Journal: bioRxiv

Article Title: Cardiovascular Disease-Associated Non-Coding Variants Disrupt GATA4-DNA Binding and Regulatory Functions

doi: 10.1101/2024.09.19.613959

Figure Lengend Snippet: Expression and purification of GATA4 ZF. A) SDS-PAGE (left) and western blot (right) of GATA4 ZF (18.8 kDa). Abbreviations: wash fractions (W), eluted fractions (E), concentrated fractions (C), un-induced protein sample (U), IPTG-induced protein sample (I), supernatant fraction (S), and pellet fraction (P). B) Western Blot of full-length GATA4 expressed in HeLA cells (48.6 kDa).

Article Snippet: HeLa Cervical Adenocarcinoma Human (HeLa) cells (ATCC–CCL-2) were grown in Eagle’s Minimum Essential Medium (EMEM) (ATCC - 30-2003) with 10% Fetal Bovine Serum (FBS) at 37 °C and 5% CO 2 .

Techniques: Expressing, Purification, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Cardiovascular Disease-Associated Non-Coding Variants Disrupt GATA4-DNA Binding and Regulatory Functions

doi: 10.1101/2024.09.19.613959

Figure Lengend Snippet: CVD-associated SNPs alter gene expression and are in eQTL in cardiac tissue. A) Relative luciferase activity in HeLa cells transfected with reporter plasmids containing reference (blue with black circles) and alternate (orange with black squares) of variants rs1506537, rs56992000, rs2941506, and rs2301249. B) Cardiac tissue eQTL analysis of MRPL33 , TOP2B , PGAP3 , and CSK expressed in heart atrial appendage or left ventricle when rs1506537, rs56992000, rs2941506, and rs2301249 occur, respectively. C) University of California Santa Cruz (UCSC) Genome Browser tracks of variants rs2941506 and rs56992000 near TAD boundaries and regulatory elements.

Article Snippet: HeLa Cervical Adenocarcinoma Human (HeLa) cells (ATCC–CCL-2) were grown in Eagle’s Minimum Essential Medium (EMEM) (ATCC - 30-2003) with 10% Fetal Bovine Serum (FBS) at 37 °C and 5% CO 2 .

Techniques: Gene Expression, Luciferase, Activity Assay, Transfection

Journal: bioRxiv

Article Title: Actomyosin contractility modulates Wnt signaling through adherens junction stability

doi: 10.1101/220178

Figure Lengend Snippet: (A,B) Wnt-responsive luciferase TOPFLASH assay measuring TCF/LEF reporter activity in (A) MCF7 and (B) RKO cells following Wnt3A transfection and siPP1β and siMYPT3 transfection. Data are presented as mean ± SEM with letters above representing significant difference from corresponding column, (P<0.01). (C) Western blot analysis of total cellular levels of E-cad, β-cat, MYPT3, Wnt3A and PP1β. β-tub was used as a loading control. (D) Western blot analysis of β-cat in cytoplasmic (Cyto), membranous (Mem), chromatin-bound (CB), and whole cell extract (WCE) fractions in MCF7 cells. GAPDH, Na + /K + ATPase, and Histone 3 were used as loading controls for corresponding fractions. Complete fraction, see . (E) Quantification of relative levels of β-cat from each fraction taken from (D), normalized to Control + siScramble conditions. Data shown as mean ± SEM (n = 4 biological replicates), ns = not significant, *P<0.05.

Article Snippet: Proteins were transferred onto nitrocellulose membranes, and probed against the following primary antibodies: rabbit anti-β-catenin (1:1000, Cell Signaling), rabbit anti-E-cadherin (1:1000, Cell Signaling), mouse anti-PPP1R16A (MYPT-3) (1:500, abcam), mouse anti-Protein Phosphatase 1 beta (PP1β) (1:1000, abcam), rabbit anti-Wnt3A (1:1000, Cell Signaling), mouse anti-β-tubulin (1:1000, ABM), rabbit anti-GAPDH (1:3000, Cell Signaling), mouse anti-Na+/K+ ATPase (1:50 DSHB), rabbit anti-Histone H3 (1:1000 Cell Signaling).

