Journal: Aging Cell

Article Title: Reduction of class I histone deacetylases ameliorates ER ‐mitochondria cross‐talk in Alzheimer's disease

doi: 10.1111/acel.13895

Figure Lengend Snippet: Tac effect on InsP3R1‐VDAC1 interaction and MERCS morphology in AβO‐treated cells. HT22 cells were pre‐treated for 1 h with Tac (10 μM) and then co‐incubated with 1 μM AβO for the remaining 23 h or treated solely with Tac for 24 h. Representative confocal images of in situ PLA signal (gray) indicating a physical interaction between InsP3R1 and VDAC1 (scale bar = 20 μm). The lower panels represent enlargements of the boxed areas in the upper panels. Quantification of number of PLA puncta in about 200–240 cells from 8 image stacks per condition (a). WT hippocampal neurons were pre‐treated for 1 h with Tac (0.5 μM) and then co‐incubated with 1 μM AβO for the remaining 23 h or treated solely with Tac for 24 h. NFAT transcriptional activity evaluated by luciferase reporter assay in hippocampal neurons (b) and HT22 cells (c). Data are the mean ± SEM of 4 independent experiments, run in triplicates to quadruplicates. Schematic representation of MERCS structural parameters: MERCS length and distance, mitochondrial perimeter and Feret's diameter (d). HT22 cells were pre‐treated for 1 h with Tac (10 μM) and then co‐incubated with 1 μM AβO for the remaining 23 h. Representative TEM images of mitochondria (in purple) in close contact with ER (in blue) (scale bar = 200 nm) in which distances ≤25 nm were considered as contacts (e). MERCS length (f) and distance (g), number of MERCS per mitochondria (h), mitochondrial area (i) and perimeter (j), Feret's diameter (k) and mitochondrial aspect ratio (l). Representative TEM images of mitochondrial cristae (in green) (scale bar = 200 nm) (m). Number of cristae per mitochondria (n), and cristae area (o). About 70–90 individual mitochondria from 10 randomly selected cells obtained from approximately 30 images were assessed in 26–38 independent TEM images. Statistical analysis: Kruskal–Wallis test followed by uncorrected Dunn's multiple comparisons test, one‐way ANOVA followed by uncorrected Fisher's LSD multiple comparison test; * p < 0.05; ** p < 0.01; *** p < 0.0001; **** p < 0.0001 when compared to control.

Article Snippet: NFAT luciferase reporter was obtained from Addgene.

Techniques: Incubation, In Situ, Activity Assay, Luciferase, Reporter Assay, Comparison, Control

Journal: Light, Science & Applications

Article Title: Near-infrared light activatable chemically induced CRISPR system

doi: 10.1038/s41377-025-01917-8

Figure Lengend Snippet: a Custom LED illumination setup. b Fluorescence of eGFP-expressing HEK293FT cells treated with rapamycin dimer, transfected with full-length dCas9. At the top of the setup there is a holder for a 35 mm glass-bottom dish. The 3D-printed double slit screen is located in the focal plane of the illumination setup. c Fluorescence of eGFP-expressing HEK293FT cells treated with rapamycin dimer, transfected with split dCas9 without NIR illumination. d Fluorescence of eGFP-expressing HEK293FT cells treated with rapamycin dimer, transfected with split dCas9 under 30 seconds of NIR illumination. e 3D-printed double-slit screen with each slit being 500 μm wide and 2 mm long. f NIR illumination using double slit screen. g Corresponding pattern of the fluorescence of eGFP-expressing cells

Article Snippet: The plasmids encoding Cas9 (PX851, #62883 and PX852, #62884), split dCas9 (PX855, #62887 and PX856, #62888), dCas9-VP64 (#61425), an eGFP-expressing surrogate reporter (#60718), and a sgRNA targeting the eGFP reporter (#60719) were obtained from Addgene.

