Journal: Cell reports

Article Title: Single-Cell RNA-Sequencing-Based CRISPRi Screening Resolves Molecular Drivers of Early Human Endoderm Development

doi: 10.1016/j.celrep.2019.03.076

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies used in this study include: SOX17 antibody (1:300; R&D Systems, AF1924), OCT3/4 antibody (1:100; Santa Cruz Biotechnology, sc5279), FOXA2 antibody (1:300; Millipore EMD, 07–633), CDX2 antibody (1:300; BioGenex, MU392A-UC), HNF4A antibody (1:500; Abcam, ab41898), HHEX antibody (1:500; R&D Systems, MAB83771), TBX3 antibody (1:300; Santa Cruz Biotechnology, sc17871), PROX1 antibody (1:300; R&D Systems, AF2727), EPCAM antibody (1:1000; Biolegend, 324202).

Techniques: Recombinant, Staining, Transfection, DNA Library Preparation, Methylation, Software, Fluorescence, Microscopy

Journal: Cancer Research

Article Title: A Runaway PRH/HHEX-Notch3–Positive Feedback Loop Drives Cholangiocarcinoma and Determines Response to CDK4/6 Inhibition

doi: 10.1158/0008-5472.can-19-0942

Figure Lengend Snippet: Figure 2. Effects of PRH manipulation on CCA cell biology. A, Proliferation of CCLP1 cells stably transfected with PRH shRNA or scrambled control; n ¼ 3; P ¼ 0.03. B, Proliferation of CCLP1 and CCSW1 cells infected with Ad myc-PRH or empty virus control; n ¼ 3; P ¼ 0.03 (CCLP1); P ¼ 0.02 (CCSW1). C, Proliferation of AKN1 and primary BECs overexpressing GFP-PRH-myc (stable) or myc-PRH (transient, 48 hours); n ¼ 3; P ¼ 0.003 (AKN1); P ¼ 0.006 (BEC). D, Morphology of CCLP1 cells stably transfected with PRH shRNA or scrambled control. E, Western blot showing increased expression of E-cadherin protein and decreased expression of vimentin protein after PRH knockdown. Lamin A/C was used as loading control. F, Migration of CCLP1 PRH knockdown cells through transwell filters in a 10% serum gradient; n ¼ 3; P ¼ 0.03. G, Invasion of CCLP1 cells through Matrigel; n ¼ 4; P ¼ 0.03. H, Western blotting for myc-PRH and EMT-associated proteins E-cadherin and vimentin in AKN1 cells and primary BECs. I and J, As in F and G for AKN1 cells and primary BECs. K, Morphology of equal numbers of AKN1 cells stably transfected with plasmids expressing GFP (control) or a GFP-PRH-Myc–tagged fusion protein (GFP-PRH-Myc). Scale bars, 50 mm in length. , P < 0.05.

Article Snippet: Stable cell lines were generated by transfecting PRH shRNA in pRS (Origene, TR312464), Notch3 shRNA in pLKO (Sigma-Aldrich, TRCN0000363316), CDH1 cDNA in pCDNA3 (hE-cadherinpcDNA3 was a gift from Dr. Barry Gumbiner, Seattle Children's Hospital, Seattle, WA; Addgene plasmid #45769), or EGFP-PRHmyc in pEGFP-C1.

Techniques: Stable Transfection, Transfection, shRNA, Control, Infection, Virus, Western Blot, Expressing, Knockdown, Migration

Journal: Cancer Research

Article Title: A Runaway PRH/HHEX-Notch3–Positive Feedback Loop Drives Cholangiocarcinoma and Determines Response to CDK4/6 Inhibition

doi: 10.1158/0008-5472.can-19-0942

Figure Lengend Snippet: Figure 4. Notch3 expression is regulated by PRH. A, Western blot showing Notch3 ICD protein expression in PRH knockdown CCLP1 cells. B, NOTCH3 gene expression in CCLP1 and CCSW1 cells infected with Ad myc-PRH, Ad myc-PRH DNA-binding deficient N187A mutant, or empty virus control. C, Western blot for CCLP1 samples in B. D, Notch3 ICD rotein expression correlates with PRH protein expression in four human CCA cell lines, two human primary BEC isolates, and an immortalized BEC line. E, Western blot showing elevated expression of Notch3 ICD upon PRH overexpression in both AKN1 cells and primary BECs. F, Western blot showing increased expression of E-cadherin and reduced expression of PRH proteins after Notch3 knockdown. Lamin A/C was used as loading control. G, Proliferation of CCLP1 cells stably transfected with Notch3 shRNA or scrambled control; n ¼ 3; P ¼ 0.04. H, Morphology of CCLP1 cells stably transfected with Notch3 shRNA or scrambled control. I, DEGs detected in Notch3 KD compared with PRH KD experiments. Hypergeometric test, P ¼ 10229 for upregulated genes and P ¼ 1031 for downregulated genes. J, Hallmark GSEA of genes differentially expressed after Notch3 KD. Red bars, gene sets that are also enriched after PRH knockdown. , P < 0.05.

