Journal: NPJ Regenerative Medicine

Article Title: Effective protection of photoreceptors using an inflammation-responsive hydrogel to attenuate outer retinal degeneration

doi: 10.1038/s41536-023-00342-y

Figure Lengend Snippet: a Inflammation-responsive hydrogel is formed by crosslinking of the DBCO conjugated with hyaluronic acid and azide of cathepsins-cleavable crosslinker. b The EZH2 inhibitor containing hydrogel response to cathepsins overexpressed in inflammation environment of the retina; and suppress the inflammation via disease-dependent released EZH2 inhibitor from the hydrogel.

Article Snippet: Subsequently, the prepared hydrogel, EZH2 inhibitor (Tazemetostat) of 1-2 μL was intravitreally injected into 5-week-old mice with a 33 G Hamilton needle (#NANOFIL, World Precision Instruments, Sarasota, Florida, USA).

Techniques:

Journal: NPJ Regenerative Medicine

Article Title: Effective protection of photoreceptors using an inflammation-responsive hydrogel to attenuate outer retinal degeneration

doi: 10.1038/s41536-023-00342-y

Figure Lengend Snippet: a Dextran-loaded hydrogels were incubated in PBS; control media; non-activated microglia conditioned media (NM CM); and activated microglia conditioned media (AM CM) and imaged by in vivo imaging systems (IVIS). b Relative fluorescence intensity (Region of interest; ROI) of remained dextran at each time point (mean ± SD; n = 3). c EZH2 inhibitor cumulative release profile from the hydrogel in control media; NM CM and AM CM. The supernatant containing EZH2 was measured at room temperature using UV absorbance at 256 nm (mean ± SD; n = 3). Data points within Day 1 show the short-term release profile. d In vitro release kinetics of EZH2 inhibitor from the hydrogel in PBS at 37 °C with three different cathepsins concentrations (i.e.; 0; 10; and 100 ng/mL) (mean ± SD; n = 3). Statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (*** p < 0.001).

Article Snippet: Subsequently, the prepared hydrogel, EZH2 inhibitor (Tazemetostat) of 1-2 μL was intravitreally injected into 5-week-old mice with a 33 G Hamilton needle (#NANOFIL, World Precision Instruments, Sarasota, Florida, USA).

Techniques: Incubation, Control, In Vivo Imaging, Fluorescence, In Vitro

Journal: NPJ Regenerative Medicine

Article Title: Effective protection of photoreceptors using an inflammation-responsive hydrogel to attenuate outer retinal degeneration

doi: 10.1038/s41536-023-00342-y

Figure Lengend Snippet: a mRNA level of inflammatory markers in microglia and inflammatory activated microglia (mean ± SD; n = 3). b Cathepsin L; S; and B activity in the protein level (mean ± SD; n = 3). c mRNA level of inflammatory markers in inflammatory microglia treated Gel only; Drug only; and Drug & Gel (mean ± SD; n = 3). Statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (* p < 0.1, ** p < 0.01, and *** p < 0.001). d Protein level of EZH2 associated markers in inflammatory microglia. e Schematic illustration of EZH2 inhibition associated anti-inflammatory pathway signaling.

Article Snippet: Subsequently, the prepared hydrogel, EZH2 inhibitor (Tazemetostat) of 1-2 μL was intravitreally injected into 5-week-old mice with a 33 G Hamilton needle (#NANOFIL, World Precision Instruments, Sarasota, Florida, USA).

Techniques: Activity Assay, Inhibition

Journal: Journal of Oncology

Article Title: Trained Immunity of IL-12-, IL-15-, and IL-18-Induced CD 3 +CD 56 + NKT-Like Cells

doi: 10.1155/2022/8724933

Figure Lengend Snippet: CDK4/6 inhibitors are involved in the formation of cytokine-induced trained CD3+CD56+ NKT-like cells. (a) Overview of experimental design. CD3+CD56+NKT-like cells were preactivated with rhIL-12 (10 ng/mL), IL-15 (1 ng/ml), IL-18 (50 ng/ml) ± CDK4/6 inhibitor (10um/L), or control (1 ng/ml of IL-15 alone) for 16 hours. Flow cytometry analysis was performed at the indicated time point after preactivation. (b) The gating strategy for CD3+CD56+NKT-like cells and percentages of IFN- γ + cell populations; overlaid histograms show repressed expression of IFN- γ ; percentages of IFN- γ + cell populations declined by preactivated with the CDK4/6 inhibitor/DNA-demethylating agent/Myc inhibitor/EZH2 inhibitor; n = 8. (c) Western blot analysis of the expression of CDK4, CDK6, cyclin D1, p-Rb, and E2F-1. ∗ P > 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 (error bars, mean ± SEM). (d) The formation of cytokine-trained CD3+CD56+ NKT-like cells，cells stimulated by IL-12/15/18 via the CDK4/6 signaling pathway induce active proliferation and differentiate into trained CD3+CD56+ NKT-like cells with enhanced function molecules expression (IFN- γ , TNF- α , GzmB, and Ki67).

