YTHmut. UTR, untranslated region; CDS, coding sequence. ( G ) Scatter plot of TDP43 targets, determined by fold enrichment in precipitated RNA from HaloTag-TDP43 cells (expressing APOBEC1-YTH and APOBEC10YTHmut) compared to cells transfected with HaloTag. Red dots signify transcripts showing > 2-fold enrichment in both APOBEC1-YTH and APOBEC1-YTHmut expressing cells. TARDBP , yellow dot, identified as high confidence target. ( H ) Stacked bar graph showing percentage of m6A modified RNA in TDP43 targets (red) and non-targets (black). ( I ) Cumulative distribution of RNA methylation in TDP43 targets (red) and non-targets (black). p = 1.87×10 −55 by Kolmogorov Smirnov test. ( J ) Euler diagram depicting overlap between TDP43 targets identified in this study, and those identified by TDP43 cross linking and immunoprecipitation followed by RNA-sequencing (CLIP-seq) in HEK293T cells (Hallegger et al ., 2021) . **p=1.5×10 −117 , hypergeometric test. ( K ) Pie charts demonstrating the percentage of methylated RNA among TDP43 targets (pink) and non-targets (grey). **p<1×10 −5 chi-square test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Immunoprecipitation, Transfection, Immunoaffinity Purification, Labeling, RNA Sequencing, Activity Assay, Expressing, Sequencing, Modification, Methylation

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) TARDBP gene map, illustrating TDP43 binding region (TBR), the location of the DRACH motif (pink square), and the C-T transition (red box) identified by DART-seq within this domain, representing an m6A site. ( B ) Schematic of the TARDBP minigene reporter, consisting of the mCherry ORF upstream of TARDBP exon 6 and 3.4 Kb of the TARDBP 3’ UTR. The A residue adjacent to the detected C-T transition via DART-seq in the WT reporter (mCherry-TBR) was mutated to a G, precluding methylation the mutant reporter (mCherry-mTBR). Red, methylated residue; blue line, DRACH motif; dagger, C-T transition from DART-seq. ( C ) HaloTag-TDP43 was isolated by immunoaffinity purification from HaloTag-TDP43 HEK293T cells expressing mCherry-TBR or mCherry-mTBR, and reporter RNA detected in elution fractions by qRT-PCR. ( D ) Outline of TDP43 autoregulation assay. Excess TDP43 binds to the reporter, triggering reporter splicing, destabilization, and reduced mCherry fluorescence. ( E ) Primary rodent neurons were transfected with WT (mCherry-TBR) or mutant (mCherry-mTBR) reporters, together with EGFP or TDP43-EGFP. After 7d, mCherry expression was assessed by fluorescence microscopy. Scale bar= 20 µm. Normalized RFP (mCherry) intensity in primary neurons expressing WT mCherry-TBR reporter ( F ) or mutant mCherry-mTBR ( G ) reporter together with EGFP or TDP43(WT)-EGFP. Cherry-TBR+GFP n= 160, Cherry-TBR+TDP43(WT)-GFP n= 58, Cherry-mTBR+GFP n= 105, Cherry-mTBR+TDP43(WT)-GFP n= 44. Data in C plotted as mean ± SD, collected from 3 biological replicates. ns= not significant, *p< 0.05, **p< 0.01; one-way ANOVA with Tukey’s test. Data in F and G plotted as mean ± SD, color coded by biological replicate. ns = not significant, *p < 0.05; Welch’s t-test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Binding Assay, Residue, Methylation, Mutagenesis, Isolation, Immunoaffinity Purification, Expressing, Quantitative RT-PCR, Fluorescence, Transfection, Microscopy

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Genome-wide analysis of RNA methylation via epitranscriptomic array. RNA was extracted from control (n= 3) and sporadic ALS (sALS) patient (n= 4) spinal cord samples, prior to m6A RNA immunoprecipitation. The resulting samples were separated into methylated and non-methylated RNA, then labeled with distinct fluorescent dyes (red and green stars) prior to hybridization, allowing relative quantification of methylation at each annotated locus. ( B ) Principal component analysis (PCA) plot comparing methylation levels of control (grey) and ALS (red) patient samples. ( C ) Hierarchical clustering of mRNA methylation profiles from control and ALS mRNA samples. ( D ) Volcano plot depicting fold change in mRNA methylation levels in ALS compared to control spinal cord. ( E ) Hierarchical clustering of lncRNA methylation profiles from control ALS lncRNA samples. ( F ) Volcano plot showing fold change in lncRNA methylation levels in ALS compared to control spinal cord. In D and F , grey horizontal vertical lines represent p= 0.05 and fold change (FC)= 2. ( G ) Euler diagram demonstrating overlap (n= 322, p= 5.09×10 −119 , hypergeometric test) among TDP43 substrates and methylated transcripts identified in HEK293T cells, in additional to hypermethylated transcripts determined via m6A array in sALS spinal cord. Comparisons were limited to the subset of transcripts expressed in both HEK293T cells and human spinal cord (nTPM>2). ( H ) Based on comparisons with the GEO transcription factor loss-of-function database via Enrichr , there was strong enrichment for TDP43-regulated genes not only among the set of 2034 transcripts hypermethylated in sALS spinal cord, but also among the 322 TDP43 targets that were also hypermethylated in sALS (A1 in G ). Combined score = (log 10 p * Z-score). ( I ) Immunohistochemical staining for m6A in control and sALS spinal cord sections. Scale bars= 50 µm. ( J ) Quantification of m6A antibody reactivity in spinal cord neurons from control (n= 110 neurons) and sALS (n= 277 neurons) sections. Plot shows mean +/- SD, color coded by patient. ****p< 0.0001 via Mann-Whitney test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Genome Wide, Methylation, Control, RNA Immunoprecipitation, Labeling, Hybridization, Quantitative Proteomics, Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Representative images of rodent primary neurons transfected with plasmids expressing Cas9-2A-EGFP and sgRNA targeting the neuronal protein NeuN or negative control (LacZ). 5d after transfection, neurons were fixed and immunostained for NeuN (red). White dashed circles indicate nucleus stained with Hoechst (blue). ( B ) NeuN antibody reactivity measured in EGFP-positive neurons expressing sgLacZ (n= 565) or sgNeuN (n= 654), ****p < 0.0001 by Mann-Whitney. ( C ) Schematic depicting m6A writers (green), erasers (red), and readers (orange) targeted by CRISPR/Cas9. ( D ) Primary neurons expressing EGFP and TDP43-mApple were assessed at regular 24h intervals by fluorescence microscopy, and their survival assessed by automated image analysis. Individual neurons are assigned unique identifiers (yellow number) and tracked until their time of death (red), indicated by cellular dissolution, blebbing, or neurite retraction. Scale bar= 20µm. ( E ) Cumulative hazard plot depicting risk of death for neurons expressing TDP43(WT) + non-targeting (NT) (red line), mApple + NT (grey line), or TDP43(WT) + Atxn2 sgRNA (purple line). †p<2.0 ×10 −16 , Hazard ratio (HR)= 3.45; ***p= 5.81 ×10 −4 , HR= 0.80). ( F ) Forest plot showing HR for TDP43-overexpressing neurons upon knockdown of m6A writers (green), erasers (dark red), and readers (orange), in comparison to nontargeting (NT) control. Dashed line indicates HR= 1, representing the survival of the reference condition, neurons expressing TDP43-mApple and NT sgRNA. Values >1 indicate increased toxicity, whereas values <1 denote relative protection. Error bars represent 95% CI. ( G ) Alkbh5 knockout significantly increases TDP43 associated toxicity. †p=3.11 ×10 −5 , HR= 1.59; ***p= 2.65×10 −11 , HR= 2.03. ( H ) Ythdf2 knockout significantly extends survival in TDP43-expressing neurons. ***p <2.0 ×10 −16 , HR= 1.69; †p= 6.2 ×10 −6 , HR= 0.71. ( I ) YTHDF2 overexpression is toxic to neurons. ***p= 3.07×10 −5 , HR= 1.30. ( J ) METTL3/14 overexpression enhances TDP43-dependent toxicity in neurons. †p = 5.53 ×10 −4 , HR= 1.32; ***p =4.16 ×10 −6 , HR= 1.31. p values in E, G-J determined via Cox proportional hazards analysis, with a minimum 3 of biological replicates.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Transfection, Expressing, Negative Control, Staining, MANN-WHITNEY, CRISPR, Fluorescence, Microscopy, Dissolution, Knockdown, Comparison, Control, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Immunostaining of YTHDF2 in control and sALS patient spinal cord samples. Scale bar= 50 µm. ( B ) Quantification of YTHDF2 immunoreactivity in spinal cord neurons from control (n= 117 neurons) and sALS (n= 193 neurons) samples. Plot shows mean +/- SD, color coded by sample. ****p< 0.0001 via Mann-Whitney test. ( C ) Strategy used to create isogenic iPSCs expressing native TDP43(WT)-Dendra2 or TDP43(M337V)-Dendra2. ( D ) Representative images of untransduced (grey) and transduced (green) iNeurons expressing shRNA against YTHDF2 (shYTHDF2) and a GFP reporter. Time of death (red circles) for each cell is used to determine cumulative risk of death, plotted in ( E ) and ( F ). Scale bar= 20µm. shRNA-mediated knockdown of YTHDF2 significantly extended the survival of TDP43(M337V)-Dendra2 iNeurons ( E ; †p= 8.42×10 −12 , HR= 6.25; ***p= 4.82×10 −9 , HR=0.32; #p= 0.08, HR= 1.84) as well as mutant C9ORF72 iNeurons ( F , †p= 1.42×10 −11 , HR= 2.85; ***p= 1.42×10 −16 , HR= 0.32). ns= not significant. Values in ( E , F ) calculated by Cox proportional hazards analysis, with a minimum 3 biological replicates.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Immunostaining, Control, MANN-WHITNEY, Expressing, shRNA, Knockdown, Mutagenesis

Journal: PLoS ONE

Article Title: Recombinant Neuregulin 1 Does Not Activate Cardiomyocyte DNA Synthesis in Normal or Infarcted Adult Mice

doi: 10.1371/journal.pone.0115871

Figure Lengend Snippet: (A) Use of BrdU to monitor cardiomyocyte DNA synthesis in non-injured adult mice receiving 9 consecutive daily injections of NRG1β1 (BrdU was delivered using a mini-osmotic pump). Left panel shows anti-β-galactosidase immune reactivity, middle panel shows anti-BrdU immune reactivity, and right panel shows the merged image. Arrow indicates a BrdU positive cardiomyocyte nucleus, arrowhead indicates a BrdU positive non-cardiomyocyte nucleus. Bar = 10 microns. (B) BrdU incorporation in the nuclei of the small intestine microvilli epithelial cells of an NRG1β1-treated mouse. Note the absence of BrdU signal in the muscularis mucosae zone (asterisk). Bar = 10 microns. (C) Use of 3 H-Thy to monitor cardiomyocyte DNA synthesis in non-injured adult mice receiving 9 consecutive daily injections of NRG1β1 ( 3 H-Thy was delivered as a single bolus 1 hour after the last NRG1β1 treatment). Arrow indicates a 3 H-Thy positive cardiomyocyte nucleus, arrowhead indicates a 3 H-Thy positive non-cardiomyocyte nucleus. Bar = 10 microns.

