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: Cell Stem Cell

Article Title: NuRD Suppresses Pluripotency Gene Expression to Promote Transcriptional Heterogeneity and Lineage Commitment

doi: 10.1016/j.stem.2012.02.020

Figure Lengend Snippet: NuRD Controls Transcriptional Heterogeneity in ESCs (A) Expression levels for indicated proteins were measured in ESC cultures by antibody staining and immunofluorescence microscopy. Log of relative fluorescence is plotted along the x axis, with the proportion of cells indicated along the y axis. Data is shown for wild-type (WT) and Mbd3 −/− (KO) ESCs grown in self-renewing conditions (+LIF, left-hand panels) or after 48 hr in the absence of LIF (−LIF, right-hand panels). n > 4,000 cells for each line. (B) Flow cytometry analysis showing expression profiles of Zfp42-GFPd2 in a wild-type (WT: ZFP42-GFPd2) or Mbd3 −/− (KO: Zfp42-GFPd2) background grown in standard media with 10% serum either with or without LIF (+LIF or –LIF, respectively). (C) Expression levels for indicated proteins were measured as in (A) for wild-type (WT) and Mbd3 −/− (KO) ESCs maintained in 2i/LIF (2i, left-hand panels) or in the absence of inhibitors and LIF for 24 hr (N2B27, right-hand panels). n > 1,800 cells for each line. (D) Flow cytometry analysis showing expression profiles of Zfp42-GFPd2 in a wild-type (WT: ZFP42-GFPd2) or Mbd3 −/− (KO: Zfp42-GFPd2) background in 2i/LIF (2i) or in defined media without inhibitors and LIF (N2B27). See also Figure S3 .

Article Snippet: To produce the Zfp42-GFPd2 allele ( ) in Mbd3 −/− and control cells, the coding region of Zfp42 was replaced by a destabilized GFPd2 (Clontech) by homologous recombination in Mbd3 Flox/− ESCs and in Mbd3 −/− ESCs.

Techniques: Expressing, Staining, Immunofluorescence, Microscopy, Fluorescence, Flow Cytometry

Journal: Cell Stem Cell

Article Title: NuRD Suppresses Pluripotency Gene Expression to Promote Transcriptional Heterogeneity and Lineage Commitment

doi: 10.1016/j.stem.2012.02.020

Figure Lengend Snippet: NuRD Is a General Regulator of Transcriptional Heterogeneity (A) Gene expression in Zfp42-GFPd2-low cells is expressed relative to expression in Zfp42-GFPd2-high cells ( Marks et al., 2012 ). Included are genes identified by bioinformatic analysis ( Zfp42 , Tbx3 , Klf4 , Zfp57 , Esrrb , Nr0b1 , Tcfcp2l1 , Aes , and Zfp296 ) as well as control pluripotency-associated genes ( Klf5 , Nanog , and Pou5f1 ) and one gene shown to be subject to NuRD-dependent transcriptional silencing in ESCs but not display transcriptional heterogeneity ( Htra1 ; Reynolds et al., 2012 ). The latter two sets of genes do not display transcriptional heterogeneity in this assay. Ppia is a control housekeeping gene. (B) Expression of indicated genes in Mbd3 −/− ESCs expressed relative to levels in wild-type ESCs. (C) Expression analysis for Zfp57 and Esrrb in wild-type (WT) and Mbd3 −/− (KO) ESCs in serum and LIF conditions as in Figure 3 A above. (D) ChIP was performed with anti-Mbd3 (top panels) and anti-Mi2β antibodies (bottom panels) as well as control IgG antibodies across the transcription start sites of Esrrb (left panels) and Zfp57 (right panels) in wild-type (WT) or Mbd3 −/− (KO; anti-Mbd3 ChIP only) ESCs grown in serum and LIF conditions. Immunoprecipitates were probed with primer pairs located across the indicated gene promoters and plotted as percentage of input (y axis). Numbers along the x axis indicate distance relative to major ES transcription start site for indicated genes in ESCs. See also .

