Journal: PLOS Biology

Article Title: Early transcriptional and epigenetic divergence of CD8 + T cells responding to acute versus chronic infection

doi: 10.1371/journal.pbio.3001983

Figure Lengend Snippet: ( A ) Heatmaps representing relative gene expression of the three Division 1 clusters, Div1 ARM-EFF , Div1 ARM-MEM , and Div1 CL13 , divided by category; rows represent selected genes and columns represent each of three Division 1 clusters. ( B ) Enriched pathways among the three Division 1 clusters. ( C ) Representative flow cytometry plots (left) displaying expression of Ezh2, CD25 (IL-2Rα), and T-bet protein among gated Division 1 (second CFSE peak) P14 T cells. Bar graphs indicate the frequencies (middle) or MFI (right) of P14 T cells responding to LCMV-Arm (blue) or LCMV-Cl13 (red) expressing each molecule. Data are shown as mean ± SEM. ( D , E ) RNA velocities of Div1 ARM (D, top) or Div1 Cl13 (E, top) subclusters derived from scVelo projected onto a UMAP-based embedding. Putative driver genes derived from scVelo that may regulate the CD8 + Division 1 T cell response to LMCV-Arm (D, bottom) vs. LCMV-Cl13 (E, bottom), represented as heatmaps. Individual lines above each heatmap represent single cells; colors correspond to subcluster identities among Div1 ARM (D, top) or Div1 Cl13 (E, top) cells. * p < 0.05, ** p < 0.01, *** p < 0.0001, **** p < 0.0001 (Student’s t test). Data are representative of 2 to 3 independent experiments. The raw data for the panels in this figure are located in file. CFSE, carboxyfluorescein succinimidyl ester; LCMV-Arm, LCMV-Armstrong; LCMV-Cl13, LCMV-Clone 13; MFI, mean fluorescence intensity; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: The MSCV-mouse-Ezh2-IRES-GFP (Ezh2 overexpression, Ezh2-OE) vector was a gift from Martine Roussel (Addgene plasmid #107146; http://n2t.net/addgene:107146 ; RRID:Addgene 107146).

Techniques: Expressing, Flow Cytometry, Derivative Assay, Fluorescence

Journal: PLOS Biology

Article Title: Early transcriptional and epigenetic divergence of CD8 + T cells responding to acute versus chronic infection

doi: 10.1371/journal.pbio.3001983

Figure Lengend Snippet: ( A ) Experimental setup. Control CD45.1 + (wild-type, “WT”) and Ezh2-deficient CD45.1.2 + ( Ezh2 fl / fl Cd4 Cre+ , “Ezh2 KO”) CD8 + P14 T cells were cotransferred into congenically distinct CD45.2 + recipient mice prior to infection with LCMV-Cl13; recipient mice were killed at 5–7 days post-infection and splenocytes analyzed by flow cytometry. ( B ) Representative flow cytometry plots (left) displaying expression of PD1, TCF1, TOX, Granzyme A, IL-2, or TNF protein among gated donor WT or Ezh2 KO P14 T cells. Bar graphs indicate the frequencies (middle) or MFI (right) of WT (blue) or Ezh2 KO (red) P14 T cells responding to LCMV-Cl13. ( C ) Experimental setup. CD8 + P14 T cells were transduced with an empty vector control (EV, CD45.1 + ) or Ezh2 overexpression (Ezh2-OE, CD45.1.2 + ) construct prior to adoptive cotransfer into CD45.2 + recipient mice prior to infection with LCMV-Cl13; recipient mice were killed at 5–7 days post-infection and splenocytes analyzed by flow cytometry. ( D ) Representative flow cytometry plots (left) displaying expression of PD1, TCF1, TOX, and Granzyme A among gated donor EV or Ezh2-OE P14 T cells. Bar graphs indicate the frequencies (middle) or MFI (right) of EV (blue) or Ezh2-OE (red) P14 T cells expressing each molecule. ( E , F ) Control CD45.1 + (wild-type, “WT”) and IFNAR1-deficient CD45.1.2 + ( Ifnar1 -/- , “IFNAR1 KO”) CD8 + P14 T cells were labeled with CFSE (E only) and cotransferred into congenically distinct CD45.2 + recipient mice prior to infection with LCMV-Cl13; recipient mice were killed at 2 (E) or 5 days (F) post-infection and splenocytes analyzed by flow cytometry. Representative flow cytometry plots (left) displaying expression of Ezh2 protein among gated Division 1 (second CFSE peak) WT vs. IFNAR1 KO P14 T cells. Bar graphs indicate the frequencies (middle) or MFI (right) of WT (blue) or IFNAR1 KO (red) P14 T cells expressing each molecule. ( F ) Representative flow cytometry plots (left) displaying expression of Ezh2, PD1, TOX, and TIM3 protein among gated WT vs. IFNAR1 KO P14 T cells. Bar graphs indicate the frequencies (middle) or MFI (right) of WT (blue) or IFNAR1 KO (red) P14 T cells expressing each molecule. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.0001, **** p < 0.0001 (paired t test). Data are representative of 2 to 3 independent experiments. The raw data for the panels in this figure are located in file. Fig 3A, 3C and 3E created with BioRender.com . CFSE, carboxyfluorescein succinimidyl ester; EV, empty vector; IL-2, interleukin 2; LCMV-Cl13, LCMV-Clone 13; MFI, mean fluorescence intensity; TNF, tumor necrosis factor; WT, wild-type.