Article Snippet: Experimental mice were treated with recombinant human NRG1β1 (corresponding to the EGF domain, amino acid residues 176–256, #396-HB, R&D Systems, Minneapolis, MN), at a dose of 2.5 micrograms per mouse per IP injection, dissolved in saline containing 0.1% Bovine Serum Albumin (BSA); control mice received vehicle alone.

Techniques: DNA Synthesis, BrdU Incorporation Assay

Journal: PLoS ONE

Article Title: Recombinant Neuregulin 1 Does Not Activate Cardiomyocyte DNA Synthesis in Normal or Infarcted Adult Mice

doi: 10.1371/journal.pone.0115871

Figure Lengend Snippet: Cardiomyocyte DNA synthesis in adult MHC-nLAC mice following vehicle or NRG1β1 injection.

Article Snippet: Experimental mice were treated with recombinant human NRG1β1 (corresponding to the EGF domain, amino acid residues 176–256, #396-HB, R&D Systems, Minneapolis, MN), at a dose of 2.5 micrograms per mouse per IP injection, dissolved in saline containing 0.1% Bovine Serum Albumin (BSA); control mice received vehicle alone.

Techniques: DNA Synthesis, Injection, Control

Journal: PLoS ONE

Article Title: Recombinant Neuregulin 1 Does Not Activate Cardiomyocyte DNA Synthesis in Normal or Infarcted Adult Mice

doi: 10.1371/journal.pone.0115871

Figure Lengend Snippet: (A) Western blot demonstrating the levels of total Erk1/2 p42/p44, P-Erk1/2[Thr 202 /Thy 204 ] and RLC in mice treated with NRG1β1 or vehicle (hearts harvested and processed 90 minutes after treatment). Densometric quantitation revealed that NRG1β1 treatment resulted in a 987% increase in the level of ERK1 phosphorylation, a 5727% increase in the level of ERK2 phosphorylation, and a 21% increase in the level of phosphorylated RLC vs. vehicle-treated mice (p<0.01, Student’s t-test). (B) Non-cardiomyocyte 3 H-Thy nuclear labeling index in non-injured adult mice following 9 consecutive daily injections of NRG1β1 (5 sections analyzed from each of 4 independent mice) or vehicle (4 sections analyzed from each of 4 independent mice). *: p<0.05 vs. vehicle treated animals, Student’s t-test.

Article Snippet: Experimental mice were treated with recombinant human NRG1β1 (corresponding to the EGF domain, amino acid residues 176–256, #396-HB, R&D Systems, Minneapolis, MN), at a dose of 2.5 micrograms per mouse per IP injection, dissolved in saline containing 0.1% Bovine Serum Albumin (BSA); control mice received vehicle alone.

Techniques: Western Blot, Quantitation Assay, Phospho-proteomics, Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel interaction interfaces mediate the interaction between the NEIL1 DNA glycosylase and mitochondrial transcription factor A

doi: 10.3389/fcell.2022.893806

Figure Lengend Snippet: Affinity pull-down experiments reveal an interaction between recombinantly purified NEIL1 and TFAM. (A) Flag-tagged, full-length NEIL1 (NEIL1-FL) was used to pull down TFAM in the presence and absence of a specific DNA (SD) sequence containing an abasic site in a buffer containing 100 mM NaCl. TFAM was observed in the elution fractions in both the presence and absence of SD. mtSSB was used as a positive control as we previously documented the interaction between NEIL1 and mtSSB. (B) The purified proteins were treated with Benzonase prior to complex formation to eliminate nucleic acid contamination followed by the pull-down experiment. TFAM was observed in elution fractions containing either 100 mM NaCl in the buffer, or 100 mM KCl in the buffer (C) in the both Benzonase treated or non-treated samples indicating that there is a direct interaction between the two proteins.

Article Snippet: The membrane was probed using a NEIL1 rabbit polyclonal antibody (1:1000; Proteintech #12145-1-AP).

Techniques: Purification, Sequencing, Positive Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel interaction interfaces mediate the interaction between the NEIL1 DNA glycosylase and mitochondrial transcription factor A

doi: 10.3389/fcell.2022.893806

Figure Lengend Snippet: Affinity pull-down experiments display an interaction between NEIL1 and TFAM using recombinantly purified proteins. (A) Flag tagged NEIL1 was used to pull down TFAM in the presence of RNA in a buffer containing 100 mM NaCl. TFAM is observed in the elution fractions in the absence and presence of RNA. (B) Flag tagged NEIL1 was used to pull down TFAM in the presence of RNA in a buffer containing 100 mM KCl. Under these conditions, TFAM is also observed in the elution fractions in the absence and presence of RNA. (C) The reverse pull-down experiment using Flag-tagged TFAM was performed. Full-length NEIL1 (NEIL1-FL) and a truncated NEIL1 enzyme lacking 100 residues from disordered C-terminal region (NEIL1-Δ100) were observed in the elution fractions in the presence and absence of DNA. Non-specific binding of untagged TFAM, NEIL1-FL, and NEIL1-Δ100 with Flag beads was not detected as shown in elution fractions when the interaction partner is absent.

Article Snippet: The membrane was probed using a NEIL1 rabbit polyclonal antibody (1:1000; Proteintech #12145-1-AP).

Techniques: Purification, Binding Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel interaction interfaces mediate the interaction between the NEIL1 DNA glycosylase and mitochondrial transcription factor A

doi: 10.3389/fcell.2022.893806

Figure Lengend Snippet: Far-western analysis indicates that NEIL1 interacts with TFAM via multiple binding sites present at both the N- and C-terminal domains. (A) A map of the His-tagged polypeptides of NEIL1 lacking portions of the C-terminal disordered tail and the GST-tagged C-terminal polypeptides of NEIL1 lacking the N-terminal portion of the enzyme. (B) Far-western analysis to determine the minimal region of NEIL1 required for interaction with TFAM. All proteins used in this study were expressed in E. coli , purified to homogeneity, and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis stained with Coomassie blue. 50 pmol of NEIL1 and the truncated enzymes, bovine serum albumin (negative control), glutathione S-transferase (negative control), and 1 pmol of TFAM (positive control) were loaded onto the gel. Far-western analysis was performed where proteins were transferred to a PVDF membrane, denatured, slowly renatured on the membrane, incubated with 10 pmol/ml purified TFAM, and probed with an anti-TFAM antibody to detect an interaction.

Article Snippet: The membrane was probed using a NEIL1 rabbit polyclonal antibody (1:1000; Proteintech #12145-1-AP).

Techniques: Western Blot, Binding Assay, Purification, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Negative Control, Positive Control, Membrane, Incubation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel interaction interfaces mediate the interaction between the NEIL1 DNA glycosylase and mitochondrial transcription factor A

doi: 10.3389/fcell.2022.893806

Figure Lengend Snippet: Hydrogen-deuterium exchange experiments for the NEIL1-DNA complex reveals regions of NEIL1 involved with DNA binding. (A) Woods plot representing the distribution of NEIL1 regions displaying differential levels of solvent protection in the presence of DNA. Percent change in deuteration for peptides after various time points (30 s–30 m) between NEIL1 and the NEIL1-DNA complex, where a negative percentage indicates less deuteration and more protection as a result of complex formation between NEIL1 and the DNA. Each horizontal line in the plot represents an individual peptide with residue range on the X-axis and deuteration level i.e., level of protection on the Y -axis. (B) Domain map and cartoon representation of crystal structure of NEIL1-DNA complex (PDB ID:5itt) are displayed. The active site and void-filling residues, and DNA binding motifs are indicated in the domain map. In the structure, the DNA is colored grey, and each region is color-matched to the domain map above.

Article Snippet: The membrane was probed using a NEIL1 rabbit polyclonal antibody (1:1000; Proteintech #12145-1-AP).

Techniques: Binding Assay, Solvent, Residue

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel interaction interfaces mediate the interaction between the NEIL1 DNA glycosylase and mitochondrial transcription factor A

doi: 10.3389/fcell.2022.893806

Figure Lengend Snippet: Hydrogen-deuterium exchange experiments for the NEIL1-DNA complex reveals regions of NEIL1 involved with DNA binding. (A) Volcano plot quantifying the significant change in deuteron uptake for each peptide at a given time point. The upper left quadrant displays peptides (solid blue circles) at various time points, representing a significant decrease in deuteron uptake upon DNA binding to NEIL1 relative to NEIL1 alone at a p -value of <0.05 (please refer to the legend for for a detailed description of the statistical tests used). (B) Representative uptake plots are shown from the HDX-MS time course for two of the significant peptides, 2–28 and 164–180, that lie within the significant quadrant in panel (A) above. (C) Interaction map showing NEIL1 residues that interact with the DNA in the crystal structure of the NEIL1-DNA complex (PDB ID:5itt). The residues within the blue oval circles indicate those present in peptides with a significant decrease in deuteration, as observed in the volcano plot.

Article Snippet: The membrane was probed using a NEIL1 rabbit polyclonal antibody (1:1000; Proteintech #12145-1-AP).

Techniques: Binding Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel interaction interfaces mediate the interaction between the NEIL1 DNA glycosylase and mitochondrial transcription factor A

doi: 10.3389/fcell.2022.893806

Figure Lengend Snippet: Hydrogen-deuterium exchange analysis of the TFAM-DNA and TFAM-NEIL1-DNA complexes reveals putative TFAM regions that interact with NEIL1 in the presence of DNA. (A) Woods plot representing percent change in deuteration for peptides after various time points (15 s–30 m) between the TFAM-DNA and TFAM-NEIL1-DNA complexes, where a positive percentage indicates more deuteration and less protection observed when NEIL1 is present within the TFAM-NEIL1-DNA complex. Each horizontal line in the plot represents an individual peptide with residue range on the X -axis and deuteration level i.e., level of protection on the Y -axis. (B) Volcano plot displaying TFAM peptides with a statistically significant increase in deuteration ( p -value < 0.05; please refer to the legend for for a detailed description of the statistical tests used) in the TFAM-NEIL1-DNA complex indicated as solid red circles (left panel). On the right panel, the peptides with a significant increase in deuteration are mapped on the crystal structure of the TFAM-DNA complex (PDB ID:4nnu) and are highlighted in red. The domain map above also displays the two regions (red) that show the greatest difference in deuterium uptake upon the addition of NEIL1. (C) Representative uptake plots are shown from the HDX-MS time course for peptides 57–68 and 81–102 that lie within the significance quadrant of the volcano plot in (B) .

Article Snippet: The membrane was probed using a NEIL1 rabbit polyclonal antibody (1:1000; Proteintech #12145-1-AP).

Techniques: Residue

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel interaction interfaces mediate the interaction between the NEIL1 DNA glycosylase and mitochondrial transcription factor A

doi: 10.3389/fcell.2022.893806

Figure Lengend Snippet: Isotopic mass distribution spectra reveal bimodal deuterium exchange upon the addition of NEIL1 to the TFAM-DNA complex. Isotopic mass distribution spectra from representative HDX-MS experiments for the peptide containing TFAM residues 57–68 at various time points as indicated (15 s–10 m). The distribution pattern for TFAM alone (black) displays greater deuterium exchange when compared to the TFAM-DNA complex (blue), which appears to exchange less deuterium. The addition of NEIL1 to the sample mixture (TFAM-NEIL1-DNA complex; red) reveals a bimodal isotopic mass distribution, which likely results from the presence of multiple species (protein-protein; protein-DNA; protein-protein-DNA; or protein alone) within the sample. The grey line within each plot indicates an m/z value of 514.29 corresponding to the non-deuterated peptide.

