Journal: bioRxiv

Article Title: Whole-genome analysis of noncoding genetic variations identifies multigranular regulatory element perturbations associated with Hirschsprung disease

doi: 10.1101/2020.04.08.032045

Figure Lengend Snippet: Functional impacts of a HSCR-associated SNP (rs2435357) and the deletion of a novel S-HSCR enhancer on RET expression. ( a ) ATAC-seq and ChIP-seq profiles of hPSC and hNC in the intron 1 of RET show that rs2435357 is residing in a hNC-specific ATAC-seq peak. ( b ) Location of rs2435357 in the RET gene locus and in the sgRNA used for CRISPR/Cas9-mediated HDR for editing the C allele to the HSCR-associated risk allele T . The electrographs of Sanger sequencing show the successful introduction of the risk allele at rs2435357 in the UE-rs2435357 hPSC line. ( c ) Differentiation strategy to generate human neural crest (hNC) and neuronal progenitor (hNP), and immunostaining of SOX10 and TUJ1 in hNC and hNP of the control and the mutant (UE-rs2435357) lines. Scale bars: (hNC): 100 μ m; (hNP): 200 μ m. RT-qPCR analysis showing the comparable ELAVL4 expression level in hNP in the control ( n =5) and the mutant (UE-rs2435357) ( n =3) lines. t- test, ns : not significant. ( d ) RT-qPCR analysis showing RET expression in the hPSC and hNP stages of the control ( n =5) and the mutant (UE-rs2435357) ( n =3). t- test, ns : not significant. ( e ) Publicly available Hi-C data from GM12878 cells are shown using the RET locus as anchor. The putative enhancer in intron 1 of RASGEF1A is marked in yellow. ( f ) ATAC-seq and ChIP-seq data from hPSC and hNC at the RASGEF1A intron 1 locus. ( g ) The design of sgRNAs used for the CRISPR/Cas9 system for deleting the DNA fragment in RASGEF1A intron 1. Genotyping reveals the specific deletion of RASGEF1A intron 1 in UE-RASGEF1A-int1-KO hPSC line. WT: wildtype; KO: knockout. ( h ) Immunostaining of SOX10, TUJ1 and HU in hNC and hNP of the control and the mutant (UE-rs2435357) lines, respectively. Scale bars: (hNC): 100 μ m; (hNP): 200 μ m. ( i ) RT-qPCR reveals the expression level of RET in the hPSC and hNP stages of the control ( n =4-5) and the mutant (RASGEF1A-int1-KO) ( n =6-7). t- test, ns : not significant.

Article Snippet: Fixed cells were blocked with blocking solution (1% BSA, 0.1% Triton X-100 in PBS) at room temperature for 1 h. The blocked cells were then incubated with primary antibodies (mouse anti-SOX10 (1:500, R&D Systems, MAB2864), rabbit anti-TUJ1 (1:1000, Abcam, ab18207) and mouse anti-HU (1:1000, Life Technologies, #A-21271) at 4 °C for 1 overnight.

Techniques: Functional Assay, Expressing, ChIP-sequencing, CRISPR, Sequencing, Immunostaining, Mutagenesis, Quantitative RT-PCR, Hi-C, Knock-Out

Journal: Nature Communications

Article Title: DNA methylation signatures of Alzheimer’s disease neuropathology in the cortex are primarily driven by variation in non-neuronal cell-types

doi: 10.1038/s41467-022-33394-7

Figure Lengend Snippet: Characteristics of the BDR samples profiled in this study

Article Snippet: Briefly, following tissue homogenization and nuclei purification using sucrose gradient centrifugation we used a FACS Aria III cell sorter (BD Biosciences) to simultaneously collect populations of NeuN+ (neuronal-enriched) (R&D systems, Cat No: NL2864R, dilution: 1:10) and SOX10+ (oligodendrocyte-enriched) (Millipore, Cat No: MAB377X, dilution: 1:1000) immunolabeled populations from bulk DLPFC tissue prior to genomic profiling, with the double-negative fraction and an aliquot of the “total” nuclei fraction (analogous to “bulk” cortex) also being collected from each tissue sample (Supplementary Fig. ).