Article Snippet: CDK4/6 inhibitor (HY-16297A, MCE), Myc inhibitor (HY-13865, MCE), DNA-demethylating agent (HY-A0004, MCE), and EZH2 (HY-15555, MCE) were obtained from Selleckchem.com .

Techniques: Control, Flow Cytometry, Expressing, Western Blot

Journal: OncoImmunology

Article Title: The tumor microenvironment disarms CD8+ T lymphocyte function via a miR-26a-EZH2 axis

doi: 10.1080/2162402x.2016.1245267

Figure Lengend Snippet: Fig. 3. EZH2 is a bona fide target of miR-26a. A. Schematic representation of putative

Article Snippet: In EZH2 inhibition experiments, pMel-1 CTLs were pretreated with 1-10 nM of the EZH2 inhibitor GSK-343 (Selleck Chemicals, TX, USA) or an ethanol vehicle control for 48 hours in vitro before flow cytometry analysis or intratumoral injection.

Techniques:

Journal: OncoImmunology

Article Title: The tumor microenvironment disarms CD8+ T lymphocyte function via a miR-26a-EZH2 axis

doi: 10.1080/2162402x.2016.1245267

Figure Lengend Snippet: Fig. 4. In a mouse tumor model, the miR-26a-EZH2 axis is exploited by the TME for

Article Snippet: In EZH2 inhibition experiments, pMel-1 CTLs were pretreated with 1-10 nM of the EZH2 inhibitor GSK-343 (Selleck Chemicals, TX, USA) or an ethanol vehicle control for 48 hours in vitro before flow cytometry analysis or intratumoral injection.

Techniques:

Journal: OncoImmunology

Article Title: The tumor microenvironment disarms CD8+ T lymphocyte function via a miR-26a-EZH2 axis

doi: 10.1080/2162402x.2016.1245267

Figure Lengend Snippet: Fig. 5. The human lung cancer microenvironment exploits the miR-26-EZH2 axis to

Article Snippet: In EZH2 inhibition experiments, pMel-1 CTLs were pretreated with 1-10 nM of the EZH2 inhibitor GSK-343 (Selleck Chemicals, TX, USA) or an ethanol vehicle control for 48 hours in vitro before flow cytometry analysis or intratumoral injection.

Techniques:

Journal: Science immunology

Article Title: Regulatory T cell control of systemic immunity and immunotherapy response in liver metastasis

doi: 10.1126/sciimmunol.aba0759

Figure Lengend Snippet: (A) Percentage of Foxp3+ CD4 Tregs within the indicated Subcutaneous (SQ) tumors. (B) SQ tumor growth curves of liver-tumor mice treated with anti-CTLA-4 antibody clone 9H10, 9H10 plus anti-PD-1 antibody, or isotype control. CR= complete rejection with no measurable SQ tumor at endpoint. (C) Survival curves of indicated groups. (D) Percentage of KSP tetramer+ CD8+ TILs and percentage that are positive for PD-1, CTLA-4, ICOS, IFNγ, and TNFα in mice treated with 9H10 versus control. (E) Day 14 SQ tumor sizes from liver-tumor bearing mice treated with EZH2 inhibitor CPI-1205(n=10), anti-PD-1 antibody (n=9), or a combination of both (n=10) compared to control (n=8). (F) Survival curves of indicated groups. (G) Percentage of KSP tetramer+ CD8+ TILs and percentage that are positive for PD-1, CTLA-4, ICOS, and IFNγ in mice treated with anti-PD-1 plus CPI-1205 versus anti-PD-1 alone. (H) Activated CD8+ T cell to Treg ratio within the SQ tumor sample of the indicated treatment groups. (I) SQ tumor growth curves from the MC38 tumor rechallenge experiment of the indicated groups. All data are shown as mean +/− s.e.m. All experiments besides E and F were n=5 or 10 and were representative of three or more independent experiments. Survival curves were analyzed by Log-rank tests, tumor growth curves were analyzed by two-way ANOVA with Sidak’s multiple comparisons, all others were analyzed by unpaired t tests. Asterisks indicating significance determined between groups are * p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Article Snippet: EZH2 inhibitor administration Mice were treated twice daily with CPI-1205 (Constellation Pharmaceuticals) by oral gavage with 300 mg/kg CPI-1205 made in a vehicle of 0.5% methylcellulose and 0.2% Tween 80 per the manufacturer’s instruction.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Methyltransferase inhibitors restore SATB1 protective activity against cutaneous T cell lymphoma in mice