Article Snippet: The membrane was probed using a NEIL1 rabbit polyclonal antibody (1:1000; Proteintech #12145-1-AP).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel interaction interfaces mediate the interaction between the NEIL1 DNA glycosylase and mitochondrial transcription factor A

doi: 10.3389/fcell.2022.893806

Figure Lengend Snippet: Estimation of relative mitochondrial mRNA expression reveals that NEIL1 is necessary for efficient transcription by TFAM upon DNA damage. (A) Left, the relative mRNA expression of four mitochondrial genes encoding Cytochrome b ( CYB ), NADH dehydrogenase subunit 1 ( ND1 ), Cytochrome c oxidase I ( CO1 ), and 12S ribosomal RNA ( RNR1 ) were estimated by qRT-PCR in untreated Hap1 cell lines where the expression of NEIL1 is either intact (i.e., wild-type, WT) or knocked out (i.e., KO). Right, estimation of mitochondrial copy number by qPCR in the WT and KO cell lines. (B) Left, the relative mRNA expression of the above four mitochondrial genes in the WT and KO Hap1 cells treated with 125 μM MMS for 3 days prior to gene expression analysis. Right, estimation of mitochondrial copy number by qPCR in the WT and KO cell lines after MMS treatment. Statistical analysis was performed in GraphPad Prism using a Student’s t-test where ns, not significant; * p < 0.05; ** p < 0.01; and *** p < 0.001.

Article Snippet: The membrane was probed using a NEIL1 rabbit polyclonal antibody (1:1000; Proteintech #12145-1-AP).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel interaction interfaces mediate the interaction between the NEIL1 DNA glycosylase and mitochondrial transcription factor A

doi: 10.3389/fcell.2022.893806

Figure Lengend Snippet: A model representing the interaction between NEIL1 and TFAM in the presence and absence of nucleic acid binding partners. Two scenarios are possible, when NEIL1, TFAM, and DNA are mixed in a 1:1:1 M ratio. In the tug-of-war model, the two proteins compete to form protein-DNA complexes, whereas, in the complex formation model, a small fraction of both proteins interact in the presence and absence of DNA, forming a complex. Species containing protein-DNA complexes or unbound-protein/DNA are also possible in this scenario. The HDX-MS data alone are insufficient to distinguish between the two proposed models but support for the complex formation model is also provided by pull-down, far-western, MALS, and SAXS analyses.

Article Snippet: The membrane was probed using a NEIL1 rabbit polyclonal antibody (1:1000; Proteintech #12145-1-AP).

Techniques: Binding Assay, Western Blot

Journal: Scientific Reports

Article Title: Inter-laboratory comparison of gene expression biodosimetry for protracted radiation exposures as part of the RENEB and EURADOS WG10 2019 exercise

doi: 10.1038/s41598-021-88403-4

Figure Lengend Snippet: Overview of methodological details of either qRT-PCR or microarrays used by the contributing teams.

Article Snippet: After several washing steps, DNA residuals were digested on the membrane (RNAse-free DNAse Set, Qiagen, Hilden, Germany), which was then washed.

Techniques: Isolation, Control, Concentration Assay, cDNA Synthesis, Reverse Transcription, Multiplex Assay, TaqMan Assay, Real-time Polymerase Chain Reaction, Quantitative Proteomics

Journal: Clinical Epigenetics

Article Title: Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients

doi: 10.1186/s13148-014-0036-2

Figure Lengend Snippet: ABCA1 expression and methylation level in IOSE cells and ovarian cancer cell lines. (A) Total RNA was isolated from ovarian cells and converted into cDNA for amplification with specific primers for ABCA1 . The relative level of expression after quantitative real-time RT-PCR was compared to IOSE cells (set as one fold). Each bar represents mean ± SD. (B) CP70 cells were treated with TSA (0.5 μM, 12 h), GSK343 (1 μM, 3 days), or 5aza (0.5 μM, 3 days). The expression level of ABCA1 was determined by RT-PCR. Treatment of 5aza, but not TSA or GSK, resulted in robust re-expression of ABCA1 in CP70 cells. Each bar represents mean ± SD. (C) The methylation status of the ABCA1 promoter and TSS region was analyzed by bisulfite pyro-sequencing from −90 to +190 (black line underneath). The upper panel shows the ABCA1 promoter and TSS region and the corresponding CpG sites (vertical bar), and the lower panel illustrates DNA methylation at the interrogated CpG site (circle) in IOSE cells, two NOSE samples, and ovarian cancer cell lines with intensity of gray color indicating methylation level.

Article Snippet: The immunohistochemistry procedure followed a standard protocol, using a rabbit polyclonal anti-human ABCA1 antibody (NB400-105, Novus Biologicals).

Techniques: Expressing, Methylation, Isolation, Amplification, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Sequencing, DNA Methylation Assay

Journal: Clinical Epigenetics

Article Title: Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients

doi: 10.1186/s13148-014-0036-2

Figure Lengend Snippet: Effects of ABCA1 knockdown on cell growth. Real-time RT-PCR expression of ABCA1 in (A) MCP3 and (B) HeyC2 cells infected by lentivirus against shGFP (control) or shABCA1. Each bar represents mean ± SD. The growth of ABCA1 knockdown (C) MCP3 and (D) HeyC2 cells was examined by soft agar assay. Quantitative analysis of the soft agar assay is also shown. *** P < 0.001; * P < 0.05.

Article Snippet: The immunohistochemistry procedure followed a standard protocol, using a rabbit polyclonal anti-human ABCA1 antibody (NB400-105, Novus Biologicals).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Infection, Control, Soft Agar Assay

Journal: Clinical Epigenetics

Article Title: Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients

doi: 10.1186/s13148-014-0036-2

Figure Lengend Snippet: Effects of ABCA1 knockdown on cholesterol level and ovarian cancer growth in vivo . The cholesterol level of ABCA1 knockdown (A) MCP3 and (B) HeyC2 cells was measured by cholesterol quantitation assay. (C) The effect of ABCA1 knockdown on tumor growth in vivo was determined by the nude mice model. HeyC2 cells stably infected with shABCA1 (red arrow) or shGFP (green arrow) were injected subcutaneously into athymic nude mice. One week later, tumor volumes were measured daily. From day 19, the volume of tumors with ABCA1 knockdown was significantly reduced as compared to vector controls (* P < 0.05; ** P < 0.005). Data were expressed as mean ± SD ( n = 3).

Article Snippet: The immunohistochemistry procedure followed a standard protocol, using a rabbit polyclonal anti-human ABCA1 antibody (NB400-105, Novus Biologicals).

Techniques: Knockdown, In Vivo, Quantitation Assay, Stable Transfection, Infection, Injection, Plasmid Preparation

Journal: Clinical Epigenetics

Article Title: Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients

doi: 10.1186/s13148-014-0036-2

Figure Lengend Snippet: Association between methylation of ABCA1 and clinicopathological features of 76 ovarian cancer patients

Article Snippet: The immunohistochemistry procedure followed a standard protocol, using a rabbit polyclonal anti-human ABCA1 antibody (NB400-105, Novus Biologicals).

Techniques: Methylation

Journal: Clinical Epigenetics

Article Title: Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients

doi: 10.1186/s13148-014-0036-2

Figure Lengend Snippet: Association between ABCA1 methylation and tumor progression. Dot plot showing the association between ABCA1 methylation in different (A) stages and (B) grades in 76 ovarian cancer patient samples. Methylation of ABCA1 was determined by bisulfite pyro-sequencing. Low stage and low grade represented FIGO I and II and grade 1–2, respectively. While high stage and high grade represented FIGO III and IV and grade 3, respectively. * P < 0.05 by the Mann-Whitney U test. Kaplan-Meier analysis of ABCA1 methylation for (C) progression-free survival and (D) overall survival in 76 ovarian cancer patient samples is shown. Patients were grouped according to methylation of ABCA1 of 3%, which is based on the methylation level of IOSE cells. Patients with “high” ABCA1 methylation (>3% methylation) have significant shorter overall survival ( P = 0.019) but not progression-free survival than patients with “low” ABCA1 methylation. Log-rank P values are shown.

Article Snippet: The immunohistochemistry procedure followed a standard protocol, using a rabbit polyclonal anti-human ABCA1 antibody (NB400-105, Novus Biologicals).

Techniques: Methylation, Sequencing, MANN-WHITNEY

Journal: Clinical Epigenetics

Article Title: Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients

doi: 10.1186/s13148-014-0036-2

Figure Lengend Snippet: Univariable analysis of survival by the Cox proportional hazards model

Article Snippet: The immunohistochemistry procedure followed a standard protocol, using a rabbit polyclonal anti-human ABCA1 antibody (NB400-105, Novus Biologicals).

Techniques: Methylation

Journal: Clinical Epigenetics

Article Title: Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients

doi: 10.1186/s13148-014-0036-2

Figure Lengend Snippet: Multivariate analysis of survival by the Cox proportional hazards model

Article Snippet: The immunohistochemistry procedure followed a standard protocol, using a rabbit polyclonal anti-human ABCA1 antibody (NB400-105, Novus Biologicals).

Techniques: Methylation

Journal: Clinical Epigenetics

Article Title: Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients

doi: 10.1186/s13148-014-0036-2

Figure Lengend Snippet: Association between expression of ABCA1 and survival in ovarian cancer patients. Expression of ABCA1 in 55 ovarian cancer patient samples was determined by IHC in tissue microarray. (A) Representative image of ovarian cancer showing high (left panel) and low (right panel) ABCA1 expression on the cell membrane or cytoplasm (×400). (B) Kaplan-Meier analysis found that patients with low ABCA1 expression have shorter progression-free survival than patients with high ABCA1 expression ( P = 0.038). (C) Similar results can be observed in TCGA ovarian cancer RNA-Seq dataset that patients with low expression of ABCA1 are associated with shorter overall survival ( P = 0.0008). Log-rank P values are shown.

Article Snippet: The immunohistochemistry procedure followed a standard protocol, using a rabbit polyclonal anti-human ABCA1 antibody (NB400-105, Novus Biologicals).

Techniques: Expressing, Microarray, Membrane, RNA Sequencing

Journal: Clinical Epigenetics

Article Title: Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients

doi: 10.1186/s13148-014-0036-2

Figure Lengend Snippet: Association between expression of ABCA1 and clinicopathological features of 55 ovarian cancer patients

Article Snippet: The immunohistochemistry procedure followed a standard protocol, using a rabbit polyclonal anti-human ABCA1 antibody (NB400-105, Novus Biologicals).