Techniques: Purification

Journal: Nature Communications

Article Title: DNA methylation signatures of Alzheimer’s disease neuropathology in the cortex are primarily driven by variation in non-neuronal cell-types

doi: 10.1038/s41467-022-33394-7

Figure Lengend Snippet: Using linear regression models controlling for major covariates (see Methods) we show that a levels of tau pathology (measured using Braak NFT stage) are significantly associated with the proportion of NeuN+ cells (effect size = −2.74, SE = 0.705, P = 1.15E–04), SOX10+ cells (effect size = 1.60, SE = 0.423, P = 1.72E–04) and NeuN–/SOX10– cells (effect size = −2.00, SE = 0.687, P = 0.004) in the DLPFC ( N = 597 donors) using cell proportion estimates derived from “bulk” DNA methylation data. b In contrast no associations ( P > 0.008) between levels of tau pathology and cell proportion estimates derived from “bulk” DNA methylation data were observed in the OCC ( N = 598 donors). Boxplots of the estimated proportion of each cell-type across Braak NFT stages are shown, where the middle box represents the interquartile range (IQR), the middle line represents the median, and the whisker lines represent the minimum (quartile 1 –1.5 × IQR) and the maximum (quartile 3 + 1.5 × IQR). Tau pathology (Braak NFT stage) is shown on the x- axis split by cell-type and estimated cell proportions are shown on the y -axis. A similar pattern of results was found for levels of amyloid pathology as shown in Supplementary Fig. .

Article Snippet: Briefly, following tissue homogenization and nuclei purification using sucrose gradient centrifugation we used a FACS Aria III cell sorter (BD Biosciences) to simultaneously collect populations of NeuN+ (neuronal-enriched) (R&D systems, Cat No: NL2864R, dilution: 1:10) and SOX10+ (oligodendrocyte-enriched) (Millipore, Cat No: MAB377X, dilution: 1:1000) immunolabeled populations from bulk DLPFC tissue prior to genomic profiling, with the double-negative fraction and an aliquot of the “total” nuclei fraction (analogous to “bulk” cortex) also being collected from each tissue sample (Supplementary Fig. ).

Techniques: Derivative Assay, DNA Methylation Assay, Whisker Assay

Journal: Nature Communications

Article Title: DNA methylation signatures of Alzheimer’s disease neuropathology in the cortex are primarily driven by variation in non-neuronal cell-types

doi: 10.1038/s41467-022-33394-7

Figure Lengend Snippet: We compared effect sizes for the 334 overlapping tau-associated DMPs identified in our “bulk” cortex meta-analysis with those at the same sites in an analysis of purified DLPFC nuclei populations from low (Braak NFT stage 0 to II) and high (Braak NFT stage >V) tau-pathology donors. Shown is a comparison of effect sizes between the meta-analysis (bulk, N = 2013 individuals]) and the a total nuclei (bulk) nuclei fraction ( N = 26) (direction of effect = 87% concordant, sign-test P = 7.24E–46); b NeuN+ (neuron-enriched) nuclei fraction ( N = 27) (direction of effect = 60% concordant, sign-test P = 7.59E–05), c SOX10+ (oligodendrocyte-enriched) nuclei fraction ( N = 28) (direction of effect = 67% concordant, sign-test P = 2.15E–10), and d double-negative (microglia- and astrocyte-enriched) nuclei population ( N = 21) (direction of effect = 96% concordant, sign-test P = 1.2E–75). The x- axis shows effect sizes from the bulk cortex meta-analysis and the y -axis shows effect sizes for those same DMPs in each purified nuclei population. Gray dashed line represents y = x . e Bar-plots of the mean absolute relative effect sizes in each purified nuclei population compared to the bulk cortex across the 334 tau-associated DMPs, with error bars denoting the 95% confidence intervals.

