Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: A. Experimental scheme. B. Spearman correlation between expression profiles of Mbd3f/- system Calculated over all differential genes (n=8,042), showing an average correlation of R=0.93 between consecutive samples. C. As in B, but between Mbd3flox/- and Gatad2a-/- systems. D. Overlap between targets of OSKM in promoters and enhancers. Pixel shade indicates Jaccard Index. E. Correlation between consecutive samples in Mbd3f/- system (MEF-day1, day1-day2, day2..day8-iPS), measured over all ESPGs promoters (promoters with differential chromatin pattern, n=3,593, top), or all differential enhancers (n=40,174, bottom), for each chromatin mark. Negative controls were calculated between MEF and IPS, are marked with solid border. F. Overlap between binding targets of Oct4, Sox2, Klf4 or Myc, and previously published binding data of the same factors, calculated in ES and iPS samples. Percentage out of our measured binding targets is presented, along Fisher exact test p-values. G. Global transcriptional pattern of 8,042 differential genes (FC>4 & maximal FPKM value>1), sorted by their temporal pattern in Mbd3f/-system (the same gene order was applied for the other reprogramming systems). Heatmap represents unit-transformation of FPKM values. H. PCA analysis of all samples, alongside samples from previous publications (Polo et al., 2012). PCA was calculated on the same set of genes and normalization as in G. I. GO categories enriched among the genes that are active in each day. Gene is defined to be active in samples where RPKM is above 0.5 of the gene max value. P-values were calculated with Fisher exact test, and FDR corrected. Categories with corrected p-value<0.01 in at least two-time points are presented. Gray Shades represent FDR corrected p-values

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: Expressing, Binding Assay, Transformation Assay

Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: A. ChIP-seq landscape of two examples. Promoters are marked in red, enhancers are marked in green. Signals are normalized to sample size (RPM). B. Overlap between binding of OSKM and active enhancers, in each day of reprogramming. Enhancer is defined as active in a specific day if its ATAC-seq z-score is above 1.5 STD in that day. Gray shades indicate Fisher exact test p-value for overlap between compared samples. Note that OSKM do not bind the enhancers that are active in MEF (D0, marked in red); these enhancers are not significantly bound by OSKM at any day during reprogramming. C. Number of enhancers bound by each of OSKM factors in each day of reprogramming. Upper row: out of enhancers that are bound by the factor in late stages (day8, iPS, ESC). Bottom row: out of enhancers that are bound by the factor in early stages (day1-day3). D. Probability to observe co-localized binding of transcription factors in promoters (gray) and enhancers (black). Calculated in days 1,8 and iPS (Error bars indicate S.E.M). Right – Myc binds 32% of promoters, and 8% of active enhancers. E. Significant motifs enriched in promoters and enhancers that are bound by each of Oct4, Sox2, Klf4 and c-Myc at different days of reprogramming, as detected by Homer/4.7 software. P-values, indicated by color shade, were reported by Homer, and are FDR corrected. Motifs which are significantly enriched (corrected p<10-30) in at least one time point are presented. F. a. Motifs enriched in differential enhancers that are active in each day of reprogramming (ATAC-seq z-score >1.5). P-values, indicated by color shade, were reported by Homer, and are FDR corrected. Motifs which are significantly enriched (corrected p<10-50) in at least one-time point are presented. G. Motifs found in “closed” vs. “open” binding targets of the indicated transcription factor. Accessibility of targets was calculated based on ATAC-seq. Motifs found in OSK binding targets calculated in Mbd3f/- day1. Motifs that are different between open and closed binding targets are marked in black line. Complementary motifs to canonical motif appear in reverse order. H. Spearman correlation between ATAC-seq profiles of the two efficient reprogramming systems: Mbd3f/- MEF and C/EBPaTg B cell systems calculated over 40,174 differential enhancers.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: ChIP-sequencing, Binding Assay, Software

Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: A. Distribution of low (<0.02), mid (0.02-0.98) and high (>0.98) methylated CpG sites, along reprogramming. Average and SEM are indicated in red plot. B. Methylation level measured in covered enhancers (n=18,072), in Mbd3f/-, Gatad2a-/- and WT-2 systems. Enhancers are clustered into eight clusters using k-means. Cluster 8 consists of enhancers that undergo fast demethylation, compared to clusters 3 and 7. C. Average methylation measured in promoters of genes that were highly methylated (>80%) in day0. Genes that change their expression level (red) are compared to genes that do not change their expression level (gray). Wilcoxon p-value indicates places where methylation of differential genes is significantly lower than methylation of non-differential genes. D. Left: Enrichment of enhancer clusters, as shown in panel B, for OSK binding, DNA accessibility, and super enhancers, showing that cluster 8 is highly enriched for OSK binding and overlaps with super enhancers. Color shades represent FDR corrected enrichment p-value. Right: Enrichment of the same enhancer clusters to transcription factor binding, taken from hmChip database. Cluster size is indicated on the right. E. Experimental scheme summary. Reprogramming efficiency was measured by Oct4-GFP+ cells percentage in Tet1/2/3 null(Δ) and Tet1/2/3fl/fl with and without Gatad2a expression, after 8 days. **p<0.01, ***p<0.001 (Student’s t-test), n=6, error bars indicate SD. F. Secondary MEF harboring Mir290-RGM and Nanog GFP-reporter were sorted after reprogramming to 3 different populations: RGM-SE-Mir290-tdTomato positive cells (sorted at day 5), Nanog-GFP and Mir290-RGM positive cells (sorted at d10-14), and "double negative" cells (sorted at d5). The cells were seeded as single cell-per-well, and were treated with medium either supplemented with Dox or lacking Dox. On day 14 colonies were inspected for GFP and mCherry (RGM) markers.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: Methylation, Expressing, Binding Assay

Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: A. Experimental flow describing three experimental perturbation settings: (i) Mbd3f/- MEFs were virally infected with cMyc over-expression (OE) cassette, OSK-OE cassette or both cassettes. Gene expression was measured on day4 following infection. (ii) Mbd3f/- MEFs carrying OSK Dox-dependent cassette were treated for knockdown of c-Myc, n-Myc and l-Myc. Gene expression was measured on days 3 and 7, and colony formation was measured on day 11. (iii) Mbd3f/- MEFs carrying OSKM Dox-dependent cassette were treated with inhibitor of cMyc (10058-F4) and with Dox. Gene expression and colony formation were measured on day 3. B. Distribution of Expression fold change (FC) compared to WT MEF of up/down regulated ESPGs (down regulated ESPGs are enriched for somatic genes), and CAPGs. Presented perturbations are over-expression of OSK cassette, over-expression of c-Myc cassette, or over-expression of the two cassettes together. (*p<10-5, **p<10-20, Wilcoxon test). C-D. Reprogrammed colony formation in Myc knockdown ort small molecule inhibition, measured 11-14 days after Dox. E. Distribution of expression fold change (FC, in log2 scale) compared to MEF of up/down regulated ESPGs and CAPGs. Presented perturbations are Myc knockdown, inhibition of Myc activity with small molecular inhibitor (10058-F4). (*p<10-5, **p<10-20, Wilcoxon test). F. Experimental scheme. G. IPSC Reprogramming efficiency in different cells expressing both endogenous and/or exogenous cMyc and nMyc. H. FACS analysis for surface expression of fibroblast surface marker Thy1 on the indicated Mbd3flox/- cell types. Dotted line indicates positive threshold for detection. I. Representative pictures of Mbd3fl/- cells harboring mCherry-NLS and ΔPE-GOF18 Oct4-GFP cassettes after 13 days of reprogramming in the presence of MYCi. Scale = 100μM. J. Left panel - iPSC reprogramming efficiency by applying highly efficient mouse B cell and WT CMP reprogramming protocols by OSKM in the presence or absence of MYC small molecule inhibitor (MYCi). Right panel – Human iPSC reprogramming efficiency by applying OKS lentiviral transduction in the presence of absence of MYCi. K. Expression fold-change distribution (log2 scale) of selected GO categories in Myc over-expression or Myc knockdown, showing that upon over-expression of Myc, processes such as ribosomal biogenesis and chromosome segregation are induced. L. Fraction of Myc targets in significantly induced and repressed GO categories, compared to what is expected by random (dashed line). M. Overlap between differential genes detected in Myc perturbation experiments, and differential genes detected in previous published perturbations (Scognamiglio et al., 2016). Fisher exact test p-values are presented. N. Expression fold change of selected chromatin modifiers.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: Infection, Over Expression, Expressing, Inhibition, Activity Assay, Marker, Transduction

Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: Key Resources Table

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: Recombinant, Protease Inhibitor, Sample Prep, Methylation, Transgenic Assay, Negative Control, Software