Techniques: Expressing

Journal: Acta Neuropathologica Communications

Article Title: Restoration of miR-193a expression is tumor-suppressive in MYC amplified Group 3 medulloblastoma

doi: 10.1186/s40478-020-00942-5

Figure Lengend Snippet: Expression of miR-193a, MYC, and the methylation status of the miR-193a promoter region, in the four molecular subgroups of medulloblastomas. Induction of miR-193a expression by MYC, and upon treatment with a DNA methylation inhibitor in medulloblastoma cells. a MiR-193a expression levels in the four molecular subgroups WNT, SHH, Group 3, and Group 4 of 763 medulloblastomas from the MAGIC cohort. b Schematic showing location of the CpG island, E-box, the transcription start site (TSS) relative to the pre-miR-193a start site (+ 1) on chromosome 17 and the mutations introduced in the E-box. c Relative luciferase reporter activity of the miR-193a promoter construct in the presence or absence of MYC and upon the site-directed mutagenesis of the MYC binding site in the miR-193a promoter constructs (Mut 1, Mut 2), upon transient transfection into the HEK293FT cells. d Western blot analysis showing MYC expression in the HEK293FT cells transfected with the MYC expressing plasmid construct. γ-tubulin was used as a loading control. e Induction of miR-193a and MYC expression in the HEK293FT cells transfected with the MYC expressing construct evaluated by real-time RT-PCR analysis. f and g . Expression levels of MYC and the methylation status of a CpG probe (cg22536383) in the miR-193a promoter region, in the four molecular subgroups of medulloblastomas from the MAGIC cohort, respectively. Higher β values indicate higher methylation at the CpG residue. h Fold change in the expression levels of miR-193a and WIF1 in the 5-aza-2′-deoxycytidine treated medulloblastoma cells evaluated by the real-time RT-PCR assay. **, *** and ns indicates p < 0.001, p < 0.0001 and non-significant, respectively

Article Snippet: Medulloblastoma cell lines Daoy and D283 were obtained from the American Type Culture Collection, MA, USA [ ].

Techniques: Expressing, Methylation, DNA Methylation Assay, Luciferase, Activity Assay, Construct, Mutagenesis, Binding Assay, Transfection, Western Blot, Plasmid Preparation, Control, Quantitative RT-PCR, Residue

Journal: Acta Neuropathologica Communications

Article Title: Restoration of miR-193a expression is tumor-suppressive in MYC amplified Group 3 medulloblastoma

doi: 10.1186/s40478-020-00942-5

Figure Lengend Snippet: Effect of miR-193a expression on the proliferation and radiation sensitivity of the medulloblastoma cells evaluated by the MTT assay and the cell cycle analysis by the flow cytometry assay. Medulloblastoma cells expressing the parental pTRIPZ vector alone (Vector control) and the P1/P2 populations expressing doxycycline-inducible miR-193a-pTRIPZ construct were treated with doxycycline before evaluation by the MTT assay or the flow cytometry analysis. a Growth curves of the indicated medulloblastoma cells evaluated by the MTT assay. b Cell cycle analysis of indicated medulloblastoma cells, stained with Propidium iodide and, evaluated by flow cytometry. c Western blot analysis of PARP, a marker of apoptotic cell death, in the indicated medulloblastoma cells. The blot was also probed with anti-GAPDH antibody to serve as a loading control. d Y-axis denotes the surviving fraction of the indicated medulloblastoma cells upon irradiation at a dose ranging from 2 Gy to 6 Gy. VC: Vector control; **, *** and ns indicate p < 0.001, p < 0.0001, respectively

Article Snippet: Medulloblastoma cell lines Daoy and D283 were obtained from the American Type Culture Collection, MA, USA [ ].

Techniques: Expressing, MTT Assay, Cell Cycle Assay, Flow Cytometry, Plasmid Preparation, Control, Construct, Staining, Western Blot, Marker, Irradiation

Journal: Acta Neuropathologica Communications

Article Title: Restoration of miR-193a expression is tumor-suppressive in MYC amplified Group 3 medulloblastoma

doi: 10.1186/s40478-020-00942-5

Figure Lengend Snippet: Effect of miR-193a expression on the anchorage-independent growth and the tumorigenicity of medulloblastoma cells. a Y-axis denotes the number of soft agar colonies formed by the indicated medulloblastoma cells, upon doxycycline treatment as a percentage of the untreated control cells. b , c In vivo bioluminescence images of the orthotopic tumors at day 2 and day 21 post-injection of doxycycline-treated vector control cells (Vector control) or miR-193a expressing medulloblastoma cells (MiR-193a) in the cerebellar region of the NOD/SCID mice. d Y-axis denotes the fold change in the tumor growth on day 21 compared to that on day two, as evaluated by the change in the average luminescence of the tumor area. e Kaplan Meier survival analysis of the mice injected with the doxycycline-treated vector control or miR-193a expressing medulloblastoma cells of the indicated cell line. P1, P2: Medulloblastoma cells expressing miR-193a upon doxycycline treatment. **, *** indicate p < 0.001, p < 0.0001 respectively. H.R. = Hazard Ratio

Article Snippet: Medulloblastoma cell lines Daoy and D283 were obtained from the American Type Culture Collection, MA, USA [ ].

Techniques: Expressing, Control, In Vivo, Injection, Plasmid Preparation

Journal: Acta Neuropathologica Communications

Article Title: Restoration of miR-193a expression is tumor-suppressive in MYC amplified Group 3 medulloblastoma

doi: 10.1186/s40478-020-00942-5

Figure Lengend Snippet: Identification of genes targeted by miR-193a by the transcriptome sequencing of the HD-MB03 medulloblastoma cells expressing miR-193a, validation of known and novel miR-193a targets. a GSEA analysis showed enrichment of miR-193a targets (Net Enrichment Score = 1.67; p = .003) in the genes differentially expressed upon the miR-193a expression as identified by the transcriptome sequencing. Heat map generated by this GSEA analysis shows downregulation of known and putative miR-193a targets in the P1, P2, the two independent cell populations expressing miR-193a as compared to the parental control [C] and the vector control [VC] cells. The expression values are represented as colors, where the range of colors (red, pink, light blue, dark blue) shows the range of expression values (high, moderate, low, lowest). b Y-axis denotes the relative activity of the luciferase reporter of the 3′-UTR construct of the indicated gene before and after miR-193a expression. Mut: Mutant 3′-UTR construct. c Mutations introduced in the miR-193a binding site by the site-directed mutagenesis in the 3′-UTR constructs of the indicated gene. d Real-time RT-PCR analysis of miR-193a target genes, upon doxycycline induction of miR-193a expression, in the P1 and P2 populations as compared to the doxycycline-treated vector control cells. e Western blot analysis of miR-193a targets MAX, CCND1, MCL1, and that of the cell cycle inhibitor p16 (CDKN2A) in the P1, P2 populations upon miR-193a expression as compared to the doxycycline-treated vector control cells. The expression levels of GAPDH, a housekeeping gene, were used as a loading control. The numbers below each blot indicate the fold change in the expression levels of the indicated gene in the P1, P2 population as compared to that in the vector control cells. *, **, *** and ns indicate p < 0.01, p < 0.001, p < 0.0001 and non-significant, respectively

Article Snippet: Medulloblastoma cell lines Daoy and D283 were obtained from the American Type Culture Collection, MA, USA [ ].

Techniques: Sequencing, Expressing, Biomarker Discovery, Generated, Control, Plasmid Preparation, Activity Assay, Luciferase, Construct, Mutagenesis, Binding Assay, Quantitative RT-PCR, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Restoration of miR-193a expression is tumor-suppressive in MYC amplified Group 3 medulloblastoma

doi: 10.1186/s40478-020-00942-5

Figure Lengend Snippet: Interaction network analysis of the pathways significantly enriched in the genes downregulated upon miR-193a expression, and the change in the expression levels of the core histone marks in the medulloblastoma cells upon miR-193a expression. a The 439 genes significantly downregulated (padj < 0.05, log2 fold change < 0.8) upon miR-193a expression were analyzed for their interaction and functional enrichment (p adj < 0.01) in the pathways from the KEGG and Reactome database using the ClueGO application in the Cytoscape software. MiR-193a targets E2F1, MAX, KMT2A are highlighted in the interaction network. The fill color of the node and that of the label indicate the statistical significance of the enrichment of the pathway. The higher size of the node indicates a higher number of interacting genes. b . Western blot analysis of the histone marks, H3K4me3, H3K27ac, and H3K27me3 in the medulloblastoma cells. The numbers below each band indicate the fold change in the expression levels of the histone mark in the P1, P2 populations expressing miR-193a as compared to the vector control cells after normalization using the total histone H3 levels as the loading controls

Article Snippet: Medulloblastoma cell lines Daoy and D283 were obtained from the American Type Culture Collection, MA, USA [ ].

Techniques: Expressing, Functional Assay, Software, Western Blot, Plasmid Preparation, Control

Journal: The Journal of biological chemistry

Article Title: Resolving the polycistronic aftermath: Essential role of topoisomerase IA in preventing R-loops in Leishmania.

doi: 10.1016/j.jbc.2024.107162

Figure Lengend Snippet: Figure 1. Topoisomerase IA, an essential protein of L. donovani. A, sequence comparison of E. coli TOPIA, M. tuberculosis TOPIA, T. brucei TOPIA and L. donovani TOPIA showing start and end residue of TOPRIM domain, Active site tyrosine and DNA binding domain region. B, homology modeled structure of LdTOPIA with the active site residues Tyr357, Glu135, Asp131, and Asp 133 exhibited in the zoomed image. C, microscopic images of (−Tet) tetracycline uninduced (top) and (+Tet) induced (bottom) LtT7TR parasites expressing antisense LdTOPIA construct, Scale bar: 25 μm (ii) Graphical representation of percentage viable LtT7TR parasites in (−Tet) and (+Tet) condition for indicated time points. (n = 5 mean ± SD, 3 biological replicates. p vs. respective control (0h)). D, relative quantitation of LtTOPIA, LtTOPIL, and Ltβ-Tub mRNA expression levels in (+Tet) parasites measured by qPCR and plotted as normalized values over 24 h (n = 3 mean ± SD, 3 biological replicates. p versus tetracycline treated for 24 h). E, flow cytometric analysis of cell-cycle arrest in antisense LtTOPIA transfected LtT7TR parasites without (−Tet, green) or with (+Tet, red) induction at indicated timepoints (representative image of n = 3). F, graphical representation of the cell cycle phases (G0-G1, S, G2-M, and 4N) for antisense LtTOPIA transfected LtT7TR parasites without (−Tet) or with (+Tet) induction for indicated time points (n = 5, mean ± SD, 3 biological replicates for each time).

Article Snippet: The DNA relaxation assay was performed using E. coli DNA gyrase (NEB), Human TOPII (Sigma), and LdTOPIA using DNA relaxation assay buffer with negative and positive supercoil DNA (pBluescript) at 37 C. The assay mixtures were run in 1% TBE agarose gel at constant 25V, 6h in 1× TBE buffer followed by 90 rotation and run in the second direction at constant 80V, 2 h in 1× TBE buffer containing 4 μg/ml chloroquine (44).