Techniques: Luciferase, TOPFlash assay, Activity Assay, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: Capturing trophectoderm-like stem cells enables step-wisely remodeling of placental development

doi: 10.1101/2025.08.25.672082

Figure Lengend Snippet: (A) The morphology of ESCs, TBLCs and ESCs, TBLCs in TS medium after 3 days of induction. Scale bars, 250 μm. (B) FACS analysis of the percentage of CDX2 + cells from ESCs and TBLCs, as well as ESCs and TBLCs cultured in TS medium, using V6.5 cell line. (C) FACS analysis of the percentage of CD40 + cells from ESCs and TBLCs, as well as ESCs and TBLCs cultured in TS medium, using TC1 cell line. (D) FACS analysis of the percentage of CD40 + TELCs obtained from the TBLCs after induction with different molecules, including FGF4, Activin A, TGFβ1 and BMP4. The corresponding cell morphology is displayed in the lower panel. (E) Scatterplots displaying the transcriptome comparison of TELCs before and after CD40-based FACS using RNA-seq. Upregulated (FC>2) and downregulated (FC<0.5) genes are shown in red and blue, respectively. (F) The morphology of TBLCs of different passages and long-term culture in TX and TS medium, also the morphology of TBLCs after CD40 FACS after induction. Scale bars, 250 μm. (G) Western blotting was used to detect OCT4, CDX2 and EOMES in TELSCs from different passages. β-Tubulin was used as a loading control. (H) The morphology 8C embryos cultured in TX medium. Scale bars, 250 μm. (I) FACS analysis of the percentage of CD40 + cells in TELSC em s at different passages. (J) Immunofluorescence staining of TFAP2C and PEG10 in TBLCs, TELSCs and TELSC em s. Scale bars, 50 μm. (K) Cell cycle analysis of ESCs, TELSCs and TELSC em s. (L) Heatmap indicating the relative expression of TBLCs, TELSCs and TELSC em s. The representative genes and enrichment of GO terms of these genes is shown. (M) Heatmap indicating the relative expression of characteristic genes in TELSCs, TELSC em s and TSCs. Bubble chart showing the relative expression of these genes in mouse embryos. (N) Heatmap indicating the relative expression of characteristic genes in TELSCs, TSCs cultured in TX medium and TSCs cultured in TS medium. Heatmap on the right demonstrating the expression of each cluster in mouse embryos. The representative genes and enrichment of GO terms of these genes is shown. (O) The scatter plot displays differentially expressed genes between TELSCs and TSCs cultured in various media. The bar graph summarizes the number of differentially expressed genes identified under each comparison condition. (P) GSEA analysis of ESCs, TBLCs, TELCs and TELSCs based on “embryonic placenta development” and “placenta development” geneset. (Q) Heatmap indicating the differentially expressed genes in Hippo pathway of TELSCs and TBLCs. (R) Heatmap indicating the relative expression of characteristic genes in TELSCs, TSCs cultured in TX medium and TSCs cultured in TS medium. Bubble chart showing the relative expression of these genes in mouse embryos. (S) Phase contrast images of TBLCs cultured in TS medium for 24h supplemented with Verteporfin at the indicated concentration. Scale bars, 100 µm. (T) Heatmap indicating the differentially expressed genes of TELCs and TBLCs induction in TS medium plus verteporfin. Bubble chart showing the relative expression of these genes in mouse embryos. (U) GSEA analysis of TELCs, TBLCs induction in TS medium and in TS medium plus verteporfin based on TE geneset. (V) The morphology of TELSCs cultured in TS medium, TS medium plus ITS-X and TS medium plus TGFβ1. (W) Heatmap indicating the differentially expressed genes of TELSCs, TBLCs induction in TX medium withdraw ITS-X, in TS medium and in TS medium plus ITS-X. Heatmap on the right demonstrating the expression of each cluster in mouse embryos. The representative genes and enrichment of GO terms of these genes is shown. (X) GSEA analysis of TBLCs induction in TX medium withdraw ITS-X and in TX medium based on “Positive regulation of stem cell proliferation” and “Positive regulation of cell cycle” geneset.

Article Snippet: All TSLs were cultured on Matrigel-coated plates, in 30% TS medium (RPMI 1640 (GIBCO, 11875119), 20% FBS, 1% GlutaMax (GIBCO, 35050061), 1% penicillin-streptomycin (GIBCO, 15140163), 1% sodium pyruvate (GIBCO, 11360070)) and 70% MEF-conditioned TS medium supplemented with 25 ng/ml human recombinant FGF4 (MCE, HY-P7014) and 1 μg/ml heparin (STEMCELL, 7980).

Techniques: Cell Culture, Comparison, RNA Sequencing, Western Blot, Control, Immunofluorescence, Staining, Cell Cycle Assay, Expressing, Concentration Assay