doi: 10.1172/JCI135711

Figure Lengend Snippet: (A) Schematic depiction of primer sites for the regulatory and control regions for ChIP-PCR analysis in B and C. (B) ChIP quantified by real-time q-PCR with anti-H3K27me3 (clone C36B11) (left) or anti-H3K9me3 (Abcam, ab8898) (right) versus the control region (approximately 14 kb from promoter) and control IgG pull-downs from HuT78 Sézary cells calculated against 2.5% input values. Regions amplified at predicted occupied region approximately 4.8 kb for H3K27me3 and approximately 5.6 kb for H3K9me3 from SATB1 promoter. Representative of 2 independent experiments. (C) ChIP quantified by real-time qPCR with anti-H3K9me3 (ab8898) and control IgG pull-downs from HuT78, Jurkat, and RAJI cells lines calculated against 2.5% input values. Regions amplified based on ChIP-seq data for H3K9me3 occupied regions near the SATB1 promoter. (D–G) HuT78 cells were treated with vehicle or increasing concentrations of the EZH2 inhibitor GSK126 for 48 hours in duplicate for MTT assay (D), SUV39H1 inhibitors chaetocin (E) and F5446 (F), or the HDAC inhibitor romidepsin (G) in duplicate, and MTT assays were performed after 72 hours. Representative of 2 independent experiments. (H–J) ChIP-PCR experiments on HuT78 cells treated with IC50 values of GSK126 (H), chaetocin (I), and romidepsin (J) pulled down with anti-H3K27me3 (clone C36B11), anti-H3K9me3 (Abcam, ab8898), or anti-H3K27ac (Abcam, ab4729), respectively. Representative of 2 independent experiments. (K) RNA was extracted from HuT78 cells identically treated for 48 hours was reversed transcribed, and SATB1 mRNA expression was quantified by q-PCR normalized to human GAPDH mRNA. Data pooled from 4 independent experiments are shown. (L) q-PCR quantification of SATB1 mRNA expression normalized to TATA-binding protein (TBP) mRNA 72 hours after treatment with the indicated doses of chaetocin, F5446, or romidepsin. Pooled from 4 independent experiments. (M) Histogram of annexin V staining on HuT78 cells treated with the IC50 value of chaetocin, F5446, or romidepsin. Two-tailed Student’s t test (B, C, and H–L): *P < 0.05; **P ≤ 0.01; ****P ≤ 0.0001.

Article Snippet: For viability analysis, HuT78 cells were seeded at 40,000 cells/well in triplicate in 96-well plates in the presence of vehicle control (ethanol or DMSO) or the EZH2 inhibitor GSK126 (Cayman Chemical, 15415), chaetocin (Abcam, ab144534), romidepsin (FK228, Depsipeptide; Selleck, S3020-5mg), F5446, and incubated at 37°C and 5% CO 2 for 48 or 72 hours.

Techniques: Control, Amplification, ChIP-sequencing, MTT Assay, Expressing, Binding Assay, Staining, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: Methyltransferase inhibitors restore SATB1 protective activity against cutaneous T cell lymphoma in mice