Journal: bioRxiv

Article Title: The N-terminal region of malaria vaccine candidate Plasmodium falciparum asparagine-rich merozoite antigen is immunodominant and targeted by polyreactive antibodies

doi: 10.64898/2025.12.11.693633

Figure Lengend Snippet: A) The percentage of IgM + and IgG + B cells with confirmed antigen-specificity obtained for PfARMA and four other merozoite proteins. B) Schematic of the predicted structural features of PfARMA. The asparagine-rich repeat region is shown in green. The six 100-amino acid (aa) long peptides with 50 aa overlap covering intrinsically disordered region (IDR) 1 are indicated below. C) The number of anti-PfARMA hmAbs targeting each of the three indicated regions of the protein. D) Percentage growth inhibition of P. falciparum strain 3D7 in in vitro cultures in the presence of hmAbs 5.134 and 4.104. Data points represent the average of three biological replicates, with the error bars showing the standard deviation. E) Dot-blot analysis showing reactivity of hmAb 5.134 to six IDR1 peptides and a negative control (NC) protein (PfAMA1). As a positive control for the presence of peptide and control protein, a second dot blot was stained in parallel with an anti-His antibody (bottom). F) The percentage of asparagine residues in overlapping 62-aa peptides covering the full P. falciparum ( Pf ) strain 3D7 proteome in the PhIP-seq library (n = 129,411) and among peptides bound by hmAb 5.134 (n = 303). Center line, median; box limits, upper and lower quartiles; whiskers, min/max values; +, mean. The difference between groups was tested using an unpaired student’s t-test. **** P < 0.0001.

Article Snippet: The plasmid encoding full-length PfARMA (PF3D7_1136200-bio, Addgene #47730) was modified to introduce a 6× His tag at the C-terminus.

Techniques: Inhibition, In Vitro, Standard Deviation, Dot Blot, Negative Control, Positive Control, Control, Staining

Journal: bioRxiv

Article Title: The N-terminal region of malaria vaccine candidate Plasmodium falciparum asparagine-rich merozoite antigen is immunodominant and targeted by polyreactive antibodies

doi: 10.64898/2025.12.11.693633

Figure Lengend Snippet: A) Immunofluorescence images of segmented schizonts stained with Hoechst 33342 (DNA), hmAb 5.134 against PfARMA and an antibody against a second merozoite protein (protein X), indicated on the left. Scale bar denotes 5 µm. B) Colocalization analysis of PfARMA with merozoite protein X using five different parasites for each pairwise analysis. The positive control (pos ctrl) was performed using anti-PfRAP1 primary antibody and a mix of two secondary antibodies conjugated to different fluorophores. Top: Pearson’s correlation, middle: Manders’ coefficient 1 (fraction of second protein signal that overlaps with PfARMA signal), bottom: Manders’ coefficient 2 (fraction of PfARMA signal that overlaps with the second protein signal). Differences in correlation coefficients tested for statistical significance using a Kruskal-Wallis test, followed by comparisons between all pairs of groups using Dunn’s post-hoc test, which reports P values that have been corrected for multiple comparisons (n = 21). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The plasmid encoding full-length PfARMA (PF3D7_1136200-bio, Addgene #47730) was modified to introduce a 6× His tag at the C-terminus.

Techniques: Immunofluorescence, Staining, Positive Control

Journal: bioRxiv

Article Title: The N-terminal region of malaria vaccine candidate Plasmodium falciparum asparagine-rich merozoite antigen is immunodominant and targeted by polyreactive antibodies

doi: 10.64898/2025.12.11.693633

Figure Lengend Snippet: Plasma samples from P. falciparum -unexposed U.S. donors (U, n = 15) and P. falciparum -exposed individuals with low (L, n = 46), moderate (M, n = 40), and high (H, n = 54) immunity to malaria were measured in duplicate, with the average of both readings shown for ( A ) PfMSP1 and full-length PfARMA and ( B ) fragments of PfARMA. PfMSP1 was included as a control for P. falciparum exposure and is therefore shown on a gray background. The groups with low and medium immunity were matched for age and P. falciparum exposure history. The dashed horizontal line indicates the cutoff for seropositivity (three standard deviations above the average for the entire P. falciparum -naïve group, or for the subset of P. falciparum -naïve individuals without IgG reactivity for full-length PfARMA and IDR1). Center line, median; box limits, upper and lower quartiles; whiskers, min/max values; +, mean. Differences between groups were tested using a Kruskal-Wallis test, followed by comparisons between all pairs of groups using Dunn’s post-hoc test, which reports P values that have been corrected for multiple comparisons (n = 6). Within each graph, groups sharing the same letter are not statistically significantly different from each other, while groups with different letters are statistically significantly different (P < 0.05). No statistically significant differences were observed between the groups with low and medium immunity. IDR, intrinsically disordered region; FD, folded domain.

Article Snippet: The plasmid encoding full-length PfARMA (PF3D7_1136200-bio, Addgene #47730) was modified to introduce a 6× His tag at the C-terminus.