Article Snippet: Stable cell lines were generated by transfecting PRH shRNA in pRS (Origene, TR312464), Notch3 shRNA in pLKO (Sigma-Aldrich, TRCN0000363316), CDH1 cDNA in pCDNA3 (hE-cadherinpcDNA3 was a gift from Dr. Barry Gumbiner, Seattle Children's Hospital, Seattle, WA; Addgene plasmid #45769), or EGFP-PRHmyc in pEGFP-C1.

Techniques: Expressing, Western Blot, Knockdown, Gene Expression, Infection, Binding Assay, Mutagenesis, Virus, Control, Over Expression, Stable Transfection, Transfection, shRNA

Journal: Cancer Research

Article Title: A Runaway PRH/HHEX-Notch3–Positive Feedback Loop Drives Cholangiocarcinoma and Determines Response to CDK4/6 Inhibition

doi: 10.1158/0008-5472.can-19-0942

Figure Lengend Snippet: Figure 5. Notch3- and PRH-correlated gene sets. A, qRT-PCR analysis of genes from CCLP1 cells overexpressing PRH in the presence or absence of Notch3 shRNA identifying PRH and Notch3-correlated expression signatures. B, Western blot analysis of EMT proteins E-cadherin and vimentin and cyclin D2 in CCLP1 cells overexpressing myc-PRH in the presence of absence of Notch3 shRNA. C, Proliferation of CCLP1 cells overexpressing myc-PRH in the presence or absence of Notch3 shRNA. , P < 0.05 after Bonferroni correction, compared with nontargeting shRNA/empty virus control. #, no statistically significant difference in the comparison indicated. D, Hallmark GSEA of Notch3-correlated and PRH-correlated gene sets identified from analysis of RNA-seq data. Red bars, gene sets enriched in both PRH- and Notch3-correlated sets. E, Overlap of genes with PRH-binding sites determined by ChIP-seq and genes that are differentially expressed after PRH overexpression determined by RNA-seq in CCLP1 cells. F, Comparison of the primary motif underlying PRH ChIP-seq peaks identified using HOMER with the PRH SELEX motif (derived from ref. 38). G, RNA-seq and ChIP-seq tracks of putative direct PRH target DKK1. Red tracks, myc-PRH overexpression. H, RNA-seq and ChIP-seq tracks of putative direct PRH target WNT11. Red tracks, myc-PRH overexpression.

Article Snippet: Stable cell lines were generated by transfecting PRH shRNA in pRS (Origene, TR312464), Notch3 shRNA in pLKO (Sigma-Aldrich, TRCN0000363316), CDH1 cDNA in pCDNA3 (hE-cadherinpcDNA3 was a gift from Dr. Barry Gumbiner, Seattle Children's Hospital, Seattle, WA; Addgene plasmid #45769), or EGFP-PRHmyc in pEGFP-C1.

Techniques: Quantitative RT-PCR, shRNA, Expressing, Western Blot, Virus, Control, Comparison, RNA Sequencing, Binding Assay, ChIP-sequencing, Over Expression, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Unraveling the Influence of HHEX Risk Polymorphism rs7923837 on Multiple Sclerosis Pathogenesis

doi: 10.3390/ijms23147956

Figure Lengend Snippet: mRNA and protein expression of HHEX. ( A ) mRNA expression in controls, multiple sclerosis (MS) patients, and MS patients stratified by treatment. Controls: n = 117; MS patients: n = 154; MS patients treated with IFN-β: n = 94; MS patients treated with glatiramer acetate: n = 57. ( B ) HHEX mRNA expression in controls and MS patients stratified by rs7923837 genotype. Heterozygotes were grouped with major homozygotes due to similar expression levels. Control GG + GA: n = 101; minor allele homozygous controls AA: n = 16; MS GG + GA: n = 133; MS AA: n = 18; MS GG + GA treated with IFN-β: n = 85; MS AA treated with IFN-β: n = 9; MS GG + GA treated with glatiramer acetate: n = 48; MS AA treated with glatiramer acetate: n = 9. ( C ) Expression of HHEX protein analyzed by Western blot, stratified by rs7923837 genotype. Control GG + GA: n = 24; Control AA: n = 9; MS GG + GA: n = 36; MS AA: n = 13. Mean and standard deviation are presented in all panels.