Article Snippet: Briefly, following tissue homogenization and nuclei purification using sucrose gradient centrifugation we used a FACS Aria III cell sorter (BD Biosciences) to simultaneously collect populations of NeuN+ (neuronal-enriched) (R&D systems, Cat No: NL2864R, dilution: 1:10) and SOX10+ (oligodendrocyte-enriched) (Millipore, Cat No: MAB377X, dilution: 1:1000) immunolabeled populations from bulk DLPFC tissue prior to genomic profiling, with the double-negative fraction and an aliquot of the “total” nuclei fraction (analogous to “bulk” cortex) also being collected from each tissue sample (Supplementary Fig. ).

Techniques: Purification, Comparison

Journal: bioRxiv

Article Title: An extra-glycolytic function for hexokinase 2 as an RNA-binding protein regulating SOX10 mRNA translation in melanoma

doi: 10.1101/2024.08.13.607712

Figure Lengend Snippet: (A) RT-qPCR quantification representing the % of SOX10 mRNA (left panel) or HPRT mRNA (right panel) in fractions (horizontal axes) obtained by sucrose-gradient (10-50%) ultracentrifugation of lysates from A375 cells transfected with siRNAs targeting HK2 (siHK2, light red) or control (siCTRL, grey) (n = 2 biological replicates). (B) Western blot analysis of the SOX10 protein level in distinct melanoma cell lines transfected with siRNAs targeting HK2 (siHK2, light red), GAPDH (siGAPDH, light blue) or control (siCTRL, grey). The SOX10 protein quantification is normalized to VCL expression. p -values were calculated by two-tailed unpaired t -test (SD, n = 3 biological replicates) and only significant comparisons are shown (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). (C) RT-qPCR quantification of the SOX10 mRNA in different melanoma cell lines transfected with siRNAs targeting HK2 (siHK2, light red), GAPDH (siGAPDH, light blue) or control (siCTRL, grey). p-values were calculated by ordinary one-way ANOVA (SD, n = 3 biological replicates).

Article Snippet: To establish the stable overexpression of SOX10 (Myc-DDK tagged; OriGene, #RC203545L3V) in A375 and A2058 cell lines, cells were transduced with pLenti-C-Myc-DDK-P2A-Puro vector expressing SOX10 (NM_006941) ORF nucleotide sequence (OriGene, #RC203545L3V) according to manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Transfection, Control, Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: An extra-glycolytic function for hexokinase 2 as an RNA-binding protein regulating SOX10 mRNA translation in melanoma

doi: 10.1101/2024.08.13.607712

Figure Lengend Snippet: (A) RIP experiment performed on A375 and A2058 melanoma cell lines (SD, n = 3 biological replicates). SOX10 mRNA was analysed in IgG and HK2 immunoprecipitated samples and expressed as percentage of the mRNA present in the input. GAPDH , TBP and ACT mRNAs were used as negative controls. p -values were calculated by ordinary two-way ANOVA with Dunnett’s multiple comparisons test (SD, n = 3 biological replicates) (**** p ≤ 0.0001). (B) In silico prediction of HK2- SOX10 mRNA interaction propensities using the CatRAPID algorithm. The highest interaction score observed between HK2 and the SOX10 mRNA is at the SOX10 5’UTR. Upper panel: HK2- SOX10 mRNA interaction profile. Lower panel: schematic representation of the SOX10 mRNA. (C) RIP experiment performed on A375 cells transfected with luciferase reporters containing the SOX10 5’UTR and 3’UTR sequences upstream of the Renilla reporter gene. An Empty reporter was used as control. Renilla mRNA was analyzed in IgG and HK2 immunoprecipitated samples and expressed as percentage of the mRNA present in the input. p -values were calculated by ordinary two-way ANOVA with Dunnett’s multiple comparisons test (SEM, n = 6 biological replicates) (* p ≤ 0.05). (D) Representative fluorescence images and quantification of RNA-proximity ligation assays (PLA) of HK2 protein and SOX10 mRNA in A375 cells transfected with siRNAs targeting SOX10 (siSOX10, orange) or control (siCTRL, grey) (n = 3 biological replicates). After 48h, cells were fixed, permeabilized, and incubated with anti-sense SOX10 5’UTR oligonucleotide probes and anti-HK2 antibody. Scale bar, 10 μm. HK2- SOX10 mRNA PLA signal (yellow), Phalloidin staining (green), and DAPI staining (blue). The significance for PLA values was derived from the Mann-Whitney statistical test (SD, ** p ≤ 0.01).