Article Snippet: The MSCV-mouse-Ezh2-IRES-GFP (Ezh2 overexpression, Ezh2-OE) vector was a gift from Martine Roussel (Addgene plasmid #107146; http://n2t.net/addgene:107146 ; RRID:Addgene 107146).

Techniques: Infection, Flow Cytometry, Expressing, Transduction, Plasmid Preparation, Over Expression, Construct, Labeling, Fluorescence

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 1 Epigenetic regulation of skeletal development of fetuses from HFD (high fat diet) rat dams. A, Representative images from Alizarin red/Alcian blue staining of E18.5 embryos from dams fed either control diet or HFD. Cartilage and calcification stained dark blue; arrows indicate differences of skeletal ossification in the head. B, Western blots for H3K27me3, Ezh2, p-Ezh2 and CDK1 in proteins from EOCCs (embryonic rat osteogenic calvarial cells) either from six HFD obese dams or six control diet dams. M, male; F, female. C, Real-time PCR for Ezh2 mRNA expression in total RNA from EOCCs either from six HFD obese dams or six control diet dams. *P < .05 by t-test. D, and E, representing top genes and significantly H3K27me3 enriched or decreased gene body and gene promoter using Heat Map analysis of ChIP-seq data from EOCCs either from six HFD obese dams or six control diet dams (pooled)

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Staining, Control, Western Blot, Real-time Polymerase Chain Reaction, Expressing, ChIP-sequencing

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 2 Identification of epigenetic target genes affected by maternal obesity during fetal skeletal development. A, SATB2 was significantly enriched for H3K27me3 within the gene body, especially in the promoter region based on the detection of a peak (black and red arrow head) of enrichment by Illumina DNA sequencing (male and female are mixed, and samples were pooled to three per group). B, Real-time PCR for SATB2 and ALP mRNA expression in total RNA from EOCCs either from six HFD obese dams or six control diet dams. *P < .05 by t-test. C, ChIP of rat SATB2 enhancer elements by specific anti H3K27me3 and Ezh2 antibodies. D, ChIP of enrichment of SATB2 after IP with H3K27me3 and Ezh2 antibodies. Fold enrichment relative to IgG. *P < .05 by t-test EOCCs from control vs EOCCs from HFD obese dams. E, Control EOCCs were treated with vehicle (control), NEFA mixture 400 µM with Palmitic and Oleic acid 2:1 mixture, Sigma-Aldrich), Palmitic acid (270 µM) or Oleic acid (130 µM) for 48 hours: Western blots for H3K27me3, Ezh2, p-Ezh2, SATB2 and Col 1 (collagen 1). n = 3/treatment

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: DNA Sequencing, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 4 Increased Ezh2/H3K27me3 but decreased SATB2 expression in human UC MSCs from obese mothers. A, UC MSCs from either lean or obese mothers were cultured, passage 2 cells were immune-stained with anti-Ezh2 antibody (red, white arrows) or anti-SATB2 antibody (green, yellow arrows). B, and C, Real-time PCR (Box & Whiskers graphs) and Western blots (under Box & Whiskers graphs) of Ezh2 and SATB2 mRNA and protein expression in UC MSCs either from lean or obese mothers. *P < .05 vs lean, t-test. D, ChIP of human Ezh2, SATB2 and GAPDH enhancer elements by specific anti H3K27me3 antibody, and E, ChIP of enrichment of human Ezh2, SATB2 and GAPDH after IP with H3K27me3 antibody, fold enrichment relative to IgG. *P < .01 by t-test vs lean

