Journal: bioRxiv

Article Title: A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

doi: 10.1101/815761

Figure Lengend Snippet: a, After gene editing, LMNB1-edited clones #5 and #8 were established as cell lines, expanded, and their normal karyotype confirmed. b-c, Western blot and immunocytochemical analysis of gene-edited and clone cell line demonstrating expression of typical markers for pluripotent (OCT4) and differentiated cells (Brachyury for mesoderm; SOX17 for endoderm; PAX6 for ectoderm) after directed differentiation in adherent cultures. Scale bar in c : 50 μm.

Article Snippet: On day 7, mRNA from individual EBs was extracted using TurboCapture 96 mRNA kit (Qiagen), reverse-transcribed into cDNA using Sensiscript RT kit (Qiagen), and pre-amplified using TaqMan PreAmp Master Mix (Thermo Fisher Scientific) and Taqman probes for PAX6 (Hs01088114 ) , Brachyury (Hs00610080) and SOX17 (Hs00751752), followed by qPCR quantification of PAX6, Brachyury, and SOX17.

Techniques: Clone Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

doi: 10.1101/815761

Figure Lengend Snippet: a, EB formation in the presence of DMSO, Y-27632 and CEPT. Human ESCs (WA09) cells were dissociated with Accutase and plated into 6-well ULA plates in E6 medium. Representative phase-contrast images were taken at 24 h post-plating. Scale bar, 50 μm. b, To generate single EBs, hESCs were dissociated with Accutase and plating into AggreWell plates (5,000 cells/well). Images were taken 24 h post-plating. In the presence of DMSO the vast majority cells underwent cell death and EB formation was not observed. Treatment with Y-27632 supported EB formation but significant numbers of dead cells were detected surrounding the EB. Note that CEPT enables EB formation without apparent cell death. Scale bar, 100 μm. c, Quantification of the diameter of single EBs (24 h post-plating). Data are mean ± s.d. (n = 20 EBs for Y-27632 and n = 22 for CEPT), **** P < 0.0001, two-tailed Student’s t -test. d, Single EB formation in 96-well ULA plates. Dissociated hESCs were plated into 96-well ULA plates at 2,000 cells/well. Live and dead cells were stained with calcein green AM and propidium iodide (PI) 24 h after cell seeding. Scale bars, 100 μm. e, Quantification of cell survival in single EBs at day 1 and day 7 by using the CTG 3D assay. Note the significant difference between Y-27632 and CEPT treatment at both timepoints. Data represent mean ± s.d. (n = 24 EBs for each group), **** P < 0.0001, one-way ANOVA. f, CEPT supports differentiation of single EBs into the three germ layers. Individual EBs were cultured in E6 medium to allow for spontaneous differentiation and analyzed on day 7 for the expression of PAX6, SOX17, and Brachyury using an optimized quantitative RT-PCR protocol that enabled detection of low transcript levels in single EBs (see Materials and Methods section for details). Data represent mean ± s.d. (n = 3 experiments and in each experiment 24 EBs were analyzed for each group), * P = 0.0327, two-tailed Student’s t -test. g, h, Cerebral organoids were generated by using Y-27632 and CEPT for the first 24 h. At day 30, organoids were fixed, sectioned, processed for histology (hematoxylin and eosin stain) and immunohistochemistry for FOXG1. Representative images show that CEPT treatment resulted in larger organoids and more abundant FOXG1-expressing cells. Scale bars, 400 μm.

Article Snippet: On day 7, mRNA from individual EBs was extracted using TurboCapture 96 mRNA kit (Qiagen), reverse-transcribed into cDNA using Sensiscript RT kit (Qiagen), and pre-amplified using TaqMan PreAmp Master Mix (Thermo Fisher Scientific) and Taqman probes for PAX6 (Hs01088114 ) , Brachyury (Hs00610080) and SOX17 (Hs00751752), followed by qPCR quantification of PAX6, Brachyury, and SOX17.

Techniques: Two Tailed Test, Staining, Cell Culture, Expressing, Quantitative RT-PCR, Generated, H&E Stain, Immunohistochemistry

Journal: bioRxiv

Article Title: A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

doi: 10.1101/815761

Figure Lengend Snippet: Basal expression levels of Caspase-3 in hESCs (WA09) in comparison to their lineage-restricted precursors after directed differentiation into ectoderm (PAX6), mesoderm (Brachyury), and endoderm (SOX17). Note the strong Caspase-3 expression at the pluripotent state and downregulation upon differentiation.