Article Snippet: To establish the stable overexpression of SOX10 (Myc-DDK tagged; OriGene, #RC203545L3V) in A375 and A2058 cell lines, cells were transduced with pLenti-C-Myc-DDK-P2A-Puro vector expressing SOX10 (NM_006941) ORF nucleotide sequence (OriGene, #RC203545L3V) according to manufacturer’s instructions.

Techniques: Immunoprecipitation, In Silico, Transfection, Luciferase, Control, Fluorescence, Ligation, Incubation, Staining, Derivative Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: An extra-glycolytic function for hexokinase 2 as an RNA-binding protein regulating SOX10 mRNA translation in melanoma

doi: 10.1101/2024.08.13.607712

Figure Lengend Snippet: (A) Schematic of full-length (FL) and mutated SOX10 5’UTR (Δ1-6) reporters cloned upstream of the Renilla reporter. The deleted regions of ∼50nts are represented as red dashed lines. (B) Luciferase assay performed in A375 cells co-transfected with a Firefly reporter and the Renilla reporters in (A). An empty Renilla vector and the SOX10 3’UTR Renilla reporter were used as controls. The activity of the Firefly and Renilla reporters was measured 48h after transfection. The Renilla activity was normalized by the Firefly activity, and the data shown is relative to the Empty vector. p -values were calculated by Mann-Whitney statistical test (SEM, n = 4 biological replicates) (∗ p < 0.05). (C) RT-qPCR quantification of the Renilla mRNA from the same transfected cells in (B). The Renilla expression was normalized by the Firefly expression, and the data shown is relative to the expression of the Empty vector. p -values were calculated by Mann-Whitney statistical test (SEM, n = 4 biological replicates). (D) RIP experiment performed on A375 cells transfected with Renilla luciferase reporters containing the SOX10 5’UTR FL and Δ4, Δ5 and Δ6. Renilla mRNA was analyzed in IgG and HK2 immunoprecipitated samples and expressed as percentage of the mRNA present in the input. p -values were calculated by ordinary two-way ANOVA with Dunnett’s multiple comparisons test (SEM, n = 3 biological replicates) (* p ≤ 0.05).

Article Snippet: To establish the stable overexpression of SOX10 (Myc-DDK tagged; OriGene, #RC203545L3V) in A375 and A2058 cell lines, cells were transduced with pLenti-C-Myc-DDK-P2A-Puro vector expressing SOX10 (NM_006941) ORF nucleotide sequence (OriGene, #RC203545L3V) according to manufacturer’s instructions.

Techniques: Clone Assay, Luciferase, Transfection, Plasmid Preparation, Activity Assay, MANN-WHITNEY, Quantitative RT-PCR, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: An extra-glycolytic function for hexokinase 2 as an RNA-binding protein regulating SOX10 mRNA translation in melanoma