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Association between gene expression and CES total editing rate We analyzed association between CES total editing rate and gene expression for 14,961 human genes. (a) Gene set enrichment analysis by hypergeometric test on GO-BP categories and REACTOME pathways revealed that associated genes are mainly involved in immune system response mediated by interferon I and alpha / beta. (b) When we analyze distribution of CES total editing rate and ADAR gene expression, ADAR expression levels explains ∼ 13% of observed variability. No significant effect is observed for ADARB1 expression. ADAR and ADARB1 expression levels are reported as residuals of ridge regression with technical covariates (see description of data in methods section). The graphs report adjusted p-value and R2 value from robust regression analysis. (c) The 1,122 genes associated to CES total editing rate after removing ADAR expression effect were enriched for genes mainly involved in ribonucleoprotein and RNA processing.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Genes associated with CES total editing rate are enriched for ADAR interactors (a) Reconstructed PPI network including ADARs and proteins encoded by best genes significantly associated with global editing levels (FDR < 0.01). Among these proteins, we observed 285 potential ADARs interactors, including 9 direct partners of ADARs proteins. (b) Boxplot of number of ADARs interacting genes observed in 1M random simulations. The observed number of interactions (285) resulted in empirical p-value < 1e-6. (c) ADARs interactors are strongly enriched for RNA binding proteins in GO-MF categories. (d) Distribution of degree and betweenness centrality values among network nodes are represented by violin plots. ADAR1 protein has a major role (higher values) among ADAR proteins. Among ADARs direct partners, ELAVL1, RPA1 and IFI16 showed high values of degree and betweenness centrality, suggesting a central role in the network. (e) ADAR1 interaction with RPA70 (coded by RPA1) and IFI16 determined by co-immunoprecipitation. After immunoprecipitation with ADAR1 antibody, western blot for IFI16 and RPA70 are reported. For a better discrimination two times of exposure are reported in the figure.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: RNA Binding Assay, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of cell composition on CES total editing rate and ADAR / ADARB1 expression Our analysis revealed strong associations with CES total editing rate for 4 cell type variables (a), representing proportion of neutrophils, monocytes, dendritic cells (DC) and T helper (Th). Specific cell variables resulted significantly associated also to ADAR (b) and ADARB1 (c) expression. Significance level (p) and correlation coefficient (r) are reported in each plot based on Pearson’s product-moment. Only non-zero observations are plotted.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of biological / pharmacological factors on CES total editing rate and ADAR / ADARB1 expression Our analysis revealed significant associations with CES total editing rate for blood pressure medication, BMI current, Age and Sex (a). Specific biological variables resulted significantly associated also to ADAR (b) and ADARB1 (c) expression. Significance level of association after correction for cell composition is reported (p (cell)) is reported in each plot based on Mann-Whitney-Wilcoxon or Pearson’s product-moment correlation test for binary and continuous variables, respectively. For continuous variables the Pearson correlation coefficient (r) is also reported.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of cell composition, biological and pharmacological factors on PCs of editing levels The heathmap represents strength of association between the first 5 principal components of CESs (PCs) and ADAR / ADARB1 expression (upper panel), 7 cell composition variables (middle panel) and 11 biological / pharmacological variables (lower panel). Only factors showing significant association with at least one of the first 5 PCs are represented. Significant p values (< 0.05) are colored in yellow-red scale, while p value > 0.05 are represented in grey scale. Age, BMI, blood pressure medications, smoke and alcohol all associate with PC1. Also time of blood draw seems to have a small, but consistent effect, on different PCs. For each PC, variance explained is represented by the bar plot in the upper side.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Association study for SNPs and CES total editing rate (a) Manhattan plot representing the association between 573,801 SNPs and CES total editing rate, where black line represents threshold for the top 100 SNPs (p value ∼ 10e-4). (b) Detailed view of genotyped SNPs located in the region at chromosome 7 that showed significant association with CES total editing rate. Known GWAS associations for human phenotypes from GRASP database are reported in the lower panel. (c) The top associated SNP (rs856554) showed a significant effect on global editing level, while no significant correlation was observed with ADAR and ADARB1 expression. (d) Real-time expression analysis of ADAR and ADARB1 mRNA after B-EBV transfection of LOC730338. Not transfected cells were used as control samples. Data are reported as 2 -ΔΔ ct (expression level of control sample is equal to 1) and represent mean values and standard errors obtained from at least 3 independent evaluations. Unpaired t test was used for statistical analysis (*p< 0.05).

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing, Transfection

Journal: bioRxiv

Article Title: The histone demethylase KDM5 is essential for larval growth in Drosophila