Article Snippet: To produce the Zfp42-GFPd2 allele ( ) in Mbd3 −/− and control cells, the coding region of Zfp42 was replaced by a destabilized GFPd2 (Clontech) by homologous recombination in Mbd3 Flox/− ESCs and in Mbd3 −/− ESCs.

Techniques: Expressing

Journal: Cell Stem Cell

Article Title: NuRD Suppresses Pluripotency Gene Expression to Promote Transcriptional Heterogeneity and Lineage Commitment

doi: 10.1016/j.stem.2012.02.020

Figure Lengend Snippet: Variable Gene Expression Correlates with Variable Activator Activity (A) ESCs expressing GFPd2 from the Zfp42 locus (Zfp42-GFPd2) were separated according to GFP intensity (x axis) and side scatter (y axis). Gate R5 contains the GFP-low sorted fraction and gate R4 contains the GFP-high fraction used for ChIP. (B) ChIP was performed using anti-Mi2β or a mouse IgG control antibody in Zfp42-GFPd2-low and Zfp42-GFPd2-high cells as shown in (A). (C) Western blots showing relative levels of Stat3 and phospho-Stat3 (pStat3) or indicated NuRD components in sorted Zfp42-GFPd2-high (GFP High) and Zfp42-GFPd2-low (GFP Low) cells. Protein sizes are shown at left in kDa. α-Tubulin is shown as a loading control. (D) Western blots for indicated NuRD components in ESCs maintained in serum and LIF (SL) or 2i/LIF (2i) conditions. Protein sizes are shown at left in kDa. α-Tubulin is shown as a loading control. (E and F) ChIP for Stat3 (E) or Mi2β (F) at the Socs3 promoter as measured in Zfp42-GFPd2-low and -high populations.

Article Snippet: To produce the Zfp42-GFPd2 allele ( ) in Mbd3 −/− and control cells, the coding region of Zfp42 was replaced by a destabilized GFPd2 (Clontech) by homologous recombination in Mbd3 Flox/− ESCs and in Mbd3 −/− ESCs.

Techniques: Expressing, Activity Assay, Western Blot

Journal: Journal of Hematology & Oncology

Article Title: Novel potent molecular glue degraders against broad range of hematological cancer cell lines via multiple neosubstrates degradation

doi: 10.1186/s13045-024-01592-z

Figure Lengend Snippet: MGD-28 exhibited a significant degradation effect on IKZF1/2/3 and CK1α via a Cullin-CRBN dependent pathway. A-B . Flow cytometry plot showing wild type and CRBN –/– NCI-H929 cells treated with indicated doses of MGD-4 , MGD-28 or pomalidomide (Pom) for 3 days, and cells were stained with Annexin V-PE and DAPI ( A ). Apoptosis was measured by flow cytometry using Annexin V as a marker ( B ). Error bars denote standard deviations (independent experiments, n = 3). Student’s t test, ns (no significance), ** p < 0.01. C . Viability of wild type and CRBN −/− NCI-H929 cells treated with MGD-28 for 96 h. Data shown are a representative graph of three independent experiments; mean ± SD of triplicates. D . MV-4-11 cells viability in the absence and presence of pomalidomide (Pom, 10 µM) for 96 h. Data shown are a representative graph of three independent experiments; mean ± SD of triplicates. E . Western blot analysis of CK1α, IKZF1, IKZF2 and IKZF3 degradation in NCI-H929 cells CRBN knockout, pre-treated with MLN4924, MG132 or pomalidomide for 1 h and treated with MGD-28 for 24 h. F . Western blot analysis of p53, p21 and MDM2 in NCI-H929 cells treated with different doses of MGD-28 for 24 h. G-H . PCR analysis of CDKN1A/p21 ( G ) and MDM2 ( H ) in MV-4-11 cells treated with different doses of MGD-28 , lenalidomide (Len, 10 µM) or pomalidomide (Pom, 10 µM) for 24 h. Error bars denote standard deviations (independent experiments, n = 3). Student’s t test, ns (no significance), ** p < 0.01. I . Western blot analysis of CK1α, p53, and p21 in NCI-H929 cells overexpressing CK1α WT, CK1α G40N, or vector control treated with lenalidomide (Len, 10 µM) or MGD-28 (1 µM). J . NCI-H929 cells overexpressing CK1α WT, CK1α G40N, or vector were treated with MGD-28 . Data shown are a representative result of three independent experiments; mean ± SD of triplicates.