Article Snippet: On day 7, mRNA from individual EBs was extracted using TurboCapture 96 mRNA kit (Qiagen), reverse-transcribed into cDNA using Sensiscript RT kit (Qiagen), and pre-amplified using TaqMan PreAmp Master Mix (Thermo Fisher Scientific) and Taqman probes for PAX6 (Hs01088114 ) , Brachyury (Hs00610080) and SOX17 (Hs00751752), followed by qPCR quantification of PAX6, Brachyury, and SOX17.

Techniques: Expressing, Comparison

Journal: bioRxiv

Article Title: Variants in the DDX6-CXCR5 autoimmune disease risk locus influence the regulatory network in immune cells and salivary gland

doi: 10.1101/2023.10.05.561076

Figure Lengend Snippet: (A) Schematic of the five prioritized SNPs arranged in genomic space. SNP rs57494551 is in the first intron of DDX6 . The other four SNPs span 978 bp in a shared promoter/enhancer region between DDX6 and CXCR5 . Risk alleles are indicated by red font; non-risk alleles by black. SNPs rs57494551 and rs4938572 (blue boxes) are representative SNPs. HindIII restriction sites (red dotted line), CTCF site (green box), and positions of 3C-qPCR primers #1-3 are also indicated. (B-F) Publicly reported cell type-specific functional annotations (horizontal rectangles), select eQTLs (top triangles), and chromatin-chromatin interactions (bottom triangles) are shown for (B) rs57494551, (C) rs4936443, (D) rs4938572, (E) rs7117261, and (F) rs4938573 across 10 different immune cell types or disease-specific tissues (GM12878 EBV B cells and primary human B cells, CD4+ T cells, CD8+ T cells, monocytes, macrophages, neutrophils, salivary gland tissue, kidney tissue, and whole blood).

Article Snippet: Taqman reactions were performed on a QuantStudio 6 qPCR machine (Thermo Fisher, #A43168) following manufacturer instructions using ∼25ng of DNA, TaqMan Genotyping Master Mix (ThermoFisher, #4371355), and TaqMan SNP Genotyping probes for rs57494551 (ThermoFisher, #4351379, Assay ID#C_90471390_10) and rs4938572 (ThermoFisher, #4351379, Assay ID#C_28012712_10) ( Supplemental Table 1 ).

Techniques: Functional Assay

Journal: bioRxiv

Article Title: Variants in the DDX6-CXCR5 autoimmune disease risk locus influence the regulatory network in immune cells and salivary gland

doi: 10.1101/2023.10.05.561076

Figure Lengend Snippet: (A-D) Radiolabeled electromobility shift assays (EMSA) were performed to assess the binding affinity of ribonucleoproteins isolated from EBV B or A253 cells to oligonucleotides containing the non-risk (NR) or risk (R) allele of (A-B) rs57494551 or (C-D) rs4938572. Probes incubated in the absence of nuclear lysate were used as negative control (Lanes 1, 2). Cold competitors were used to assess non-specific binding (Lanes 5, 6). Images shown in (A) and (C) are representative of n>6 biological replicates. (B, D) Bands indicated in (A, C) by the orange or green circles were quantified by densitometry and analyzed using paired t-test (n>6); p-values indicated. (E) Summary analysis of the allele-specific nuclear protein affinities of the five prioritized SNPs in EBV B, Daudi, Jurkat, THP1, and A253 cells shown in A-D and Supplemental Figures 4-9 . Increases in binding relative to NR are shown in red; decreases relative to NR in blue; no change relative to NR in grey; no detected band in black; data not available in white.

Article Snippet: Taqman reactions were performed on a QuantStudio 6 qPCR machine (Thermo Fisher, #A43168) following manufacturer instructions using ∼25ng of DNA, TaqMan Genotyping Master Mix (ThermoFisher, #4371355), and TaqMan SNP Genotyping probes for rs57494551 (ThermoFisher, #4351379, Assay ID#C_90471390_10) and rs4938572 (ThermoFisher, #4351379, Assay ID#C_28012712_10) ( Supplemental Table 1 ).