doi: 10.1101/297804

Figure Lengend Snippet: (A) Lethality of kdm5 K06801 , kdm5 10424 and kdm5 140 homozygous mutant animals generated from a cross between five female and five male heterozygous parents balanced using CyO-GFP. The column labeled total flies indicates the number of progeny (adult) flies scored from at least three independent crosses. Expected number of progeny is based on Mendelian frequencies and taking into account the lethality of CyO homozygotes, i.e 33% of total adult flies. * p <0.01 (chi-squared test). (B) Position of the NP4707 , 10424 and K06801 P element insertions and molecular mapping of the kdm5 140 deletion. Ab indicates the region used to generated the rabbit polyclonal anti-KDM5 antibody ( S ecombe et al . 2007 ). (C) RT-PCR using primers to the 5’ end of the gene using RNA from whole 3 rd instar larvae. Animals homozygous for kdm5 K06801 or kdm5 10424 show low levels of transcript while kdm5 140 shows none. kdm5 mRNA normalized to wildtype ( w 1118 ) using rp49 . **** p <0.0001. (D) RT-PCR using primers to the 3’ end of the gene using RNA from whole 3 rd instar larvae. kdm5 140 has wildtype levels of the 3’ end of the transcript. **** p <0.0001. ns = not significant. (E) Western from wildtype ( w 1118 ) and kdm5 140 homozygous mutant wing imaginal discs showing KDM5 and alpha tubulin. kdm5 140 animals have no detectable full length or truncated KDM5. *ns indicates non-specific band. (F) Schematic of strain genotype for rescue of kdm5 140 with a genomic rescue transgene. Flies are homozygous for the kdm5 140 mutation on the 2 nd chromosome and homozygous for an 11kb genomic rescue transgene on the 3 rd chromosome. (G) Western blot showing KDM5 protein levels from 3 rd instar larval wing imaginal discs from wildtype ( w 1118 ) and kdm5 140 homozygotes that also have two copies of the kdm5:HA genomic rescue transgene. Anti-KDM5 (top), anti-HA (middle) and anti-histone H3 loading control (bottom). (H) kdm5 140 lethality is rescued by a transgene encoding the kdm5 locus. These data were generated by crossing female and male flies heterozygous for kdm5 140 and homozygous the wildtype genomic rescue transgene (intercross of kdm5 140 /CyO-GFP; kdm5:HA / kdm5:HA males and females).

Article Snippet: Antibodies used were anti-pH3 (Cell signaling #9701, 1/1000), anti-histone H3 (Active Motif #39763 or #39163, 1/5000), anti-alpha Tubulin (Developmental Studies Hybridoma Bank, University of Iowa; 1:5000).

Techniques: Mutagenesis, Generated, Labeling, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: bioRxiv

Article Title: The Aryl Hydrocarbon Receptor Controls IFNγ-Induced Immune Checkpoints PD-L1 and IDO via the JAK/STAT Pathway in Lung Adenocarcinoma

doi: 10.1101/2024.08.12.607602

Figure Lengend Snippet: A) Expression of Cd274 , Ido1 , Ido2 , Cyp1a1 , and Cyp1b1 mRNA was quantified by RT-qPCR in control and CMT167 AhR-KO cells (left two bars in each plot) or after 72 hours of treatment with 10 µM benzo(a)pyrene (B(a)P)(right two bars in each plot). Data from three independent experiments, each in duplicate or triplicate are presented as Gapdh -normalized means + SE. B) Protein extracted from cells treated as in ( A ) was probed by western immunoblotting for PD-L1 and, as a loading control, β-actin. One of three representative western blots is shown on the left and β-actin-normalized protein band densities are on the right. Band density data are presented as means from three experiments, each in triplicate, + SE. C) The percentage of Kyn + CMT167 WT or CMT167 AhR-KO cells treated with vehicle or B(a)P as in ( A ) was quantified by flow cytometry. Data from two experiments, each in triplicate, are presented as means + SE from. *p<0.05, **p<0.01, ****p<0.0001 (Student’s t-test, equal variance).

Article Snippet: The following TaqMan assays were purchased from Thermo Fisher Scientific: Cd274 (Mm03048248_m1), Ido1 (Mm00492590_m1), Ido2 (Mm00524210_m1), Cyp 1a1 (Mm00487218_m1), Cyp1b1 (Mm00487229_m1), Jak2 (Mm01208489_m1), Stat1 (Mm012 57286_m1), gapdh (Mm99999915_g1), CD274 (Hs00204257_m1), IDO1 (Hs00984148_m1), J AK2 (Hs01078136_m1), STAT1 Hs01013996_m1), STAT3( Hs00374280_m1 ), Muc1 ( Mm00449604_m1 ), Col5a1 ( Mm00489299_m1 ), Thbs1 ( Mm01335418_m1 ), Egfr ( Mm01187858_m1 ), Itgb2 ( Mm00434513_m1 ), Cd109 ( Mm00462151_m1 ), Ccl2 ( Mm00441242_m1 ), Ccl5 ( Mm01302427_m1 ), GAPDH (Hs99999905_m1).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Flow Cytometry