Article Snippet: The following primary antibodies were used: CRBN (#71810, 1:1000), IKZF1 (#14859, 1:1000), IKZF2 (#42427, 1:1000), IKZF3 (#15103, 1:500) and were all from Cell Signaling Technology, Boston, MA, USA; CK1α (#ab108296, 1:1000) and ZNFX1 (#ab179452, 1:500) were purchased from Abcam, MA, USA; BRD9 (#24785-1-AP, 1:1000), GSPT1 (#10763-1-AP, 1:1000), STAT5 (#13179-1-AP, 1:500), c-Myc (#10828-1-AP, 1:1000), MDM2 (#27883-1-AP, 1:1000), P53 (#1044-1-AP, 1:1000), P21 (#10355-1-AP, 1:500), DTWD1 (#26810-1-AP, 1:1000), MBD3 (#14258-1-AP, 1:1000), MNT (#23742-1-AP, 1:1000) and MBD1 (#29998-1-AP, 1:1000) were purchased from Proteintech, Rosemont, IL, USA; ZMIZ2 (#GTX118779, 1:1000) were purchased from GeneTex, San Antonio, TX, USA; ZFP91 (#CSB-PA504466, 1:1000) was purchased from Cusibio, Houston, TX, USA; Anti-rabbit IgG (HRP-conjugated, Cell Signaling Technology, #7074, 1:5000), anti-mouse IgG (HRP-conjugated, Cell Signaling Technology, #7076, 1:5000) were used as secondary antibodies.

Techniques: Flow Cytometry, Staining, Marker, Western Blot, Knock-Out, Plasmid Preparation, Control

Journal: Cell reports

Article Title: ZBTB7A is a modulator of KDM5-driven transcriptional networks in basal breast cancer

doi: 10.1016/j.celrep.2024.114991

Figure Lengend Snippet: (A) GSEA on genes ranked by log2(FC) for ±10 μM C70 for 7 days. The analysis was performed in all three cell lines with wild-type (i.e., ROSA26-g1) or ZBTB7A KO. (B) Oxygen consumption rate (OCR) in ROSA26-g1 and ZBTB7A KO SUM149 cells ± pre-treatment with 10 μM C70 for 6 days. Values are the mean ± standard deviation. N = 5 for all conditions except ZBTB7A KO + C70, which had one outlier well removed ( N = 4). (C) Ridge plot depicting flow cytometry for total ROS detection with the Total Reactive Oxygen Species (ROS) Assay Kit and for mitochondrial cardiolipins with nonyl acridine orange (NAO). SUM149 cells were treated with or without 10 μM C70 for 5 days. One millimolar H 2 O 2 (7 h for ROS and 2 h for NAO) was used as a positive control. (D) Heatmap of DEGs upon C70 treatment in either SUM149 ROSA26-g1 or ZBTB7A -g1 ( p adj < 0.05). Genes are ordered based on k-means clustering (k = 5) and samples are ordered based on hierarchical clustering. (E) RNA Z scores of cluster 3 and 5 genes from (D). Box plots represent mean, first and third quantile, and min and max values. (F and G) Overrepresentation analysis for (F) MSigDB transcription factor targets and (G) MSigDB Hallmark pathways within each gene cluster specified by (D). (H) Plot of NF-κB target genes associated with KDM5 + ZBTB7A peaks, ZBTB7A unique peaks, or both (KDM5 + ZBTB7A and ZBTB7A unique). Target genes were defined by the union of MSigDB transcription factor target gene sets (GGGNNTTTCC_NFKB_Q6_01, NFKAPPAB_01, NFKAPPAB65_01, NFKB_C, NFKB_Q6_01, and NFKB_Q6). The p value was determined by the t test. Box plots represent mean, first and third quantile, and min and max values. (I) Immunoblot for phospho-NF-κB p65 (Ser536) in SUM149 ROSA26-g1 and ZBTB7A KO cells ± 10 μM C70 for 7 days. Cells treated with 20 ng/mL TNF-α for 5 min were used as positive control. Image is the left side part of a larger blot with additional lanes. (J) Immunoblot for NF-κB targets MMP9, MIA, and IL-27-RA in SUM149 ROSA26-g1 or ZBTB7A KO cell lines ± 10 μM C70 for 6 days. Tubulin was used as loading control. (K) Diagram of proposed interaction between ZBTB7A and KDM5 inhibition on NF-κB signaling. See also and .