Article Snippet: Cells were stained with rabbit anti-human HHEX (HPA055460, Atlas Antibodies, Bromma, Sweden) as primary antibody, and a combination of biotinylated anti-rabbit antibody (BA-1000, Vector Laboratories, Newark, CA, USA) and streptavidin-Alexa Fluor 555 (S32355, Invitrogen, Waltham, MA, USA) supplemented with Draq5 (ab108410, Abcam, Cambridge, United Kingdom).

Techniques: Expressing, Control, Western Blot, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Unraveling the Influence of HHEX Risk Polymorphism rs7923837 on Multiple Sclerosis Pathogenesis

doi: 10.3390/ijms23147956

Figure Lengend Snippet: Subcellular localization of HHEX by confocal microscopy and epistatic interaction HHEX - BCL6 . ( A ) Immunofluorescence of HHEX by confocal microscopy shows a higher localization of the transcription factor in the nucleus in rs7923837*AA multiple sclerosis (MS) patients. One confocal plane of each condition is shown. ( B ) Cytoplasmic/nuclear HHEX ratio measured by immunofluorescence and stratified by rs7923837 genotypes. Control carriers of the major allele GG + GA: n = 320 cells from 8 subjects; minor allele homozygous controls AA: n = 128 cells from 4 subjects; MS GG + GA: n = 599 cells from 17 subjects; and MS AA: n = 256 cells from 8 subjects. Red lines represent the median of the distribution (Control GG + GA median = 0.48; Control AA median = 0.56; MS GG + GA median = 0.52; MS AA median = 0.39). ( C ) HHEX mRNA expression stratified by the MS-risk polymorphism located near BCL6 , rs2590438. Control carriers of the major allele TT + GT: n = 38; minor allele homozygous controls GG: n = 9; MS TT + GT: n = 40; and MS GG: n = 6. Mean and standard deviation are presented.

Article Snippet: Cells were stained with rabbit anti-human HHEX (HPA055460, Atlas Antibodies, Bromma, Sweden) as primary antibody, and a combination of biotinylated anti-rabbit antibody (BA-1000, Vector Laboratories, Newark, CA, USA) and streptavidin-Alexa Fluor 555 (S32355, Invitrogen, Waltham, MA, USA) supplemented with Draq5 (ab108410, Abcam, Cambridge, United Kingdom).

Techniques: Confocal Microscopy, Immunofluorescence, Control, Expressing, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Unraveling the Influence of HHEX Risk Polymorphism rs7923837 on Multiple Sclerosis Pathogenesis

doi: 10.3390/ijms23147956

Figure Lengend Snippet: Glycolytic profile and mitochondrial mass of PBMCs from multiple sclerosis (MS) patients and controls stratified by the genotypes of HHEX rs7923837. ( A ) Energy phenotype of the studied groups. Minimum and maximum values of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were confronted to obtain an estimation of the metabolic range of the cells. ( B – D ) Glycolytic analysis comparing carriers of the major allele in HHEX rs7923837 and minor-allele homozygotes from both MS patients and controls. Control GG + GA: n = 11; Control AA: n = 7; MS GG + GA: n = 18; MS AA: n = 7. ( E – H ) Increase in mitochondrial mass measured by flow cytometry with Mitotracker Green TM after activation with PHA. The ratio of the median fluorescence intensity in the presence/absence of PHA was calculated for PBMCs ( E ) and the indicated lymphocyte subpopulations ( F – H ). Control GG + GA: n = 9; Control AA: n = 5; MS GG + GA: n = 17; MS AA: n = 8. Mean and standard deviation are presented in panels ( B – H ).

Article Snippet: Cells were stained with rabbit anti-human HHEX (HPA055460, Atlas Antibodies, Bromma, Sweden) as primary antibody, and a combination of biotinylated anti-rabbit antibody (BA-1000, Vector Laboratories, Newark, CA, USA) and streptavidin-Alexa Fluor 555 (S32355, Invitrogen, Waltham, MA, USA) supplemented with Draq5 (ab108410, Abcam, Cambridge, United Kingdom).

Techniques: Control, Flow Cytometry, Activation Assay, Fluorescence, Standard Deviation