doi: 10.1172/JCI135711

Figure Lengend Snippet: (A) CD4+CD26– isolated T cells from peripheral blood apheresis of Sézary patients (n = 4) were cultured in R10 media with 100 U/mL human recombinant IL-2 and treated with increasing doses of SUV39H1/2 inhibitors chaetocin and F5446 (72 hours) as well as romidepsin (72 hours) and GSK126 (48 hours) versus the vehicle control (DMSO) prior to MTT analysis. IC50 values (nM) were calculated for each patient sample for the respective treatments. (B) Summary of IC50 values for chaetocin, F5446, romidepsin, and GSK126 for each malignant sample (n = 4). One-way ANOVA with Tukey’s multiple-comparison test: *P < 0.05; **P ≤ 0.01. (C) RNA was extracted from primary CD4+CD26– Sézary patient cells that were treated with chaetocin, F5446, romidepsin, or vehicle control (DMSO) for 24–36 hours, and SATB1 mRNA expression was quantified by q-PCR normalized to human TBP mRNA. Data pooled from 3 patient samples with 2 independent experiments shown (n = 6). Two-tailed Student’s t test: ****P ≤ 0.0001. (D) Chromatin immunoprecipitation quantified by real-time q-PCR with anti-H3K27me3 (clone C36B11) or control IgG isotype immunoprecipitation from isolated CD4+ T cells from peripheral blood of Sézary patients (n = 2) calculated against 2.5% input values. Regions amplified at the predicted occupied region (approximately 4.8 kb)of the SATB1 promoter. Representative of 2 independent experiments. Two-tailed Student’s t test: *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001. (E) Similar to D except with anti-H3K9me3 (Abcam, ab8898) at the approximately 5.6-kb region versus the control region (n = 2). Representative of 2 independent experiments. Two-tailed Student’s t test: *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001. (F) Primary Sézary CD4+CD26– cells (n = 2) were transduced with retrovirus containing human SATB1 and sorted for GFP+ and GFP– cells. Western blot was performed with antibodies against human p-STAT5 and STAT5 protein in cells endogenously expressing SATB1 versus cells with ectopic expression of SATB1 (n = 2). (G) Primary Sézary cells were labeled with CellTrace Violet and were serum starved for 24 hours prior to stimulation with anti-CD3/anti-CD28 beads and cultured in complete medium with 100 U/mL rhIL-2 for 5 days. Proliferation was assessed by CellTrace Violet dilution using FACS for cells ectopically (n = 2) or endogenously (n = 2) expressing SATB1. Experiment was performed on 2 replicates per patient (n = 4). Two-tailed Student’s t test: **P ≤ 0.01.

Article Snippet: For viability analysis, HuT78 cells were seeded at 40,000 cells/well in triplicate in 96-well plates in the presence of vehicle control (ethanol or DMSO) or the EZH2 inhibitor GSK126 (Cayman Chemical, 15415), chaetocin (Abcam, ab144534), romidepsin (FK228, Depsipeptide; Selleck, S3020-5mg), F5446, and incubated at 37°C and 5% CO 2 for 48 or 72 hours.

Techniques: Isolation, Cell Culture, Recombinant, Control, Comparison, Expressing, Two Tailed Test, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Transduction, Western Blot, Labeling

Journal: Molecular cell

Article Title: Large DNA Methylation Nadirs Anchor Chromatin Loops Maintaining Hematopoietic Stem Cell Identity

doi: 10.1016/j.molcel.2020.04.018

Figure Lengend Snippet: (A). The experimental scheme. Cord blood CD34+ cells are treated with EZH2 inhibitor for 7 days in the ex vivo culture condition. The CD34+CD38- cells are isolated for in situ HiC, RNA-seq and ChIP-seq.

Article Snippet: EZH2 inhibitor-EPZ6438 , Selleckchem , S7128.

Techniques: Ex Vivo, Isolation, In Situ, RNA Sequencing, ChIP-sequencing

Journal: Molecular cell

Article Title: Large DNA Methylation Nadirs Anchor Chromatin Loops Maintaining Hematopoietic Stem Cell Identity

doi: 10.1016/j.molcel.2020.04.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: EZH2 inhibitor-EPZ6438 , Selleckchem , S7128.

Techniques: Recombinant, Protease Inhibitor, Transplantation Assay, DNA Methylation Assay, Purification

Journal: STAR Protocols

Article Title: Denaturing cross-linking immunoprecipitation to identify footprints for RNA-binding proteins

doi: 10.1016/j.xpro.2021.100819

Figure Lengend Snippet: The method for selection of clonal cell lines for dCLIP experiments Representative Western blotting for clonal cell lines derived from mouse embryonic fibroblasts with stable expression of BirA and Avi-GFP-EZH2 or Avi-GFP alone (control). Protein extracts prepared from each clonal cell line were probed with specific antibodies against EZH2 and GAPDH (loading control) proteins. Following densitometry analysis, the intensity ratios between Avi-GFP-EZH2 and endogenous EZH2 and between total EZH2 (transgenic + endogenous) and GAPDH were computed. Note that expression of transgenic EZH2 resulted in reduced expression of the endogenous EZH2, thus leveraging a total amount of EZH2 protein between control and Avi-GFP-EZH2 expressing cells. Clones# 1C, C14 (highlighted in Bold), that exhibited total EZH2 / GAPDH ratio closest to the control cell lines, were chosen for subsequent dCLIP experiments.