doi: 10.1101/2024.08.13.607712

Figure Lengend Snippet: (A) Western blot analysis of the ectopic SOX10 Myc-DDK expression in A375 and A2058 melanoma cells. VCL was used as loading control (n = 3 biological replicates). (B) A375 and A2058 cells from (A) were plated 24h after transfection with siRNAs targeting HK2 (siHK2, light red) or control (siCTRL, grey). The colonies were stained with crystal violet after 10 days, and the clonogenic cell growth was measured. Upper panel: percentage of area covered by crystal violet stained cell colonies. Lower panel: representative images of three independent experiments. p -values were calculated by ordinary two-way ANOVA with Turkey’s multiple comparison test (SD, n = 3 biological replicates), and only significant differences within the same cell line are shown (* p ≤ 0.05, **** p ≤ 0.0001). (C) Cell proliferation assay performed in A375 and A2058 cells from (A). Cells were plated 24h after transfection with siRNAs targeting HK2 (Empty, light blue; SOX10, light orange) or control (Empty, dark blue; SOX10 dark orange). p -values were calculated by ordinary two-way ANOVA with Turkey’s multiple comparison test (SD, n = 3 biological replicates), and only significant differences within the same cell line are shown (**** p ≤ 0.0001).

Article Snippet: To establish the stable overexpression of SOX10 (Myc-DDK tagged; OriGene, #RC203545L3V) in A375 and A2058 cell lines, cells were transduced with pLenti-C-Myc-DDK-P2A-Puro vector expressing SOX10 (NM_006941) ORF nucleotide sequence (OriGene, #RC203545L3V) according to manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Transfection, Staining, Comparison, Proliferation Assay

Journal:

Article Title: The Waardenburg Syndrome Type 4 Gene, SOX10 , Is a Novel Tumor-associated Antigen Identified in a Patient with a Dramatic Response to Immunotherapy

doi:

Figure Lengend Snippet: IFN-γ release by TIL clone M37 cocultured with T2 cells pulsed with decreasing concentrations of peptides SOX10: 331–340 and 332–340. The flu A matrix protein M1 peptide, FluM1: 58–66, was used as a negative control.

Article Snippet: RT-PCR Assay RT-PCR for expression of SOX10 in normal tissues was performed using the Human Rapid-Scan Plate (OriGene Technologies, Inc., Rockville, MD). qRT-PCR was also performed on normal tissues and fresh tumors.

Techniques: Negative Control

Journal:

Article Title: The Waardenburg Syndrome Type 4 Gene, SOX10 , Is a Novel Tumor-associated Antigen Identified in a Patient with a Dramatic Response to Immunotherapy

doi:

Figure Lengend Snippet: Real-time relative qRT-PCR analysis of SOX10 mRNA expression relative to that of β-actin in 19 normal tissues and 5 fresh melanoma tissues. All measurements were normalized to the SOX10:β-actin ratio for the 624mel cell line, which was used as a reference in all experiments.

Article Snippet: RT-PCR Assay RT-PCR for expression of SOX10 in normal tissues was performed using the Human Rapid-Scan Plate (OriGene Technologies, Inc., Rockville, MD). qRT-PCR was also performed on normal tissues and fresh tumors.

Techniques: Quantitative RT-PCR, Expressing

Journal:

Article Title: The Waardenburg Syndrome Type 4 Gene, SOX10 , Is a Novel Tumor-associated Antigen Identified in a Patient with a Dramatic Response to Immunotherapy

doi:

Figure Lengend Snippet: A, real-time qRT-PCR analysis of the mRNA copy number encoding the SOX10 gene normalized to mRNA encoding the β-actin housekeeping gene. Melanocyte and melanomas (mel) had increased expression of SOX10 mRNA compared with other cell lines. HUVEC, human umbilical vein endothelial cells. B, The same cell lines in Fig. 3A were tested for recognition by CTL clone M37. A correlation existed between levels of SOX10 expression and recognition by CTL clone M37 (P < 0.001).

Article Snippet: RT-PCR Assay RT-PCR for expression of SOX10 in normal tissues was performed using the Human Rapid-Scan Plate (OriGene Technologies, Inc., Rockville, MD). qRT-PCR was also performed on normal tissues and fresh tumors.

Techniques: Quantitative RT-PCR, Expressing