Article Snippet: Antibodies used for Immunoblotting were anti-beta-Actin (Sigma, A2228), anti-alpha Tubulin (Sigma, T5168), anti-ZBTB7A (Invitrogen, 14-3309-82), anti-KDM5A (Abcam, ab70892), anti-KDM5B (Sigma, HPA027179), anti-KDM5C (Abcam, ab34718), anti-H3K4me3 (Abcam, ab8580), anti-H3 (Active Motif, 39763), anti-RHOA (Cell Signaling Technology, 2117), anti-PKN2 (Abcam, ab87812), anti-MTA1 (Cell Signaling Technology, 5647), anti-MTA2 (Cell Signaling Technology, 15793), anti-MBD2 (Abcam, ab188474), anti-MBD3 (Cell Signaling Technology, 14540), anti-CHD3 (Cell Signaling Technology, 4241), anti-CHD4 (Cell Signaling Technology, 11912), anti-phospho-p65 (Ser536) (Cell Signaling Technology, 3033), anti-p65 (Abcam, ab32536), anti-phospho-p50 (Ser337) (Invitrogen, PA5–37658), anti-p50 (Invitrogen, MA5–15870), Goat anti-Mouse IgG Secondary HRP (Invitrogen, 62–6520), Goat anti-Rabbit IgG Secondary HRP (Invitrogen, 65–6120), and Goat anti-Armenian Hamster IgG Secondary HRP (Invitrogen, PA1–32045).

Techniques: Standard Deviation, Flow Cytometry, ROS Assay, Positive Control, Western Blot, Control, Inhibition

Journal: Cell reports

Article Title: ZBTB7A is a modulator of KDM5-driven transcriptional networks in basal breast cancer

doi: 10.1016/j.celrep.2024.114991

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used for Immunoblotting were anti-beta-Actin (Sigma, A2228), anti-alpha Tubulin (Sigma, T5168), anti-ZBTB7A (Invitrogen, 14-3309-82), anti-KDM5A (Abcam, ab70892), anti-KDM5B (Sigma, HPA027179), anti-KDM5C (Abcam, ab34718), anti-H3K4me3 (Abcam, ab8580), anti-H3 (Active Motif, 39763), anti-RHOA (Cell Signaling Technology, 2117), anti-PKN2 (Abcam, ab87812), anti-MTA1 (Cell Signaling Technology, 5647), anti-MTA2 (Cell Signaling Technology, 15793), anti-MBD2 (Abcam, ab188474), anti-MBD3 (Cell Signaling Technology, 14540), anti-CHD3 (Cell Signaling Technology, 4241), anti-CHD4 (Cell Signaling Technology, 11912), anti-phospho-p65 (Ser536) (Cell Signaling Technology, 3033), anti-p65 (Abcam, ab32536), anti-phospho-p50 (Ser337) (Invitrogen, PA5–37658), anti-p50 (Invitrogen, MA5–15870), Goat anti-Mouse IgG Secondary HRP (Invitrogen, 62–6520), Goat anti-Rabbit IgG Secondary HRP (Invitrogen, 65–6120), and Goat anti-Armenian Hamster IgG Secondary HRP (Invitrogen, PA1–32045).