Journal: bioRxiv

Article Title: The Aryl Hydrocarbon Receptor Controls IFNγ-Induced Immune Checkpoints PD-L1 and IDO via the JAK/STAT Pathway in Lung Adenocarcinoma

doi: 10.1101/2024.08.12.607602

Figure Lengend Snippet: A) CMT167 Ctrl or CMT167 AhR-KO cells were untreated or treated with 100 ng/ml IFNγ. Cd274 mRNA was quantified by RT-qPCR 24h later. Ido1 , Ido2 , Cyp1a1 , and Cyp1b1 mRNA was quantified 72h later. Data are from three independent experiments, each in duplicate or triplicate, are expressed as fold change of Gapdh -normalized means + SE. B) CMT167 Ctrl or CMT167 AhR-KO cells were left untreated or treated for 24h with 100 ng/ml IFNγ and PD-L1 protein expression assayed by western immunoblotting. A representative immunoblot is on the left and β-actin normalized band densities, averaged from three independent experiments, is on the right. (Bands from IFNγ-treated cells reached saturation prior to bands from untreated cells becoming visible). C) CMT167 Ctrl or CMT167 AhR-KO cells were treated for 24h with IFNγ and IDO1 protein expression assayed by western immunoblotting. A representative immunoblot is on the left and β-actin normalized band densities, averaged from three independent experiments, are on the right. D) CMT167 Ctrl or CMT167 AhR-KO cells were treated with 1-1000 ng/ml IFNγ for 24h and Kyn released into the media quantified via colorimetric assay. Data are averaged from two independent experiments each in quadruplicate + SE. E) CMT167 Ctrl or CMT167 AhR-KO cells were treated with 100 ng/ml IFNγ and Jak2 and Stat1 mRNA quantified 24h later. RT-qPCR data are from three independent experiments, each in triplicate, and presented as Gapdh -normalized means + SE. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student’s t-test, equal variance).

Article Snippet: The following TaqMan assays were purchased from Thermo Fisher Scientific: Cd274 (Mm03048248_m1), Ido1 (Mm00492590_m1), Ido2 (Mm00524210_m1), Cyp 1a1 (Mm00487218_m1), Cyp1b1 (Mm00487229_m1), Jak2 (Mm01208489_m1), Stat1 (Mm012 57286_m1), gapdh (Mm99999915_g1), CD274 (Hs00204257_m1), IDO1 (Hs00984148_m1), J AK2 (Hs01078136_m1), STAT1 Hs01013996_m1), STAT3( Hs00374280_m1 ), Muc1 ( Mm00449604_m1 ), Col5a1 ( Mm00489299_m1 ), Thbs1 ( Mm01335418_m1 ), Egfr ( Mm01187858_m1 ), Itgb2 ( Mm00434513_m1 ), Cd109 ( Mm00462151_m1 ), Ccl2 ( Mm00441242_m1 ), Ccl5 ( Mm01302427_m1 ), GAPDH (Hs99999905_m1).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Colorimetric Assay

Journal: bioRxiv

Article Title: The Aryl Hydrocarbon Receptor Controls IFNγ-Induced Immune Checkpoints PD-L1 and IDO via the JAK/STAT Pathway in Lung Adenocarcinoma

doi: 10.1101/2024.08.12.607602

Figure Lengend Snippet: A) A549 Ctrl or A549 AhR-KO cells were untreated or treated with 100 ng/ml IFNγ for 24h and CD274 , IDO1, and CYP1B1 expression quantified by RT-qPCR. Data from four experiments, each in triplicate, are represented as fold change of GAPDH -normalized means + SE. B) The percent positive PD-L1 + cells treated as in ( A ) was quantified by flow cytometry. Data from three experiments, each in triplicate, are presented as mean percent PD-L1 + + SE. C) The baseline percent of Kyn + A549 ctrl and A549 AhR- KO cells in two experiments, each in triplicate, was determined by flow cytometry. D) A549 Ctrl or A549 AhR-KO cells were treated with 0-1000 ng/ml IFNγ for 24h and Kyn release quantified by the Kyn-specific colorimetric assay using a standard Kyn curve. Data from two experiments, each in quadruplicate, are presented as average μM Kyn + SE. E) A549 Ctrl or A549 AhR-KO cells were left untreated or treated with 100 ng/ml IFNγ for 24h and baseline or 100 ng/ml IFNγ-induced JAK2 , STAT1, and STAT3 expression quantified by RT-qPCR. Data from four experiments, each in triplicate, are presented as average fold change of Gapdh -normalized means + SE. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student’s t-test, equal variance).

Article Snippet: The following TaqMan assays were purchased from Thermo Fisher Scientific: Cd274 (Mm03048248_m1), Ido1 (Mm00492590_m1), Ido2 (Mm00524210_m1), Cyp 1a1 (Mm00487218_m1), Cyp1b1 (Mm00487229_m1), Jak2 (Mm01208489_m1), Stat1 (Mm012 57286_m1), gapdh (Mm99999915_g1), CD274 (Hs00204257_m1), IDO1 (Hs00984148_m1), J AK2 (Hs01078136_m1), STAT1 Hs01013996_m1), STAT3( Hs00374280_m1 ), Muc1 ( Mm00449604_m1 ), Col5a1 ( Mm00489299_m1 ), Thbs1 ( Mm01335418_m1 ), Egfr ( Mm01187858_m1 ), Itgb2 ( Mm00434513_m1 ), Cd109 ( Mm00462151_m1 ), Ccl2 ( Mm00441242_m1 ), Ccl5 ( Mm01302427_m1 ), GAPDH (Hs99999905_m1).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Colorimetric Assay