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Expressing, Cell Culture, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 6 Increased trabecular bone mineral density in Ezh2 osteoblastic cell specific deletion male mice. A, Sagittal views of total, trabecular and cortical bone mineral density, and Strength Strain Index (SSI) from a represented mouse from cko and their respective control mice. B, Total bone mineral content in all female mice. B, Tibia pQCT parameters, TOT-BMC (total bone mineral content), P = .0499 by one-way ANOVA; TOT-BMD (total bone mineral density), P = .0365 by one-way ANOVA; TRAB-BMD (trabecular bone mineral density), P = .0188 by one-way ANOVA; CRT-BMD (cortical bone mineral density), P = .0620 by one-way ANOVA in male cko mice compared to their respective genotypic control mice, followed by Tukey's post hoc test comparing cko with its respective control genotype group, *means P < .05 significantly different, n = 4

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Control

Journal: International Journal of Molecular Medicine

Article Title: Downregulation of hsa_circ_0026123 suppresses ovarian cancer cell metastasis and proliferation through the miR-124-3p/EZH2 signaling pathway

doi: 10.3892/ijmm.2020.4804

Figure Lengend Snippet: miR-124-3p and EZH2 are the downstream targets of hsa_circ_0026123. (A) Predicted binding sites of miR-124-3p in the hsa_circ_0000291. The mutated (MUT) version of the hsa_circ_0026123 is also shown. (B) Relative luciferase activity was determined 48 h following transfection with miR-124-3p mimic/normal control (NC) or with the hsa_circ_0026123 wild-type/Mut (WT/MUT) in 293T cells. Data are presented as the means ± SD. ** P<0.01 vs. NC. (C) Predicted binding sites of miR-124-3p with the 3′-UTR of EZH2. The MUT version of the 3′-UTR-EZH2 is also shown. (D) Relative luciferase activity was determined 48 h following transfection with miR-124-3p mimic/normal control or with the 3′-UTR-EZH2 WT/MUT in 293T cells. Data are presented as the means ± SD. ** P<0.01 vs. NC.

Article Snippet: Small interfering RNAs (siRNAs) for hsa_circ_0026123 (si-circ0026123; 5′-AAG GAG AGG AAT ACT AAT TAT CC-3′) (Shanghai GenePharma Co., Ltd.), miR-124-3p mimics (5′-UAA GGC ACG CGG UGA AUG CC-3′) (Shanghai GenePharma Co., Ltd.), miR-124-3p inhibitors (5′-CGU GUU CAC AGC GGA CCU UGA U-3′), EZH2 overexpression vector (the sequence was inserted into the pcDNA3.1 vector) (Shanghai GenePharma Co., Ltd.), and their negative controls (Shanghai GenePharma Co., Ltd.) were transfected into cultured SKOV3 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) with 50 nM according to standard procedures.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection

Journal: International Journal of Molecular Medicine

Article Title: Downregulation of hsa_circ_0026123 suppresses ovarian cancer cell metastasis and proliferation through the miR-124-3p/EZH2 signaling pathway

doi: 10.3892/ijmm.2020.4804

Figure Lengend Snippet: Knockdown of hsa_circ_0026123 inhibits ovarian cancer cell proliferation and migration via the regulation of the miR-124-3p/EZH2 axis in vitro . (A) RT-qPCR assay revealed hsa_circ_0026123 expression in SKOV3 cells following transfection with siRNA against hsa_circ_0026123 combined with or without miR-124-3p inhibitor or the EZH2 overexpression vector. Data are presented as the means ± SD. *** P<0.001 vs. the normal control (NC). (B) RT-qPCR assay showing miR-124-3p expression in SKOV3 cells. Data are presented as the means ± SD. *** P<0.001 vs. NC. ### P<0.001 vs. si-circ0026123. (C) Western blots showing EZH2 expression in SKOV3 cells. Relative protein levels were analyzed and data are presented as the means ± SD. *** P<0.001 vs. NC. ### P<0.001 vs. si-circ0026123. (D) CCK-8 assays were performed to assess the proliferation of SKOV3 cells. Data are presented as the means ± SD. *** P<0.001 vs. NC. ### P<0.001 vs. si-circ0026123. (E and F) Colony formation assay showing the proliferation of SKOV3 cells. Data are presented as the means ± SD. *** P<0.001 vs. NC. ### P<0.001 vs. si-circ0026123. (G and H) Migration of SKOV3 cells was determined by Transwell ® assays. Data are presented as the means ± SD. *** P<0.001 vs. NC. ### P<0.001 vs. si-circ0026123. (I) Western blots showing the relative protein expression levels of markers related to cancer stem cell differentiation. NC, normal control.

Article Snippet: Small interfering RNAs (siRNAs) for hsa_circ_0026123 (si-circ0026123; 5′-AAG GAG AGG AAT ACT AAT TAT CC-3′) (Shanghai GenePharma Co., Ltd.), miR-124-3p mimics (5′-UAA GGC ACG CGG UGA AUG CC-3′) (Shanghai GenePharma Co., Ltd.), miR-124-3p inhibitors (5′-CGU GUU CAC AGC GGA CCU UGA U-3′), EZH2 overexpression vector (the sequence was inserted into the pcDNA3.1 vector) (Shanghai GenePharma Co., Ltd.), and their negative controls (Shanghai GenePharma Co., Ltd.) were transfected into cultured SKOV3 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) with 50 nM according to standard procedures.