Techniques: Sequencing, Comparison, Residue, Binding Assay, Expressing, Construct, Control, Quantitation Assay, Transfection

Journal: The Journal of biological chemistry

Article Title: Resolving the polycistronic aftermath: Essential role of topoisomerase IA in preventing R-loops in Leishmania.

doi: 10.1016/j.jbc.2024.107162

Figure Lengend Snippet: Figure 3. Functional characterization of purified LdTOPIA. A, SDS-PAGE (10%) analysis of purified LdTOPIA, LdTOPIAY357A, and LdTOPIAE135A from tetracycline induced, pLew100v5 cloned LdTOPIA, LdTOPIAY357A, and LdTOPIAE135A transfected LtT7TR conditional expression system, stained with Coo- massie G-250. Plasmid DNA relaxation assay using (−SC) pBluescript and (B) LdTOPIA or its active site mutants LdTOPIAY357A and LdTOPIAE135A or (C) LdTOPIA along with Camptothecin (CPT) or Etoposide (Etop) and in presence of Mg2+ at 37 C for 25 min followed by electrophoresis in 1% agarose gel and thereafter EtBr staining for visualization. D, plasmid DNA relaxation assay using (−SC) pBluescript, increasing concentration of Mg2+ and purified LdTOPIA at 37 C for 15 min. E, bidirectional agarose gel electrophoresis using (−SC) pBluescript and reverse gyrase generated (+SC) pBluescript plasmid DNA incubated with Human TOPII and purified LdTOPIA in order to differentiate the relaxation of negative and positive topoisomers. F, electrophoretic mobility shift assay (EMSA) using 100 nM γ-32P labeled (i) single-stranded and (ii) double-stranded oligonucleotide substrates incubated with increasing concentrations of LdTOPIA (5–200) nM (G) DNA binding affinity was measured by fluorescence polarization using 50 FAM tagged ssDNA and dsDNA substrate incubated with increasing concentration of LdTOPIA. Fraction-bound values were plotted against LdTOPIA concentration (2–200) nM and KD values of LdTOPIA were calculated for ssDNA and dsDNA (n = 5, mean ± SD, 3 biological replicates).

Article Snippet: The DNA relaxation assay was performed using E. coli DNA gyrase (NEB), Human TOPII (Sigma), and LdTOPIA using DNA relaxation assay buffer with negative and positive supercoil DNA (pBluescript) at 37 C. The assay mixtures were run in 1% TBE agarose gel at constant 25V, 6h in 1× TBE buffer followed by 90 rotation and run in the second direction at constant 80V, 2 h in 1× TBE buffer containing 4 μg/ml chloroquine (44).

Techniques: Functional Assay, SDS Page, Clone Assay, Transfection, Expressing, Staining, Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis, Concentration Assay, Generated, Incubation, Electrophoretic Mobility Shift Assay, Labeling, Binding Assay

Journal: The Journal of biological chemistry

Article Title: Resolving the polycistronic aftermath: Essential role of topoisomerase IA in preventing R-loops in Leishmania.

doi: 10.1016/j.jbc.2024.107162

Figure Lengend Snippet: Figure 4. LdTOPIA can functionally complement the RFM475 (E. coli TOPIA null GyrB ts) strain. A-C, To LB media agar plates containing ampicillin, the following strains RFM475 transformed with pBAD24, pBAD24-LdTOPIA were streaked and grown at non-permissible temperature 30 C for 36 h. D, SDS- PAGE (10%) analysis of purified LdTOPIA and its deletion mutants stained with Coomassie G-250. E, plasmid relaxation assay using 10 nM of each purified protein LdTOPIA, LdTOPIAΔNLS, LdTOPIA1-684, LdTOPIA1-640 at 37 C.

Article Snippet: The DNA relaxation assay was performed using E. coli DNA gyrase (NEB), Human TOPII (Sigma), and LdTOPIA using DNA relaxation assay buffer with negative and positive supercoil DNA (pBluescript) at 37 C. The assay mixtures were run in 1% TBE agarose gel at constant 25V, 6h in 1× TBE buffer followed by 90 rotation and run in the second direction at constant 80V, 2 h in 1× TBE buffer containing 4 μg/ml chloroquine (44).

Techniques: Transformation Assay, SDS Page, Staining, Plasmid Preparation

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Family pedigree of patients 1 and 2 (P1 and P2), with segregation of the TMEFF1 mutations (red). b , Brain images for P1 and P2, with yellow arrows showing the lesions observed during HSE. c , Schematic of TMEFF1 cDNA and protein structure and the position of the two mutated residues. SP, signal peptide; TM, transmembrane domain. d , Graph showing the CADD scores of all TMEFF1 non-synonymous or essential splice-site variants reported in the homozygous state in the gnomAD database (v.4.1.0.) and their MAFs. Mutation significance cut-offs (MSCs) are shown for 95% and 99% confidence intervals. e , Amounts of TMEFF1 mRNA, as measured by RT–qPCR, in various human tissues. Data shown are from two independent experiments. GUS , β-glucuronidase.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Mutagenesis, Quantitative RT-PCR

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , CoNeS analysis of negative selection for TMEFF1 plotted against the density distribution for genes underlying autosomal dominant (AD) inborn errors of immunity (IEI), autosomal recessive (AR) IEI, and AR/AD IEI. b , VirScan test for antibodies against a wide range of viruses and other pathogens in the serum of the two patients, 8 years (P1) and 10 years (P2) after the HSE episode, and in their parents. The numerical values in the heatmap represent the number of enriched non-overlapping peptides recognized by the antibodies in the serological sample. c , Viral serological test on the two patients, 8 years (P1) and 10 years (P2) after the HSE episode, and their relatives. Samples with values below the detection threshold are indicated as N (negative). d , Electropherogram showing the TMEFF1 gDNA sequences surrounding the mutations of interest, in a healthy control (Ctrl), P1, P2, and their parents. e , Alignment of TMEFF1 protein sequences around the P44 residue or the cytoplasmic tail, across species. Numbers in parentheses indicate the amino-acid position in each organism.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Selection, Control, Residue

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Schematic representation of the experimental design of the TOPO-cloning (upper panel) and exon-trapping (lower panel) experiments. In brief, for TOPO-TA, 1418 bp of cDNA, corresponding to exons 8 to 10 and including the 3’UTR and a polyA tail, for a control or P2 was extracted, amplified and inserted into a reporter vector for cDNA sequencing. For the exon-trapping experiment, a 5540 bp gDNA sequence for a control or P2 was amplified with forward and reverse primers, as shown. All of exons 9 and 10, including most of the 3’UTR, was amplified. The amplified gDNA was then digested with the BamHI and Xho1 enzymes and inserted into a pTAG4 vector, which was then used to transfect COS-7 cells for DNA extraction. P2’s essential splice-site mutation is indicated by a lightning bolt. b , Image of the gel, showing the fragment of cDNA amplified in the exon-trapping experiment, after amplification, insertion into the pTAG4 plasmid, the transfection of COS-7 cells, extraction of mRNA and amplification of the cDNA by PCR. The fragment obtained for the patient is of slightly lower molecular weight. Representative data from three independent experiments are shown. c , Sequencing results for the cDNA extracted from COS-7 cells following transfection with the pTAG4 plasmid containing gDNA from a control and P2, after the exon-trapping experiment as described in a - b . Experiments with the patient’s cDNA indicated that a single transcript lacking the first 21 nucleotides of exon 10 was produced. The data shown are representative of three independent experiments. d , Sequencing results for the TMEFF1 cDNA extracted from primary fibroblasts from a control and P2, after the TOPO-TA experiment. Experiments with the patient’s cDNA indicated that a single transcript lacking the first 21 nucleotides of exon 10 (P2-M1) was produced, as in the exon-trapping experiment, but there were also two additional transcripts with larger deletions encompassing the entire coding sequence of exon 10 and part of the 3’UTR (P2-M2) and encompassing the entire coding sequence of exons 9 and 10 and part of the 3’UTR (P2-M3). Data representative of three independent experiments are shown. e , Immunostaining for TMEFF1 in HeLa cells transfected with plasmids containing wild-type (WT) untagged, or N-ter Myc-tagged or C-ter Myc-tagged TMEFF1 cDNA sequences, showing that the C-ter Myc-tag impairs the expression of TMEFF1 at the cell surface. The data shown are representative of three independent experiments. f , Immunoblotting for TMEFF1 in HEK293T cells transfected with plasmids containing wild-type (WT) or mutant TMEFF1 sequences without a tag (upper panel), or with a C-ter Myc-tag (lower panel). The data shown are representative of three independent experiments. g , Cell membrane labeling with two different staining kits, MemBrite (green) and wheatgerm agglutinin (WGA, white). h , TMEFF1 immunostaining in HeLa cells transfected with an empty vector (EV) or with plasmids containing wild-type (WT) or various patient-specific mutant TMEFF1 cDNA sequences. WGA: cell membrane marker. Blue indicates DAPI chromosome staining. Data representative of three independent experiments are shown.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Cloning, Control, Amplification, Plasmid Preparation, Sequencing, DNA Extraction, Mutagenesis, Transfection, Extraction, Molecular Weight, Produced, Immunostaining, Expressing, Western Blot, Membrane, Labeling, Staining, Marker

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Relative abundance of TMEFF1 cDNA isoforms generated from mRNA extracted from primary fibroblasts from a healthy control (Ctrl) and P2, as assessed by TOPO-TA cloning. Mutant isoforms are shown in red. b , Schematic representation of TMEFF1 protein structure and the impact of P1’s missense mutation, and P2’s mutation resulting in three mutant isoforms (P2-M1, P2-M2, P2-M3). c , Amounts of TMEFF1 mRNA, as measured by RT–qPCR on HEK293T cells, not transfected (NT) or transfected with an empty vector (EV) or with plasmids containing WT or various patient-specific mutant TMEFF1 cDNA sequences. Two probes, targeting exons 1–2 (left) and exons 9–10 (right) of TMEFF1 , were used. Data are presented as mean ± s.d. d , TMEFF1 protein levels, as assessed by western blotting on HEK293T cells, NT or transfected with various plasmids as in c . Protein lysates were either left untreated or were treated with peptide: N -glycosidase F (PNGase F). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. e , TMEFF1 protein levels, as assessed by flow cytometry, in permeabilized and unpermeabilized HEK293T cells (right), NT or transfected with various plasmids as in c . Cell-surface TMEFF1 expression was quantified in unpermeabilized cells (left). Data are presented as mean ± s.d. Statistical analysis was done with Kruskal–Wallis tests with Dunn’s test for multiple comparisons. NS, not significant; * P < 0.05. MFI, mean fluorescence intensity. f , TMEFF1 immunostaining in HeLa cells transfected with an EV or with plasmids containing WT or various patient-specific mutant TMEFF1 cDNA sequences. MemBrite is a cell membrane marker. Blue indicates DAPI chromosome staining. Scale bars, 20 μm. The data shown in c – f are representative of three independent experiments.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Generated, Control, TA Cloning, Mutagenesis, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Flow Cytometry, Expressing, Fluorescence, Immunostaining, Membrane, Marker, Staining