Article Snippet: Ezh2 (D2C9) XP rabbit monoclonal antibody, 1:1000 dilution , Cell Signaling Technologies Cat# 5246 , RRID: AB_10694683.

Techniques: Selection, Western Blot, Derivative Assay, Expressing, Control, Transgenic Assay, Clone Assay

Journal: STAR Protocols

Article Title: Denaturing cross-linking immunoprecipitation to identify footprints for RNA-binding proteins

doi: 10.1016/j.xpro.2021.100819

Figure Lengend Snippet: dCLIP vs Conventional CLIP of Polycomb proteins in mouse ES cells (A) Representative autoradiography dCLIP experiment. The red arrowhead indicates the Bio-tagged-CBX7 signal in two clonal cell lines, 3E and 6F, expressing nearly physiological levels of Bio-tagged-CBX7. The excised membrane area is marked in white. (B) Representative autoradiography of CLIP experiment using specific antibodies against CBX7. Rabbit IgG was used as a control. The expected size of CBX7 protein is marked with a red arrowhead. A high background signal was visible at 40 kDa level and was not cleared by up to 1M salt washes. (C) Representative autoradiography of CLIP experiment with anti-HA tag antibody. The 6C and 12D clonal cell lines express HA-tagged-CBX7. The red arrowhead marks HA-CBX7 related signal. Note the presence of strong background signal of anti-HA antibody, similar to anti-CBX7 antibody in (B). (D) Representative dCLIP experiments for EZH2. Left panel, autoradiography of dCLIP experiment. Right panel, Western blot with the anti-GFP antibody. Red arrows, GFP-Biotagged-EZH2 signal. Ezh2-4A and Ezh2-5A are two clonal 16.7 mES cell lines expressing nearly physiological levels of GFP-Biotagged-EZH2. Green arrowhead – Avi-GFP protein.

Article Snippet: Ezh2 (D2C9) XP rabbit monoclonal antibody, 1:1000 dilution , Cell Signaling Technologies Cat# 5246 , RRID: AB_10694683.

Techniques: Autoradiography, Expressing, Membrane, Control, Western Blot

Journal: STAR Protocols

Article Title: Denaturing cross-linking immunoprecipitation to identify footprints for RNA-binding proteins

doi: 10.1016/j.xpro.2021.100819

Figure Lengend Snippet:

Article Snippet: Ezh2 (D2C9) XP rabbit monoclonal antibody, 1:1000 dilution , Cell Signaling Technologies Cat# 5246 , RRID: AB_10694683.

Techniques: Virus, Recombinant, Protease Inhibitor, Immunoprecipitation, Reverse Transcription, Western Blot, Multiplex Assay, Library Quantification, Irradiation, Membrane, Electroporation

Journal: Cancers

Article Title: BET and CDK Inhibition Reveal Differences in the Proliferation Control of Sympathetic Ganglion Neuroblasts and Adrenal Chromaffin Cells

doi: 10.3390/cancers14112755

Figure Lengend Snippet: Effect of IGF, EZH2, ALK, and WNT inhibitors on the proliferation of chromaffin cells, neuroblasts, and NESTIN-expressing cells (NECs). Dose–response curves are shown for IGFR inhibitor (PPP) ( A ), EZH2 inhibitor (EPZ6438) ( B ), Alk inhibitor (Alectinib), ( C ) and Wnt inhibitor (ICG001) ( D ). Data represent the mean ± s. e. m. of at least three independent experiments.

Article Snippet: The inhibitors that were used were the BET inhibitors JQ1 (Tocris Biotechne, Wiesbaden, Germany; 4499) and GSK1324726A (iBET 726) (Selleckchem Biozol, Eching, Germany), the CDK-7 inhibitors THZ1 (Medchem Express Biotrend, Köln, Germany) and YKL-5-125 (Selleckchem), the CDK12/13 inhibitor THZ 531 (Selleckchem), the IGF1-R inhibitor picropodophyllin (PPP) (Tocris 2956), the EZH2 inhibitor EPZ 6438 (Axon Medchem, Groningen, NL; 2227), the WNT inhibitor ICG001 (Axon Medchem 1766), and the ALK inhibitor Alectinib (Selleckchem S276).

Techniques: Expressing