Journal: bioRxiv

Article Title: Drosophila melanogaster Toll-9 elicits antiviral immunity against Drosophila C virus

doi: 10.1101/2024.06.19.599730

Figure Lengend Snippet: (A) In silico prediction of signal peptide in Toll-9 protein sequence. Red solid line indicates predicted n-terminal region, orange solid line indicates the predicted center hydrophobic region, and yellow solid line indicates predicted c-terminal region of signal peptide. Black dotted line indicates the cleavage site (CS) of the signal peptide. Sec/SPI: Sec translocon transported secretory signal peptide/Signal Peptidase I Tat/SPI: Tat translocon transported Tat signal peptides/Signal Peptidase I (B) Western blot analysis demonstrating the presence of Toll-9/V5 in endosomes. Endosomal fractions were identified using Rab5 as a microsomal marker, while Actin served as a cytosolic marker. Data are representative of three independent experiments. (C) Micrographs show colocalization of Rab5-early endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (D) Micrographs show colocalization of Rab7-Late endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (E) Micrographs show colocalization of Rab5-early endosome marker (green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (F) Toll-9 (anti-V5 tag ab-Green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (G) Western blot analysis using the indicated antibodies following immunoprecipitation of V5 tag (Toll-9) using J2 dsRNA antibody from the lysate of Poly (I:C) treated Toll-9 OE and S2 cells in presence and absence of CuSO 4 (500 µM). Data are representative from three independent experiments.

Article Snippet: The cells were blocked in phosphate-buffered saline (PBS) containing 10% FBS and incubated with antibodies against Rab5 (1:50; Abcam ab31261), Rab7(1:20; Developmental Studies Hybridoma Bank Rab7), DCV Capsid (1:200; Abcam ab92954), dsRNA J2 (1:200; Jena Biosciences RNT-SCI-10010200) and V5 tag (1:200; ThermoFisher Scientific R960-25) for overnight at 4°C.

Techniques: In Silico, Sequencing, Western Blot, Marker, Immunoprecipitation

Journal: bioRxiv

Article Title: Targeted protein degradation in lysosome utilizing naturally produced bifunctional antibodies with high levels of mannose 6-phosphate glycans

doi: 10.1101/2024.09.03.611037

Figure Lengend Snippet: A . Schematic showing the procedure for detection of FLAG-mCherry and NCA-FLAG or PNCA-FLAG complexes binding toward CI-MPR. B . Bound mCherry fluorescence from (A) for NCA-FLAG (gray) or PNCA-FLAG (red). N=3 or 4. C . HepG2 cells were incubated with FLAG-mCherry and NCA-FLAG or PNCA-FLAG overnight with or without 2 mM M6P and cells were examined by fluorescent microscopy to detect mCherry signal (red). Lysosomes were stained with LysoTracker (green) and Hoechst (blue) for nuclei. D-H . Cell lines (D. HEK293T, E. Tay-Sachs Patient Fibroblasts, F. Daoy, G. HepG2, or H. MDA-MB-231) were incubated with FLAG-HexM enzyme with NCA-FLAG or PNCA-FLAG or with addition of 2 mM M6P. HexM internalization was determined by performing a HexM activity assay and activity graphed – HexM alone (gray), NCA-FLAG with HexM (Green), PNCA-FLAG with HexM (Blue), M6P added (black striped bars). Background HexM activity in untreated group wa subtracted. Dot represents each independent experiment. N=3-6. I . HEK293T CI-MPR -/- cells were generated by CRISPR-Cas9. Western blot shows CI-MPR deficiency in the cells. J . Graphs depict internalized HexM activity in CI-MPR -/- cells. Dot represents each independent experiment. N=3. Data are represented as mean values; error bars represent the standard deviation of biological replicates.

Article Snippet: HepG2 (ATCC, HB-8065), Daoy (ATCC, HTB-186) and A431 (Sigma, 85090402) cells were cultured in MEM medium (Corning, 10-009-CV) with 10% FBS.