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Levels of TMEFF1 mRNA, as determined by RT–qPCR, in various human cell lines or primary cells. b , TMEFF1 mRNA levels were determined by RT–qPCR in cortical neurons from control and TMEFF1 -KO hPSCs. c , TMEFF1 protein expression was studied by confocal microscopy on cortical neurons derived from healthy control and TMEFF1 -KO hPSCs. Cells were fixed and stained for TMEFF1 (anti-TMEFF1 antibody, green), cell membrane (wheat germ agglutinin (WGA), white) and chromosomes (DAPI, blue). Scale bar, 10 μm. d , Cortical neurons derived from hPSCs from healthy controls, TMEFF1 -KO hPSCs and TLR3 −/− hPSCs were infected with HSV-1 (MOI 0.001) and assessed for HSV-1 titres at the timepoints indicated. TCID 50 , 50% tissue culture infectious dose. e , TMEFF1 mRNA levels were determined by RT–qPCR on hPSC-derived cortical neurons for healthy controls and the two patients with TMEFF1 mutations (P1 and P2). f , Relative abundance of TMEFF1 cDNA isoforms generated from mRNA extracted from hPSC-derived cortical neurons for healthy controls and P2, as assessed by TOPO-TA cloning. g , h , hPSC-derived cortical neurons from a healthy control (H9), the patients with TMEFF1 mutations (P1 and P2) and other TLR3 −/− and IFNAR1 −/− HSE patients were infected with HSV-1 (MOI 0.001) and assessed for HSV-1 titres at the timepoints indicated, without ( g ) or with ( h ) IFNβ pretreatment for 18 h. The data shown in a , b , d , e , g and h are mean ± s.e.m. of three independent experiments. Statistical analysis: for b and e , two-tailed Mann-Whitney U -tests; for d , g and h , mean log-transformed relative values were compared between control cells and TMEFF1 -mutated cells in one-way analysis of variance (ANOVA) with Tukey tests for multiple comparisons. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Quantitative RT-PCR, Control, Expressing, Confocal Microscopy, Derivative Assay, Staining, Membrane, Infection, Generated, TA Cloning, Two Tailed Test, MANN-WHITNEY, Transformation Assay

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Electropherogram representation (left panels) of the CRISPR-Cas9-introduced compound-heterozygous TMEFF1 mutations confirmed by Sanger sequencing on genomic DNA from a gene-edited TMEFF1 KO hPSC line (TMEFF1 KO #1). Sequencing results for the parental line (Ctrl parental, BJ1) are also shown. The relative abundance of WT or mutated TMEFF1 cDNA generated from mRNA extracted from the control parental and TMEFF1 KO hPSCs was assessed by TOPO-TA cloning and is shown in the right panels. b , Representative images of cortical neurons from controls (Ctrl 1-H9, Ctrl 2 parental-BJ1) and TMEFF1 KO hPSCs. Cells were fixed and stained with DAPI (blue) and for a neuron-specific marker, microtubule-associated protein 2 (MAP2, green). c , FOXG1 and PAX6 mRNA levels, as measured by RT-qPCR, in cortical neurons from control and TMEFF1 KO hPSCs. SV40-transformed fibroblasts from healthy controls (Fibros ctrl 1, Fibros ctrl 2) were used as a negative control in this assay. d , Representative images of cortical neurons from control (H9) and various patient-specific hPSC lines (P1, P2, IFNAR1 −/− , TLR3 −/− ). Cells were fixed and stained with DAPI (blue), and for a neuron-specific marker MAP2 (green). e , FOXG1 and PAX6 mRNA levels were measured by RT-qPCR in cortical neurons derived from control and patient-specific hPSC lines. SV40-transformed fibroblasts from healthy controls (Fibros ctrl 3, Fibros ctrl 4) were used as a negative control in this assay. f , Electropherogram representation of the three TMEFF1 mutant isoforms, as detected by TOPO-cloning of cDNA from P2’s hPSC-derived cortical neurons.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: CRISPR, Sequencing, Generated, Control, TA Cloning, Staining, Marker, Quantitative RT-PCR, Transformation Assay, Negative Control, Derivative Assay, Mutagenesis, Cloning

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , TMEFF1 mRNA levels were determined by RT-qPCR, in SV40-transformed fibroblasts from the patients with TMEFF1 mutations, a TLR3 −/− HSE patient, and healthy controls treated with poly(I:C) for 2 or 4 h or left untreated (NS). b , TMEFF1 mRNA levels were measured by RT-qPCR, in SV40-transformed fibroblasts from patients with TMEFF1 mutations, an IFNAR1 −/− HSE patient, and healthy controls treated with IFN-α2b for 8 h or left untreated. c , TMEFF1 mRNA levels were measured by RT-qPCR in cortical neurons derived from control parental or TMEFF1 KO hPSCs, and hPSCs from a TLR3 −/− HSE patient, after treatment with poly(I:C) for 6 h, or without treatment. d , TMEFF1 mRNA levels were measured by RT-qPCR, in cortical neurons derived from control parental or TMEFF1 KO hPSCs, and hPSCs from an IFNAR1 −/− HSE patient treated with IFN-β for 8 h or left untreated. In a - d , two probes, targeting exons 1-2 (upper panels) and exons 9-10 (lower panels) of TMEFF1 were used. The data shown are the means ± SEM from three ( a , b ) or two ( c , d ) independent experiments. e , Abundance of TMEFF1 mRNA, as assessed by RNAseq, in healthy control neurons (Ctrls, n = 6), SNORA31 -mutated ( SNORA31 -MT, n = 8), TLR3 −/− ( n = 2) or STAT1 −/− ( n = 2) hPSC-derived cortical neurons treated with poly(I:C) or IFN-α2b, or left unstimulated (NS). Data are presented as mean ± SD. f , Abundance of TMEFF1 mRNA, as assessed by RNAseq, in healthy controls (Ctrls, n = 6), SNORA31 -mutated ( SNORA31 -MT, n = 8) or STAT1 −/− ( n = 2) hPSC-derived cortical neurons infected with HSV-1 for 24 h, or left unstimulated (NS). g , IFNB1 (upper panel) or IFNL1 (lower panel) mRNA levels were measured by RT-qPCR, in SV40-transformed fibroblasts from the patients with TMEFF1 mutations, a TLR3 −/− HSE patient, and healthy controls, after treatment with poly(I:C) for 2 or 4 h or without treatment. h , MX1 (upper panel) or IFIT1 (lower panel) mRNA levels were measured by RT-qPCR, in SV40-transformed fibroblasts from the patients with TMEFF1 mutations, an IFNAR1 −/− HSE patient, and healthy controls, after treatment with IFN-α2b for 8 h, or without treatment. The data shown in g , and h are the means ± SEM from three independent experiments. i , Basal levels of IFNAR1 (top panel), IFNAR2 (middle panel), and TLR3 (lower panel) mRNA were measured by RT-qPCR, in hPSC-derived cortical neurons from healthy controls (Ctrl 1-H9, Ctrl 2-Parental BJ1), TMEFF1 KO hPSCs, or hPSCs from TMEFF1-mutated patients. j , Levels of MX1 (upper panels) or IFIT1 (lower panels) mRNA were measured by RT-qPCR, in cortical neurons derived from control parental or TMEFF1 KO hPSCs, hPSCs from a TLR3 −/− HSE patient, and an IFNAR1 −/− HSE patient, with and without treatment with poly(I:C) for 6 h (left panels), or with IFN-β for 8 h (right panels). Statistical analysis was performed with two-tailed Mann-Whitney U tests. ns: not significant. k , Scatterplots of the mean log 2 fold-changes in RNAseq-quantified gene induction following stimulation with 100 IU/ml IFN-β for 8 h (upper panel) or HSV-1 (MOI 1) for 24 h (lower panel) in hPSC-derived CNS cortical neurons from two healthy controls (Ctrl1-H9, Ctrl2 parental-BJ1), TMEFF1-mutated patients (TMEFF1 Pts) or TMEFF1 KO hPSCS, or hPSCs from an IFNAR1 −/− HSE patient. Each point represents a single gene. Genes with an absolute fold-change in expression > 2 in response to IFN-β or HSV-1 treatment relative to NS samples in the Ctrl group are plotted. l , Heatmaps of RNA-Seq-quantified gene expression (z-score-scaled DESeq2 vst-normalization) in hPSC-derived CNS cortical neurons from healthy controls (Ctrl 1-H9, Ctrl 2-Parental BJ1) or TMEFF1 KO hPSCS, or hPSCs from an IFNAR1 −/− HSE patient, a TLR3 −/− HSE patient and TMEFF1-mutated P1 and P2 (TMEFF1 Pts), not stimulated (NS), stimulated with HSV-1 for 24 h, or stimulated with IFN-β for 8 h. Duplicates were studied for each set of conditions and mean gene expression levels were used for subsequent analyses. The heatmap includes genes with a relative fold-change in expression > 2 in response to HSV-1 or IFN-β treatment relative to NS samples in the Ctrl group.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Quantitative RT-PCR, Transformation Assay, Derivative Assay, Control, Infection, Two Tailed Test, MANN-WHITNEY, Expressing, RNA Sequencing, Gene Expression

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , RFP intensity in HeLa and HEK293T cells, as measured in an HSV-1-RFP nuclear translocation reporter assay 10 h after infection with an RFP-reporter HSV-1, on parental WT cells or IFNAR1 KO cells transfected with an empty vector (EV) or WT TMEFF1 expression construct. HeLa or HEK293T cells were labeled with an anti-TMEFF1 antibody. RFP intensity was assessed under a confocal microscope. Statistical analysis was performed with two-tailed Mann-Whitney U tests. *** p -value < 0.001. The data shown are representative of three independent experiments. b , TMEFF1 mRNA levels in HeLa (left panel) and HEK293T cells (right panel), either parental WT (top) or IFNAR1 KO (bottom), transfected with an EV or a WT TMEFF1 expression plasmid, as measured by RT-qPCR. A probe targeting exons 1-2 of TMEFF1 was used. c , TMEFF1 mRNA levels in HeLa cells transfected with an EV, or a WT or mutant TMEFF1 expression plasmid, as measured by RT-qPCR. A probe targeting exons 1-2 of TMEFF1 was used. d , Representative images of HeLa cells transfected with an EV, or a WT or mutant TMEFF1 expression plasmid, in an HSV-1-RFP nuclear translocation reporter assay. The cells were fixed and identified by DAPI staining (blue). HSV-1-RFP infection results in the expression of RFP, the levels of which were assessed in the nucleus at 10 hpi. e , TMEFF1 mRNA levels in HeLa cells transfected with an EV, or a WT full-length (FL) TMEFF1 or different domains of TMEFF1 in an expression plasmid (EX: extracellular domain; TM + IN: transmembrane and intracellular domains), as measured by RT-qPCR. Two probes, targeting exons 1-2 (upper panel) and exons 9-10 (lower panel) of TMEFF1 , were used. The data shown are representative of three independent experiments. f , TMEFF1 protein levels, as assessed by western blotting in HeLa cells transfected with an EV, or a WT FL or different domains of TMEFF1 in an expression plasmid. g , TMEFF1 immunostaining in HeLa cells transfected with an EV, or a WT FL or different domains of TMEFF1 in an expression plasmid. HeLa cells were labeled with anti-TMEFF1 antibody (green) and DAPI (blue) and TMEFF1 overexpression was assessed under a confocal microscope. h , TMEFF1 mRNA levels in HeLa cells transfected with an EV or with plasmids containing WT or gnomAD homozygous mutant (H104Y, E134V, P255S, G281V, I284F, A297V, I344V) TMEFF1 cDNAs, as measured by RT-qPCR. Two probes, targeting exons 1-2 (upper panel) and exons 9-10 (lower panel) of TMEFF1 , were used. i , TMEFF1 immunostaining in HeLa cells transfected with an EV or with plasmids containing WT or gnomAD homozygous mutant (H104Y, E134V, P255S, G281V, I284F, A297V, I344V) TMEFF1 cDNAs. HeLa cells were labeled with anti-TMEFF1 antibody (green), membrane stain (MemBrite, white), and DAPI (blue) and TMEFF1 overexpression was assessed under a confocal microscope. The data shown in c - i are representative of three independent experiments. j , TMEFF1 protein levels, as assessed by western blotting on HeLa cells transfected with an EV or with plasmids containing WT or gnomAD homozygous mutant (H104Y, E134V, P255S, G281V, I284F, A297V, I344V) TMEFF1 cDNAs. k , Measurement of RFP intensity in an HSV-1-RFP nuclear translocation reporter assay, 10 h after infection, in HeLa cells cells transfected with an EV or with plasmids containing WT or gnomAD homozygous mutant (H104Y, E134V, P255S, G281V, I284F, I344V) TMEFF1 cDNAs. HeLa cells were labeled with anti-TMEFF1 antibody and DAPI. RFP intensity was assessed under a confocal microscope. Statistical analysis was conducted with Kruskal-Wallis tests with Dunn’s test for multiple comparisons. ns: not significant, * p -value < 0.05; **** p -value < 0.0001. The data shown in j - k are representative of three independent experiments.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Translocation Assay, Reporter Assay, Infection, Transfection, Plasmid Preparation, Expressing, Construct, Labeling, Microscopy, Two Tailed Test, MANN-WHITNEY, Quantitative RT-PCR, Mutagenesis, Staining, Western Blot, Immunostaining, Over Expression, Membrane