Techniques: Control, Produced, Recombinant, Virus, XF Assay, ROS Assay, CRISPR, Mass Spectrometry, Knock-Out, Plasmid Preparation, Software, Combined Bisulfite Restriction Analysis Assay, Genome Wide

Journal: Journal of Hematology & Oncology

Article Title: Novel potent molecular glue degraders against broad range of hematological cancer cell lines via multiple neosubstrates degradation

doi: 10.1186/s13045-024-01592-z

Figure Lengend Snippet: MGD-28 exhibited a significant degradation effect on IKZF1/2/3 and CK1α via a Cullin-CRBN dependent pathway. A-B . Flow cytometry plot showing wild type and CRBN –/– NCI-H929 cells treated with indicated doses of MGD-4 , MGD-28 or pomalidomide (Pom) for 3 days, and cells were stained with Annexin V-PE and DAPI ( A ). Apoptosis was measured by flow cytometry using Annexin V as a marker ( B ). Error bars denote standard deviations (independent experiments, n = 3). Student’s t test, ns (no significance), ** p < 0.01. C . Viability of wild type and CRBN −/− NCI-H929 cells treated with MGD-28 for 96 h. Data shown are a representative graph of three independent experiments; mean ± SD of triplicates. D . MV-4-11 cells viability in the absence and presence of pomalidomide (Pom, 10 µM) for 96 h. Data shown are a representative graph of three independent experiments; mean ± SD of triplicates. E . Western blot analysis of CK1α, IKZF1, IKZF2 and IKZF3 degradation in NCI-H929 cells CRBN knockout, pre-treated with MLN4924, MG132 or pomalidomide for 1 h and treated with MGD-28 for 24 h. F . Western blot analysis of p53, p21 and MDM2 in NCI-H929 cells treated with different doses of MGD-28 for 24 h. G-H . PCR analysis of CDKN1A/p21 ( G ) and MDM2 ( H ) in MV-4-11 cells treated with different doses of MGD-28 , lenalidomide (Len, 10 µM) or pomalidomide (Pom, 10 µM) for 24 h. Error bars denote standard deviations (independent experiments, n = 3). Student’s t test, ns (no significance), ** p < 0.01. I . Western blot analysis of CK1α, p53, and p21 in NCI-H929 cells overexpressing CK1α WT, CK1α G40N, or vector control treated with lenalidomide (Len, 10 µM) or MGD-28 (1 µM). J . NCI-H929 cells overexpressing CK1α WT, CK1α G40N, or vector were treated with MGD-28 . Data shown are a representative result of three independent experiments; mean ± SD of triplicates.

Article Snippet: The following primary antibodies were used: CRBN (#71810, 1:1000), IKZF1 (#14859, 1:1000), IKZF2 (#42427, 1:1000), IKZF3 (#15103, 1:500) and were all from Cell Signaling Technology, Boston, MA, USA; CK1α (#ab108296, 1:1000) and ZNFX1 (#ab179452, 1:500) were purchased from Abcam, MA, USA; BRD9 (#24785-1-AP, 1:1000), GSPT1 (#10763-1-AP, 1:1000), STAT5 (#13179-1-AP, 1:500), c-Myc (#10828-1-AP, 1:1000), MDM2 (#27883-1-AP, 1:1000), P53 (#1044-1-AP, 1:1000), P21 (#10355-1-AP, 1:500), DTWD1 (#26810-1-AP, 1:1000), MBD3 (#14258-1-AP, 1:1000), MNT (#23742-1-AP, 1:1000) and MBD1 (#29998-1-AP, 1:1000) were purchased from Proteintech, Rosemont, IL, USA; ZMIZ2 (#GTX118779, 1:1000) were purchased from GeneTex, San Antonio, TX, USA; ZFP91 (#CSB-PA504466, 1:1000) was purchased from Cusibio, Houston, TX, USA; Anti-rabbit IgG (HRP-conjugated, Cell Signaling Technology, #7074, 1:5000), anti-mouse IgG (HRP-conjugated, Cell Signaling Technology, #7076, 1:5000) were used as secondary antibodies.