Techniques: Binding Assay, Fluorescence, Incubation, Microscopy, Staining, Activity Assay, Generated, CRISPR, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: Targeted protein degradation in lysosome utilizing naturally produced bifunctional antibodies with high levels of mannose 6-phosphate glycans

doi: 10.1101/2024.09.03.611037

Figure Lengend Snippet: A . Schematic demonstrating production of Ab-TNFα and PNCA-TNFα in CHO cells. B . Western blotting to examine the Ab-TNFα and PNCA-TNFα heavy chain N-glycosylation after Endo-H and PNGase-F treatment. C-D . CI-MPR affinity chromatograph to determine the binding of Ab-TNFα and PNCA-TNFα. To visualize the binding signal, Alexa Fluor 594 conjugated to Ab-TNFα (C) or PNCA-TNFα (D) was used for the assay. Red dash line indicates the concentration of free M6P for antibody elution from the CI-MPR column. E . Schematic depicting internalization and degradation of extracellular TNFα b PNCA-TNFα through CI-MPR to the lysosome. F . HepG2 cells were incubated with mCherry-TNFα and Ab-TNFα or PNCA-TNFα overnight with or without 2 mM M6P and cells were examined by fluorescent microscopy to detect mCherry-TNFα signal (red). Lysosomes were stained with LysoTracker (green) and Hoechst for Nuclei (blue). G . Internalized mCherry-TNFα protein was analyzed by western blot for cell lysates using antibodies to detect mCherry, TNFα, and GAPDH as a loading control. H . Western blot analysis for internalized mCherry-TNFα with the addition of 1% or 0.5% of lysosomal protease inhibitors (PI). Antibodies are used to detect mCherry, TNFα and GAPDH, as a loading control.

Article Snippet: HepG2 (ATCC, HB-8065), Daoy (ATCC, HTB-186) and A431 (Sigma, 85090402) cells were cultured in MEM medium (Corning, 10-009-CV) with 10% FBS.

Techniques: Western Blot, Glycoproteomics, Binding Assay, Concentration Assay, Incubation, Microscopy, Staining, Control

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Ter119 negative cells and Ter119 positive erythroid cells were purified from wild-type mouse bone marrow cells. G4 levels were tested by flow cytometry using the BG4 antibody that specifically recognizes G4. Quantification is on the right. (B) Bone marrow lineage-negative cells were cultured in Epo medium for 2 days. G4 levels were tested on different days using flow cytometry by the BG4 antibody. Quantification is on the right. (C) CD34+ human HSPCs were cultured in Epo medium for 21 days. The levels of G4 were measured by flow cytometry as in B at the indicated time. Cells at day 7, 14, and 21 represent proerythroblasts, polychromatic to orthochromatic erythroblasts, and orthochromatic to mature red blood cells, respectively. (D) Flow cytometric assays of G4 levels in the indicated bone marrow lineage cells purified from wild-type mice. (E) Quantification of D. (F) Gating strategy of various erythroblasts. Populations I to VI represent proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts, late orthochromatic to reticulocytes, and mature red blood cells, respectively. (G-H) Flow cytometric assay of G4 level in bone marrow erythroid populations I (G) and V (H) from the indicated mice. Quantification is on the right. (I) Bone marrow lineage negative cells from the indicated mice were cultured in Epo medium for 2 days. G4 levels on different days were measured by flow cytometry using BG4 antibody. Quantification is below the histogram. (J) CD34+ cells were transduced with lentiviral vectors expressing indicated sgRNAs and Cas9. Cells were then harvested for Western blotting of the indicated proteins at day 9 in culture. (K) Quantitative analyses of G4 levels in cells from J using flow cytometric assays. (L) Quantitative analyses of cell death in cells from J using flow cytometric assays. The dead cells are defined as propidium iodide and annexin V double positive. (M) Quantitative analyses of G4 levels in bone marrow mononuclear cells from the patient with DDX41 mutated MDS. All the error bars represent the SEM of the mean. The comparison between two groups was evaluated with 2 tailed t tests, and the comparison among multiple groups was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. ns: not significant.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Purification, Flow Cytometry, Cell Culture, Transduction, Expressing, Western Blot, Comparison

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Epo medium-cultured mouse bone marrow lineage negative HSPCs were treated with 1 μM PDS for the indicated time. Immunofluorescence assays of γ-H2AX were performed, and representative images of the erythroid cells were presented. Scale bar: 5 μm. (B) Flow cytometry assay of the cells in A. (C) Statistical quantification of γH2AX signals in B. (D) Epo medium-cultured mouse bone marrow lineage negative HSPCs were cultured for 1 day, followed by the treatment of 1 μM PDS for 6 hours. Quantitative RT-PCR analyses of indicated ribosome RNAs were performed using different primer sets. (E) Western blotting assays of indicated in cells from D. Actin was used as a loading control. (F) Same as D except that bone marrow lineage negative HSPCs from HBBCre:Ddx41 fl/fl mouse were cultured for 1 day before the quantitative RT-PCR assays. (G) Western blotting assays of the indicated proteins in F. Cells from both day 1 and day 2 cultured cells were analyzed. (H) CD34+ cells were transduced with lentiviral vectors expressing indicated sgRNAs and Cas9. Cells were then harvested for Western blotting of the indicated proteins at day 9 in culture. (I) Immunohistochemical stains of p53 in bone marrow core biopsies from the patient in normal individual. Scale bar: 100 μm. (J) Quantification of γ-H2AX in bone marrow mononuclear cells from the patient in I and 2 control individuals. All the error bars represent the SEM of the mean. The comparison between two groups was evaluated with 2 tailed t tests, and the comparison among multiple groups was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01, ns: not significant.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Cell Culture, Immunofluorescence, Flow Cytometry, Quantitative RT-PCR, Western Blot, Control, Transduction, Expressing, Immunohistochemical staining, Comparison