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , b , Measurement of HSV-1–RFP intensity in HeLa cells overexpressing an EV, WT or various patient-specific TMEFF1 mutants ( a ) or WT full-length TMEFF1 , TMEFF1 extracellular domain (EX) or transmembrane and intracellular domains (TM + IN) ( b ) at 10 h post-infection (hpi). Statistical analysis: Kruskal–Wallis tests with Dunn’s test for multiple comparisons; *** P < 0.001. a.u., arbitrary units. c , d , HEK293T cells were cotransfected with Flag-tagged NECTIN-1 and EV or WT TMEFF1 plasmids ( c ) or with WT TMEFF1 and EV or Flag-tagged NECTIN-1 plasmids ( d ), and subjected to immunoprecipitation (IP) with anti-TMEFF1 antibodies or anti-Flag antibody-conjugated agarose beads, and immunoblotting with anti-Flag or anti-TMEFF1 antibodies. e , HEK293T cells were infected with HSV-1 (MOI 1) and subjected to immunoprecipitation with mouse IgG isotype control or anti-NECTIN-1 antibody and immunoblotting with anti-NECTIN-1 or anti-TMEFF1 antibody. f , HEK293T cells were cotransfected with C-terminal Myc-tagged WT full-length or truncated (EX, TM + IN) TMEFF1 or N-terminal Myc-tagged WT full-length TMEFF1 plasmids with EV or Flag-tagged NECTIN-1 plasmids, and subjected to immunoprecipitation with anti-Myc antibody-conjugated agarose beads and immunoblotting with anti-Myc or anti-Flag antibody. g , HEK293T cells were cotransfected with the N-terminal Flag–GFP-tagged full-length or truncated (EX, TM + IN) NECTIN-1 plasmids with N-terminal Myc-tagged TMEFF1 plasmids, and subjected to immunoprecipitation with anti-Flag antibody-conjugated agarose beads and immunoblotting with anti-Myc or anti-Flag antibody. h , HEK293T cells were cotransfected with the N-terminal Flag-tagged WT full-length or EX NECTIN-1 plasmids and N-terminal Myc-tagged WT full-length or EX TMEFF1 plasmids, and subjected to immunoprecipitation with anti-Flag antibody-conjugated agarose beads and immunoblotting with anti-Myc or anti-Flag antibody. i , HEK293T cells were cotransfected with the N-terminal Flag-tagged NECTIN-1 and EV, WT or various patient-specific mutant TMEFF1 plasmids, and subjected to immunoprecipitation with anti-TMEFF1 antibody and immunoblotting with anti-TMEFF1 or anti-Flag antibody. The data shown in a – i are representative of three independent experiments.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Infection, Immunoprecipitation, Western Blot, Control, Mutagenesis

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a - h , HEK293T cells were cotransfected with N-ter Myc-tagged TMEFF1 constructs and Flag-tagged NECTIN-1 ( a ), HVEM ( b ), PILRa ( c ), gB ( d ), gC ( e ), gD ( f ), gH ( g ), or gL ( h ). The cells were then subjected to immunoprecipitation (IP) with mouse IgG isotype control or anti-TMEFF1 Ab, and immunoblotting with anti-Flag or anti-Myc Abs. i - j , N-ter Flag-tagged NECTIN-1 constructs were co-expressed with N-ter Myc-tagged WT full-length (FL) or different domains (EX, TM + IN) of TMEFF1 in expression constructs in HEK293T cells, which were subjected to IP with anti-Myc Ab-conjugated agarose beads ( i ), or anti-Flag Ab-conjugated agarose beads ( j ), and immunoblotting with anti-Myc or anti-Flag Ab. k , TMEFF1 mRNA levels, as determined by RT-qPCR, in HEK293T cells 24 h after transfection with N-ter Myc-tagged WT full-length (FL) or different domains (EX, TM + IN) of TMEFF1 in expression constructs. l , HEK293T cells were cotransfected with C-ter Myc-tagged WT FL, different domains (EX, TM + IN) of TMEFF1 in expression constructs, or an N-ter Myc-tagged WT FL TMEFF1 plasmid, together with EV or N-ter Flag-tagged NECTIN-1-expressing constructs. The cells were then subjected to IP with anti-Flag Ab-conjugated agarose beads and immunoblotting with anti-Myc or anti-Flag Ab. m , HEK293T cells were cotransfected with the N-ter Flag-GFP-tagged WT FL or different domains (EX, TM + IN) of NECTIN-1 in expression constructs together with N-ter Myc-tagged TMEFF1 constructs, and subjected to IP with anti-Myc Ab-conjugated agarose beads and immunoblotting with anti-Myc or anti-Flag Ab. For l - m , the red asterisk indicates non-specific binding. n , HEK293T cells were cotransfected with N-ter Flag-tagged NECTIN-1-expressing constructs and TMEFF1-expressing constructs containing the cDNA for the WT or homozygous TMEFF1 variants from the gnomAD database (H104Y, E134V, P255S, G281V, I284F, A297V, I344V). The cells were then subjected to IP with anti-Flag Ab-conjugated agarose beads, and immunoblotting with anti-TMEFF1 or anti-Flag antibodies. The red asterisk indicates non-specific binding. The data shown in a - n are representative of three independent experiments.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Construct, Immunoprecipitation, Control, Western Blot, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Binding Assay

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Levels of NECTIN1 mRNA, as determined by RT-qPCR, in various human cell lines or primary cells. The data shown are from two independent experiments. b , Abundance of the canonical NECTIN1 transcript (isoform 1, delta, ENST00000264025.8) as assessed by RNAseq, in cortical neurons derived from gene-edited line of TMEFF1 KO hPSCs, and from healthy controls (H9, BJ1). NECTIN1 transcripts for isoform 2 (ENST00000341398.6) and 3 (ENST00000340882.2) were undetectable. c , Electropherogram showing the TMEFF1 gDNA sequence at the sgRNA target region in WT and TMEFF1 KO HEK293T cells. d , Electropherogram showing the NECTIN1 gDNA sequence at the sgRNA target region in WT, NECTIN-1 KO, and TMEFF1 and NECTIN-1 double KO HEK293T cells. e , Levels of NECTIN1 (left panel) and TMEFF1 (center and right panels) mRNA, as determined by RT-qPCR, in WT, TMEFF1 KO, NECTIN-1 KO, and TMEFF1 and NECTIN-1 double KO HEK293T cells. Two probes, targeting exons 1-2 (center panel) and exons 9-10 (right panel), were used for TMEFF1 . f , Cell-surface NECTIN-1 protein expression assessed by flow cytometry in WT, TMEFF1 KO, NECTIN-1 KO, and TMEFF1 and NECTIN-1 double KO HEK293T cells.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Quantitative RT-PCR, Derivative Assay, Sequencing, Expressing, Flow Cytometry

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , TMEFF1 and NECTIN-1 localization in HeLa cells after cotransfection or single transfection with the TMEFF1 and NECTIN-1 plasmids. Green, TMEFF1; purple, NECTIN-1; blue, DAPI; white, MemBrite. Scale bars, 20 μm. b , HeLa cells were cotransfected with CFP-tagged TMEFF1 and YFP-tagged NECTIN-1 or HVEM, and subjected to FRET imaging. Scale bars, 20 μm. CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; Ex, excitation; Em, emission. c , Bleed-through-corrected FRET at the cell surface was quantified. d – f , Histograms of the surface His-tagged gD signal ( d ), the MFI of surface gD binding ( e ) and the percentage of surface gD-positive cells ( f ) after incubation with a His-tagged gD for 150 min in WT or TMEFF1 -KO HEK293T cells stably expressing NECTIN-1. g , HEK293T cells were cotransfected with Flag-tagged gD, Myc-tagged NECTIN-1 and EV or TMEFF1 plasmids, then subjected to immunoprecipitation with anti-Flag antibody-conjugated agarose beads, and immunoblotting with anti-Flag, anti-Myc and anti-TMEFF1 antibodies. The data shown in a – g are representative of three independent experiments. h, i , MFI of surface NECTIN-1 after gD treatment ( h ) or HSV-1 infection ( i ) relative to untreated cells (left) and MFI of total NECTIN-1 in the presence or absence of gD treatment or HSV-1 infection (right) on WT or TMEFF1 -KO HEK293T cells. j , HSV-1–RFP infection rates in WT and TMEFF1 -KO HEK293T cells 8 hours after infection. k , HSV-1–RFP intensity in WT and TMEFF1 -KO HEK293T cell nucleus 8 hours after infection. Data are shown as median ± interquartile range and are representative of five independent experiments. l , HSV-1–RFP infection rates in WT, TMEFF1 KO, NECTIN-1 KO and TMEFF1 -and- NECTIN-1 double-KO HEK293T cells, as assessed by flow cytometry 8 hours after infection. Data are shown as mean ± s.e.m. from three ( b , c , e and f ), six ( h and i ), five ( j ) or four ( l ) independent experiments. Statistical analysis was done for c , h , i , j , k and l using two-tailed Mann–Whitney U tests; * P < 0.05; *** P < 0.001.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Cotransfection, Transfection, Imaging, Binding Assay, Incubation, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Infection, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Representative gating strategy for HEK293T cells stably expressing NECTIN-1, not treated or treated with recombinant His-tagged HSV-1 gD for 150 min. b , Mean fluorescence intensity (MFI) of surface gD binding in NECTIN-1-positive or NECTIN-1-negative fractions of HEK293T cells stably expressing NECTIN-1, following incubation with different concentrations of His-tagged gD (gD-His-Tag) for 150 min. c , Histogram of surface NECTIN-1 expression in WT and TMEFF1 KO HEK293T cells incubated with recombinant His-tagged HSV-1 gD (5 µg/ml) for 150 min. The data shown are representative of three independent experiments. d , Histogram of surface NECTIN-1 expression in WT and TMEFF1 KO HEK293T cells infected with HSV-1 (MOI 10) for 45 min. The data shown are representative of three independent experiments. e , NECTIN1 mRNA levels (left panel) as determined by RT-qPCR in total RNA, and protein expression in cell total lysates as assessed by immunoblotting (right panel), in TMEFF1 KO or parental WT HEK293T cells upon gD treatment for 150 min or without treatment. f , NECTIN1 mRNA levels (left panel) as determined by RT-qPCR in total RNA, and protein expression in cell total lysates as assessed by immunoblotting (right panel), in TMEFF1 KO or parental WT HEK293T cells after infection with HSV-1 for 45 min, or without infection. g , Representative contour plots of the RFP signal in WT, TMEFF1 KO, NECTIN-1 KO, and TMEFF1 and NECTIN-1 double KO HEK293T cells infected with HSV-1-RFP (MOI 10) at 8 hpi. h , Basal mRNA levels for NECTIN-1 , HVEM , and PILRa , as assessed by RT-qPCR, in WT parental or TMEFF1 KO hPSC-derived cortical neurons and HEK293T cells (left panel), and WT HeLa cells (right panel). The data shown are the mean ± SEM from three independent experiments. i , MFI of N-ter YFP-tagged HVEM on the cell surface in NECTIN-1 KO and TMEFF1 and NECTIN-1 double KO HEK293T cells transfected with an empty vector (EV), or N-ter YFP-tagged HVEM-expressing plasmid. The data shown are the mean ± SEM from four independent experiments. j , Percentage of HSV-1-positive cells, as assessed in an assay of HSV-1 translocation to the cell nucleus 10 h after infection with an RFP-reporter HSV-1, in NECTIN-1 KO or TMEFF1 and NECTIN-1 double KO HEK293T cells transfected with an empty vector (EV) or HVEM-expressing plasmid. The data are presented as the mean ± SEM from four independent experiments. Statistical analysis was conducted with Kruskal-Wallis tests with Dunn’s test for multiple comparisons. ns: not significant.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Stable Transfection, Expressing, Recombinant, Fluorescence, Binding Assay, Incubation, Infection, Quantitative RT-PCR, Western Blot, Derivative Assay, Transfection, Plasmid Preparation, Translocation Assay