Techniques: Clinical Proteomics, Control

Journal: bioRxiv

Article Title: The N-terminal region of malaria vaccine candidate Plasmodium falciparum asparagine-rich merozoite antigen is immunodominant and targeted by polyreactive antibodies

doi: 10.64898/2025.12.11.693633

Figure Lengend Snippet: A) Plasma samples were obtained from P. falciparum -unexposed individuals living in the U.S. who were convalescent COVID-19 patients (n = 7), healthy blood donors (n = 8), systemic lupus erythematosus (SLE) patients (n = 10), or rheumatoid arthritis (RA) patients (n = 8). In all plots, the horizontal dashed line indicates the cut-off for reactivity to PfARMA (MFI = 2.6 × 10 3 ), which equals the average MFI + three standard deviations of reactivity to the other six merozoite antigens among samples from non-autoimmune donors (convalescent COVID-19 patients and healthy blood donors). Differences in reactivity to the various merozoite antigens were tested for statistical significance using a Kruskal-Wallis test, followed by comparisons between PfARMA and all other antigens using Dunn’s post-hoc test, which reports P values that have been corrected for multiple comparisons (n = 6). ** P < 0.01; *** P < 0.001. B) Dose-dependent reactivity of IgG purified from plasma of a P. falciparum -naïve U.S. donor with high anti-PfARMA IgG reactivity to PfMSP1, full-length PfARMA, and fragments of PfARMA, as measured by ELISA. IDR, intrinsically disordered region; FD, folded domain. C) Total plasma IgG concentration plotted against PfARMA IgG reactivity for P. falciparum -naïve individuals and P. falciparum -exposed individuals. The line shows the best-fit using simple linear regression, with 95% confidence intervals. D) Reactivity of recombinant IgG1, IgG2, IgG3, and IgG4 with specificity for a different P. falciparum antigen to PfARMA and PfMSP1.

Article Snippet: The plasmid encoding full-length PfARMA (PF3D7_1136200-bio, Addgene #47730) was modified to introduce a 6× His tag at the C-terminus.

Techniques: Clinical Proteomics, Purification, Enzyme-linked Immunosorbent Assay, Concentration Assay, Recombinant

Journal: bioRxiv

Article Title: The N-terminal region of malaria vaccine candidate Plasmodium falciparum asparagine-rich merozoite antigen is immunodominant and targeted by polyreactive antibodies

doi: 10.64898/2025.12.11.693633

Figure Lengend Snippet: A) Overview of the affinity purification of autoantibodies and anti-PfARMA IgG from plasma IgG from P. falciparum -naïve and P. falciparum -exposed individuals. B) Reactivity of affinity-purified autoantibodies (AAb) and the flowthrough (FT) fraction from plasma IgG of P. falciparum -naïve individuals, both tested at 20 µg/mL, to a panel of seven merozoite antigens (left) and PfARMA fragments (right). The average fold change in MFI in the autoantibody fractions relative to the flowthrough fractions is indicated in the top. C) Heatmap showing binding of anti-PfARMA plasma IgG isolated from P. falciparum -naïve and P. falciparum -exposed individuals by affinity purification, as well as anti-PfARMA hmAbs, to (macro)molecules with properties often recognized by auto- or polyreactive antibodies, measured by ELISA. Values are color-coded based on the optical density (OD) after subtraction of the background signal. OD = 0.4 was used as the cutoff for binding. DNP-BSA, dinitrophenol-bovine serum albumin; CFP, cyan-fluorescent protein; KLH, keyhole limpet hemocyanin; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.

Article Snippet: The plasmid encoding full-length PfARMA (PF3D7_1136200-bio, Addgene #47730) was modified to introduce a 6× His tag at the C-terminus.

Techniques: Affinity Purification, Clinical Proteomics, Binding Assay, Isolation, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The N-terminal region of malaria vaccine candidate Plasmodium falciparum asparagine-rich merozoite antigen is immunodominant and targeted by polyreactive antibodies

doi: 10.64898/2025.12.11.693633

Figure Lengend Snippet: A) PfARMA peptides recognized by plasma IgG from P. falciparum -exposed and P. falciparum -naïve individuals. All peptides were located in intrinsically disordered region (IDR) 1 and overlapped either a negatively charged patch (shown in red) or the asparagine-rich repeat region (shown in green). B) Non-PfARMA-derived peptides that were bound by hmAb 5.134 and one or more plasma IgG samples. The red shading indicates the percentage of asparagine residues in the peptides. Absence of reactivity to peptides is indicated with gray shading.

Article Snippet: The plasmid encoding full-length PfARMA (PF3D7_1136200-bio, Addgene #47730) was modified to introduce a 6× His tag at the C-terminus.

Techniques: Clinical Proteomics, Derivative Assay

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet:

Article Snippet: pXL010-Wnt dual (GFP-Fire) reporter (), , Addgene , Cat# 40588.

Techniques: Ubiquitin Proteomics, Recombinant, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Ligation, Western Blot, Over Expression, Plasmid Preparation, Amplification, Clone Assay, Expressing, Software, Microscopy