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Representative wide-field picture and H&E stains of bone marrow organoid in culture. (B) Whole-mount 3D imaging of the organoids. Imaris was used for cell surface rendering. Organoids were stained with indicated antibodies and subsequently imaged using a laser scanning confocal platform. (C) Confocal immunofluorescence assays of erythroid islands in the iPSC-derived bone marrow organoids (left) and a primary human bone marrow biopsy (right). CD71 was labeled with green for organoids and magenta for primary bone marrow. DAPI: blue. (D) Flow cytometry assays of the organoids using indicated antibodies for various lineages. (E) 10,000 CellVue-labeled donor CD34+ HSPCs were co-incubated with iPSC-derived bone marrow organoids for 3 days in each well of a 96-well plate, followed by an immunofluorescence assay. Representative pictures show the engraftment of donor hematopoietic cells into the organoid. Green, red, and blue represent CD71, CellVue, and DAPI-positive nuclei, respectively. The arrow points to an engrafted CellVue positive cell expressing CD71. (F) Flow cytometry of the organoids using indicated antibodies for various lineages of the engrafted cells in organoids from E. (G) Same as E, except the donor CD34+ cells were transduced with lentiviral vectors expressing Cas9 and indicated sgRNAs before co-incubation. After 3 days, the cells were collected for flow cytometric assays of erythroid and myeloid differentiation of CellVue-positive donor hematopoietic cells and negative iPSC-derived hematopoietic cells. Each data point represents cells combined from 10 organoids. The comparison was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01. (H) Schematic model of the function of DDX41 during erythropoiesis. The diagram is generated through BioRender.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Imaging, Staining, Immunofluorescence, Derivative Assay, Labeling, Flow Cytometry, Incubation, Expressing, Transduction, Comparison, Generated

Journal: bioRxiv

Article Title: Virus and Cell Specific HMGB1 Secretion and Subepithelial Infiltrate Formation in Adenovirus Keratitis

doi: 10.1101/2025.01.07.631509

Figure Lengend Snippet: (A) Cytoplasmic and nuclear extracts prepared from uninfected or HAdV-D37-infected for 2, 12, 24, and 48 hpi. were resolved on 4-20% SDS-PAGE. Western blots for HMGB1 in the nuclear (Nuc), cytoplasmic (Cyt), and supernatant (Sup) extracts with TBP (nuclear) β-actin (cytoplasmic) as loading controls in cell types THE, PHCE, HCF, A549, and HEK293 (left to right). (B) Densitometric analysis of HMGB1 band intensity of cytoplasmic and nuclear extracts in THE, PHCE, HCF, A549, and HEK293 (left to right). p values were determined by t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 ( n=3 ). (C) Quantification of extracellular HMGB1 measured from supernatants collected at indicated time points pi in THE and PHCE cells ( n=3 ). (D) Schematic representation (created using Biorender.com ) for HAdV-D37 induced translocation of HMGB1 from the cell nucleus to the cytoplasm and then into the extracellular space.

Article Snippet: A549 (CCL-185), a human lung carcinoma cell line, and HEK293 (CRL-1573), a human embryonic kidney cell line, were purchased from American Type Culture Collection (ATCC).

Techniques: Infection, SDS Page, Western Blot, Translocation Assay

Journal: bioRxiv

Article Title: Virus and Cell Specific HMGB1 Secretion and Subepithelial Infiltrate Formation in Adenovirus Keratitis

doi: 10.1101/2025.01.07.631509

Figure Lengend Snippet: (A) Western blot analysis for HMGB1 expression along with β-actin for load control from whole cell lysates of uninfected (M) or HAdV-D37-infected (V) THE cells for 2, 12, 24, and 48 hpi. qRT-PCR analysis of HMGB1 gene expression for mock and virus infected cells at the same times pi is shown as bar graphs below the Western blot. (B) Bar graph for qRT-PCR of the viral early gene E1A expression, a surrogate marker for viral entry, and normalized to human ACTG gene for quantification. (C) Viral late protein pIIIa expression in cytoplasmic and nuclear extracts prepared from uninfected or HAdV-D37-infected THE, PHCE, HCF, A549 and HEK293 for 2, 12, 24, and 48 hpi show successful infection of all cell types.

Article Snippet: A549 (CCL-185), a human lung carcinoma cell line, and HEK293 (CRL-1573), a human embryonic kidney cell line, were purchased from American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Control, Infection, Quantitative RT-PCR, Gene Expression, Virus, Marker