Techniques: Fluorescence, Expressing, Transfection

Journal: Nucleic acids research

Article Title: Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.

doi: 10.1093/nar/gkaf213

Figure Lengend Snippet: Figure 1. BLU-VIPR allows for optogenetic CRISPR using light-induced expression of gRNA. ( A ) The RGR design consists of an HH ribozyme followed by the gRNA and an HDV ribozyme. After precise self-cleavage of the ribozymes, a functional gRNA is released. ( B ) Design of construct for VPR-EL222-dependent activation of C120 promoter transcription, allowing for simultaneous expression of mCherry and gRNA after exposure to blue light (470 nm). ( C ) Comparison of mCherry reporter expression in HEK293T cells after transfection with VPR-EL222 or VP16-EL222 constructs f ollo w ed b y e xposure to blue light f or 24 h. T he e xperiment w as perf ormed three times and one representativ e image is sho wn. Scale bars are 100 μm. ( D ) Out-of-frame Cas9 reporter where the expression of tdTomato is restored after Cas9-mediated indels. ( E ) HEK293T cells were transfected with BLU-VIPR (without mCherry) containing a gRNA targeting the out-of-frame sequence in tdTomato and Cas9. After 48 h of light exposure, the cells were cultured for an additional 72 h before detection of tdTomato demonstrating successful optogenetic induction of indels by Cas9. Scale bars are 55 μm. T he e xperiment w as perf ormed three times and one representativ e image is sho wn.

Article Snippet: Reporter cell lines for Cas9 and base editors were generated using lentiviruses made from CRISPR-SP-Cas9 reporter (Addgene #62733), pLV-SI-121 (Addgene #131126), and pLV-SI112 (Addgene #131127).

Techniques: CRISPR, Expressing, Functional Assay, Construct, Activation Assay, Comparison, Transfection, Sequencing, Cell Culture

Journal: Nucleic acids research

Article Title: Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.

doi: 10.1093/nar/gkaf213

Figure Lengend Snippet: Figure 4. Optogenetic gene editing of primary mouse Cas9 + T lymphocytes in vitro . ( A ) Design of the retroviral MSCV-BLU-VIPR constructs for blue-light induced expression of gRNA after transduction of primary mouse T lymphocytes. ( B ) Cas9 + splenic mouse T lymphocytes (Thy1.2 + ) were transduced with MSCV-BLU-VIPR containing T h y1.2-specific or NTC gRNA, f ollo w ed b y e xposure to blue light and analy sis b y flo w cytometry. ( C ) After 48 h of light exposure, followed by a 72-h dark period, the T lymphocytes were stained for Thy1.2, gated for singlets and viability, and then analyzed for T h y1.2 e xpression. R epresentativ e result from tw o e xperiments is sho wn.

Article Snippet: Reporter cell lines for Cas9 and base editors were generated using lentiviruses made from CRISPR-SP-Cas9 reporter (Addgene #62733), pLV-SI-121 (Addgene #131126), and pLV-SI112 (Addgene #131127).

Techniques: In Vitro, Retroviral, Construct, Expressing, Transduction, Cytometry, Staining

Journal: Nucleic acids research

Article Title: Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.

doi: 10.1093/nar/gkaf213

Figure Lengend Snippet: Figure 5. BLU-VIPR allows for optogenetic gene editing of Cas9 + T lymphocytes in vivo . ( A ) Optogenetic setup for illumination of lymph nodes with blue light (470 nm). ( B ) Illumination of a single iLN. ( C ) Cas9 + splenic mouse T lymphocytes (CD45.1 + T h y1.2 + ) w ere transduced with MSCV-BLU-VIPR containing T h y1.2-specific or NTC gRNA. T he transduced cells w ere adoptiv ely transferred to TCR β−/ −CD45.2 + mice. After reconstitution of the T cell lymphocyte pool, we illuminated an iLN with blue light to induce gene editing of Thy1.2. ( D ) Gating strategy to determine levels of Thy1.2 by flow cytometry. ( E ) T he le v els of T h y1.2 on transduced Cas9 + T lymphocytes (CD45.1 + T h y1.1 + ) from illuminated iLNs were determined by flow cytometry. ( F ) T he le v els of T h y1.2 on transduced Cas9 + T lymphocytes (CD45.1 + T h y1.1 + ) from nonilluminated mice w ere determined b y flo w cytometry. ( G ) T he le v els of T h y1.2 on transduced Cas9 + T lymphocytes (CD45.1 + T h y1.1 + ) in blood bef ore and after illumination of an iLN w ere determined b y flo w cytometry.

Article Snippet: Reporter cell lines for Cas9 and base editors were generated using lentiviruses made from CRISPR-SP-Cas9 reporter (Addgene #62733), pLV-SI-121 (Addgene #131126), and pLV-SI112 (Addgene #131127).

Techniques: In Vivo, Transduction, Flow Cytometry, Cytometry

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