Techniques: Flow Cytometry, Staining, Marker, Western Blot, Knock-Out, Plasmid Preparation, Control

Journal: Journal of Hematology & Oncology

Article Title: Novel potent molecular glue degraders against broad range of hematological cancer cell lines via multiple neosubstrates degradation

doi: 10.1186/s13045-024-01592-z

Figure Lengend Snippet: MGD-28 exhibited a significant degradation effect on IKZF1/2/3 and CK1α via a Cullin-CRBN dependent pathway. A-B . Flow cytometry plot showing wild type and CRBN –/– NCI-H929 cells treated with indicated doses of MGD-4 , MGD-28 or pomalidomide (Pom) for 3 days, and cells were stained with Annexin V-PE and DAPI ( A ). Apoptosis was measured by flow cytometry using Annexin V as a marker ( B ). Error bars denote standard deviations (independent experiments, n = 3). Student’s t test, ns (no significance), ** p < 0.01. C . Viability of wild type and CRBN −/− NCI-H929 cells treated with MGD-28 for 96 h. Data shown are a representative graph of three independent experiments; mean ± SD of triplicates. D . MV-4-11 cells viability in the absence and presence of pomalidomide (Pom, 10 µM) for 96 h. Data shown are a representative graph of three independent experiments; mean ± SD of triplicates. E . Western blot analysis of CK1α, IKZF1, IKZF2 and IKZF3 degradation in NCI-H929 cells CRBN knockout, pre-treated with MLN4924, MG132 or pomalidomide for 1 h and treated with MGD-28 for 24 h. F . Western blot analysis of p53, p21 and MDM2 in NCI-H929 cells treated with different doses of MGD-28 for 24 h. G-H . PCR analysis of CDKN1A/p21 ( G ) and MDM2 ( H ) in MV-4-11 cells treated with different doses of MGD-28 , lenalidomide (Len, 10 µM) or pomalidomide (Pom, 10 µM) for 24 h. Error bars denote standard deviations (independent experiments, n = 3). Student’s t test, ns (no significance), ** p < 0.01. I . Western blot analysis of CK1α, p53, and p21 in NCI-H929 cells overexpressing CK1α WT, CK1α G40N, or vector control treated with lenalidomide (Len, 10 µM) or MGD-28 (1 µM). J . NCI-H929 cells overexpressing CK1α WT, CK1α G40N, or vector were treated with MGD-28 . Data shown are a representative result of three independent experiments; mean ± SD of triplicates.

Article Snippet: The following primary antibodies were used: CRBN (#71810, 1:1000), IKZF1 (#14859, 1:1000), IKZF2 (#42427, 1:1000), IKZF3 (#15103, 1:500) and were all from Cell Signaling Technology, Boston, MA, USA; CK1α (#ab108296, 1:1000) and ZNFX1 (#ab179452, 1:500) were purchased from Abcam, MA, USA; BRD9 (#24785-1-AP, 1:1000), GSPT1 (#10763-1-AP, 1:1000), STAT5 (#13179-1-AP, 1:500), c-Myc (#10828-1-AP, 1:1000), MDM2 (#27883-1-AP, 1:1000), P53 (#1044-1-AP, 1:1000), P21 (#10355-1-AP, 1:500), DTWD1 (#26810-1-AP, 1:1000), MBD3 (#14258-1-AP, 1:1000), MNT (#23742-1-AP, 1:1000) and MBD1 (#29998-1-AP, 1:1000) were purchased from Proteintech, Rosemont, IL, USA; ZMIZ2 (#GTX118779, 1:1000) were purchased from GeneTex, San Antonio, TX, USA; ZFP91 (#CSB-PA504466, 1:1000) was purchased from Cusibio, Houston, TX, USA; Anti-rabbit IgG (HRP-conjugated, Cell Signaling Technology, #7074, 1:5000), anti-mouse IgG (HRP-conjugated, Cell Signaling Technology, #7076, 1:5000) were used as secondary antibodies.

Techniques: Flow Cytometry, Staining, Marker, Western Blot, Knock-Out, Plasmid Preparation, Control