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Representative images of healthy control (Ctrl 2 parental-BJ1) and TMEFF1 -KO hPSC-derived cortical neurons, stained for endogenous TMEFF1 (green), NECTIN-1 (purple), chromosomes (DAPI, blue) and cell membrane (WGA, white) before and 10 h after infection (hpi) with an RFP reporter HSV-1 (red). The dashed grey line is located immediately beneath the WGA-stained cell membrane. The areas in white squares are enlarged in the image on the right. Scale bar, 10 μm. The images are representative of three independent experiments. NI, non-infected. b , Comparison of HSV-1 entry into heathy control (Ctrl 1-H9, Ctrl 2 parental-BJ1) and TMEFF1 -KO hPSC-derived cortical neurons in a β-lactamase assay (449/520 nm). Data are shown as median ± interquartile range and are representative of three independent experiments. c , Representative images of hPSC-derived cortical neurons in an HSV-1–RFP cell nuclear translocation reporter assay 10 h after infection. Neurons were identified by staining for microtubule-associated protein 2 (MAP2, green) and chromosomes (DAPI, blue). Scale bar, 10 μm. d , e , Percentage of HSV-1-positive ( d ) and cell nuclear RFP intensity ( e ) of healthy control and TMEFF1 -KO hPSC-derived cortical neurons 10 h after infection. f , Comparison of HSV-1 entry into healthy control (H9), IFNAR1 −/− , P1 and P2 hPSC-derived cortical neurons in the β-lactamase assay. g , h , Percentage of HSV-1-positive ( g ) and cell nuclear RFP intensity ( h ) of healthy control, IFNAR1 −/− , P1 and P2 hPSC-derived cortical neurons 10 h after infection. i , Percentage of HSV-1-positive TMEFF1 -KO cortical neurons transduced with EV, WT TMEFF1 or patient-specific TMEFF1 variant cDNA 10 h after infection. Data are shown as mean ± s.e.m. ( d , g and i ) or median ± interquartile range ( e , f and h ) from four ( d and e ) or three ( f , g , h and i ) independent experiments. Statistical analysis was done for d , g and i using two-tailed Mann–Whitney U tests, and for b , e , f and h using Kruskal–Wallis tests with Dunn’s test for multiple comparisons. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Control, Derivative Assay, Staining, Membrane, Infection, Comparison, Lactamase Assay, Translocation Assay, Reporter Assay, Transduction, Variant Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Electropherogram representation (left panels) of the CRISPR-Cas9-introduced compound-heterozygous TMEFF1 mutations confirmed by Sanger sequencing on genomic DNA from an additional gene-edited line of TMEFF1 KO hPSCs (TMEFF1 KO #2). Sequencing results for the parental line (Ctrl parental, BJ1) are also shown. The relative abundance of WT and mutated TMEFF1 cDNAs generated from mRNA extracted from the parental control and TMEFF1 KO hPSC clone #2 was assessed by TOPO-TA cloning and is shown in the panels on the right. b , TMEFF1 mRNA levels were determined by RT-qPCR on cortical neurons from control parental clones and two different clones of TMEFF1 KO human pluripotent stem cells (hPSCs). Two probes, targeting exons 1-2 (upper panel) and exons 9-10 (lower panel) of TMEFF1 , were used. The data shown are the mean ± SEM from three independent experiments. Statistical analysis was conducted with two-tailed Mann-Whitney U tests. ** p -value < 0.01. c , TMEFF1 protein expression was studied by confocal microscopy on cortical neurons derived from healthy control H9, control parental (BJ1) and TMEFF1 KO hPSCs. Cells were fixed and stained for TMEFF1 with anti-TMEFF1 antibody (green), the cell membrane was stained with WGA (white), and chromosomes were stained with DAPI (blue). d , Representative images of cortical neurons from controls (Ctrl 1-H9, Ctrl 2 parental-BJ1) and TMEFF1 KO hPSC line #2. Cells were fixed and stained with DAPI (blue) and with microtubule-associated protein 2 (MAP2, green) as a neuron-specific marker. e , FOXG1 and PAX6 mRNA levels, as measured by RT-qPCR, in cortical neurons from a control and TMEFF1 KO hPSC line #2. SV40-transformed fibroblasts from healthy controls (Fibros ctrl 1, Fibros ctrl 2) were used as a negative control in this assay.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: CRISPR, Sequencing, Generated, Control, TA Cloning, Quantitative RT-PCR, Clone Assay, Two Tailed Test, MANN-WHITNEY, Expressing, Confocal Microscopy, Derivative Assay, Staining, Membrane, Marker, Transformation Assay, Negative Control

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , Immunostaining of hPSC-derived cortical neurons from healthy control (H9) and TMEFF1 KO hPSCs (TMEFF1 KO #2) for endogenous TMEFF1 (green), NECTIN-1 (purple), AT-rich DNA (DAPI, blue) and the cell membrane (WGA, white) before and 10 h after infection with an RFP-reporter HSV-1 (red). The dashed white line is immediately beneath the WGA-stained cell membrane. b , Abundance of NECTIN1 mRNA, as assessed by RNAseq, in WT controls (Ctrl1-H9, Ctrl2 parental-BJ1) and TMEFF1 KO hPSC-derived cortical neurons infected with HSV-1 for 24 h or left unstimulated (NS). c , Immunostaining of hPSC-derived cortical neurons from a healthy control (Ctrl1-H9), an IFNAR1 −/− patient, and P1 and P2 with TMEFF1 mutations, in a reporter assay for the nuclear translocation of HSV-1, 10 h after infection with an RFP-reporter HSV-1. Neurons were fixed and identified by staining for a neuron-specific microtubule-associated protein 2 (MAP2, green) and with DAPI (blue). HSV-1-RFP infection results in the expression of RFP, which was detected in the nucleus 10 hpi. d , Replication levels for HSV-2, measles virus (MeV), or EMCV, following infection at various time points as indicated, in hPSC-derived cortical neurons from healthy controls (Ctrl1-H9, Ctrl2 parental-BJ1), or TMEFF1 KO hPSCs. RT-qPCR was performed with the SYBR green assay, to assess HSV-2 polymerase ( Pol ), MeV nucleocapsid ( N ) or EMCV 3D gene expression indicative of viral replication levels. The data are presented as the mean ± SD and are representative of four independent experiments.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Immunostaining, Derivative Assay, Control, Membrane, Infection, Staining, Reporter Assay, Translocation Assay, Expressing, Virus, Quantitative RT-PCR, SYBR Green Assay, Gene Expression

Journal: Nature

Article Title: Human TMEFF1 is a restriction factor for herpes simplex virus in the brain

doi: 10.1038/s41586-024-07745-x

Figure Lengend Snippet: a , TMEFF1 and NECTIN1 mRNA levels, as measured by RT-qPCR, in TMEFF1 KO cortical neurons transduced with an EV, WT TMEFF1 or patient-specific mutant TMEFF1-expressing lentivirus. Two probes, targeting exons 1-2 (left) and exons 9-10 (center) of TMEFF1 , were used. The data shown are representative of two independent experiments. b , Immunostaining of exogenous TMEFF1 (green), endogenous NECTIN-1 (purple), AT-rich DNA (DAPI, blue) and the cell membrane (WGA, white), for hPSC-derived TMEFF1 KO cortical neurons transduced with EV, WT TMEFF1 or patient-specific mutant TMEFF1-expressing lentiviruses. The data shown are representative of three independent experiments. c , Measurement of RFP intensity, 10 h after infection with HSV-1-RFP, in TMEFF1 KO cortical neurons transduced with EV, WT TMEFF1 or patient-specific mutant TMEFF1-expressing lentiviruses. Cortical neurons were labeled with anti-TMEFF1 antibody (green) and DAPI (blue). RFP intensity was assessed under a confocal microscope. HSV-1 infection results in the expression of RFP, which is detected in the nucleus at 10 hpi. Statistical analysis was conducted with Kruskal-Wallis tests with Dunn’s test for multiple comparisons. ** p -value < 0.01; **** p -value < 0.0001. The data are presented as the mean ± SEM from three independent experiments.

Article Snippet: RT–qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System with Applied Biosystems TaqMan assays for TMEFF1 (Hs00902905_m1, spanning exons 1–2; Hs00186495_m1, spanning exons 9–10), NECTIN-1 (Hs01591978_m1), HVEM (Tnfrsf14) (Hs00998605_g1), PILRa (Hs00956112_m1) and the β-glucuronidase (GUS, #4310888E) housekeeping gene for normalization.

Techniques: Quantitative RT-PCR, Transduction, Mutagenesis, Expressing, Immunostaining, Membrane, Derivative Assay, Infection, Labeling, Microscopy