Techniques: Binding Assay, Isolation, Incubation, Negative Control

Journal: bioRxiv

Article Title: Variants in the DDX6-CXCR5 autoimmune disease risk locus influence the regulatory network in immune cells and salivary gland

doi: 10.1101/2023.10.05.561076

Figure Lengend Snippet: (A-E) gBlocks carrying the non-risk or risk alleles of (A) rs57494551, (B) rs4936443, (C) rs4938572, (D) rs7117261, or (E) rs4938573 were cloned into a promoter-less (pGL4.14; noP) or minimal promoter (pGL4.26; minP) luciferase vector. Plasmids were transfected into EBV B, Daudi, Jurkat, THP1, A253, or 293T cells. Luciferase activity was measured after 24 hours and normalized to the Renilla transfection control and then the vector-only control; reported as Relative Luciferase Activity. Statistical comparisons were performed using a paired t-test (n>3); p-values are indicated. (F) Summary analysis of the allele-specific luciferase activity of the five prioritized SNPs in EBV B, Daudi, Jurkat, THP1, A253 and 293Tcells. Increases in luciferase activity relative to non-risk are shown in purple; decreases relative to non-risk in orange; no change relative to non-risk in grey. (G) gBlocks carrying all non-risk or all risk alleles of rs4936443, rs4938572, and rs7117261 were cloned into the promoter-less or minimal promoter above, transfected into EBV B, Jurkat, THP1, A253, or 293T cells, and luciferase activity tested as described above. Statistical comparisons were performed using a paired t-test (n>3); p-values are indicated.

Article Snippet: Taqman reactions were performed on a QuantStudio 6 qPCR machine (Thermo Fisher, #A43168) following manufacturer instructions using ∼25ng of DNA, TaqMan Genotyping Master Mix (ThermoFisher, #4371355), and TaqMan SNP Genotyping probes for rs57494551 (ThermoFisher, #4351379, Assay ID#C_90471390_10) and rs4938572 (ThermoFisher, #4351379, Assay ID#C_28012712_10) ( Supplemental Table 1 ).

Techniques: Clone Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Control

Journal: bioRxiv

Article Title: Variants in the DDX6-CXCR5 autoimmune disease risk locus influence the regulatory network in immune cells and salivary gland

doi: 10.1101/2023.10.05.561076

Figure Lengend Snippet: (A, B) Chromatin conformation capture with quantitative PCR (3C-qPCR) across the DDX6-CXCR5 region where (A) rs57494551 or (B) rs4938572 is the anchor SNP (grey dot). Relative interaction frequency (RIF) is plotted relative to the primer number in 5’-3’ genomic orientation (blue dots; see for additional detail). Primers 9-12 are shown in a text box for simplicity in (B). (C) SjD GWAS association (top panel) and publicly available epigenomic enrichment across the DDX6-CXCR5 region in GM12878 EBV B cells. Vertical grey lines indicate the locations of rs57494551 or rs4938572, respectively. Promoter-capture Hi-C looping (purple lines) contrasts the summary 3C-qPCR results (red lines); line thickness indicates relative interaction frequency (RIF) of the 3C data. (D) SjD GWAS association (top panel) across the DDX6-CXCR5 region in A253 cells. In house ATAC-seq, CUT & RUN: H3K27me3 (Epicypher), and RNA-seq data from A253 cells are shown because of limited publicly available epigenetic data on salivary gland. Vertical grey lines indicate the locations of rs57494551 or rs4938572, respectively. Summary 3C-qPCR results are shown (red lines); line thickness indicates relative interaction frequency (RIF).

Article Snippet: Taqman reactions were performed on a QuantStudio 6 qPCR machine (Thermo Fisher, #A43168) following manufacturer instructions using ∼25ng of DNA, TaqMan Genotyping Master Mix (ThermoFisher, #4371355), and TaqMan SNP Genotyping probes for rs57494551 (ThermoFisher, #4351379, Assay ID#C_90471390_10) and rs4938572 (ThermoFisher, #4351379, Assay ID#C_28012712_10) ( Supplemental Table 1 ).

Techniques: Real-time Polymerase Chain Reaction, Hi-C, RNA Sequencing