Techniques: Migration, In Vitro, Quantitative RT-PCR, Expressing, Transfection, Over Expression, Plasmid Preparation, Western Blot, CCK-8 Assay, Colony Assay, Cell Differentiation

Journal: International Journal of Molecular Medicine

Article Title: Downregulation of hsa_circ_0026123 suppresses ovarian cancer cell metastasis and proliferation through the miR-124-3p/EZH2 signaling pathway

doi: 10.3892/ijmm.2020.4804

Figure Lengend Snippet: Overexpression of EZH2 restores cell growth and migration following the upregulation miR-124-3p in vitro . SKOV3 cells were transfected with miR-124-3p mimics combined with or without the EZH2 overexpression vector. (A) RT-qPCR assay showing the expression of miR-124-3p in SKOV3 cells. Data are presented as the means ± SD. *** P<0.001 vs. NC. (B) Western blot showing EZH2 expression in SKOV3 cells. Relative protein levels were analyzed and data are presented as the means ± SD. *** P<0.001 vs. NC. ### P<0.001 vs. mimic. (C) CCK8 assays were performed to assess cell proliferation in SKOV3 cells. Data are presented as the mean ± SD. *** P<0.001 vs. NC. ### P<0.001 vs. mimic. (D and E) Cloning formation assay showing the cell proliferation of SKOV3 cells. Data are presented as the mean ± SD. *** P<0.001. ### P<0.001 vs. mimic. (F and G) Cell migration and invasion of SKOV3 cells were determined by Transwell ® assays. Data are presented as the means ± SD. *** P<0.001 vs. NC. ### P<0.001 vs. mimic. (H) Western blots showing the relative protein expression of markers related to cancer stem cell differentiation. NC, normal control.

Article Snippet: Small interfering RNAs (siRNAs) for hsa_circ_0026123 (si-circ0026123; 5′-AAG GAG AGG AAT ACT AAT TAT CC-3′) (Shanghai GenePharma Co., Ltd.), miR-124-3p mimics (5′-UAA GGC ACG CGG UGA AUG CC-3′) (Shanghai GenePharma Co., Ltd.), miR-124-3p inhibitors (5′-CGU GUU CAC AGC GGA CCU UGA U-3′), EZH2 overexpression vector (the sequence was inserted into the pcDNA3.1 vector) (Shanghai GenePharma Co., Ltd.), and their negative controls (Shanghai GenePharma Co., Ltd.) were transfected into cultured SKOV3 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) with 50 nM according to standard procedures.

Techniques: Over Expression, Migration, In Vitro, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Western Blot, Clone Assay, Tube Formation Assay, Cell Differentiation

Journal: Open Medicine

Article Title: miR-22-5p regulates the self-renewal of spermatogonial stem cells by targeting EZH2

doi: 10.1515/med-2022-0429

Figure Lengend Snippet: Primer sequences used in miRNA reverse transcription and PCR

Article Snippet: The EZH2 overexpressing plasmid was purchased by Ribobio (Guangzhou, China).

Techniques: Reverse Transcription

Journal: Open Medicine

Article Title: miR-22-5p regulates the self-renewal of spermatogonial stem cells by targeting EZH2

doi: 10.1515/med-2022-0429

Figure Lengend Snippet: miR-22-5p was significantly upregulated in the testicular tissues of patients with cryptorchidism. Testicular tissues of cryptorchidism patients (cry group) and normal testicular tissues of fertile participants (NC group) were collected. (a) The expression of miR-22-5p was detected by QRT-PCR. (b–e) The expression of EZH2 in the testicular tissues was detected by QRT-PCR, western blot, and immunohistochemistry (scale bar = 50 µm). *** p < 0.001 vs NC group.

Article Snippet: The EZH2 overexpressing plasmid was purchased by Ribobio (Guangzhou, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Open Medicine

Article Title: miR-22-5p regulates the self-renewal of spermatogonial stem cells by targeting EZH2

doi: 10.1515/med-2022-0429

Figure Lengend Snippet: miR-22-5p directly targets EZH2. (a) The target region of the EZH2 3′UTR for miR-22-5p and the mutant type of EZH2 3′UTR. (b) Effects of miR-22-5p on the activity of firefly luciferase reporters containing either wild-type (Wt) or mutant-type (Mut) 3′UTR were assessed by luciferase reporter gene assays. (c–e) Effects of miR-22-5p on EZH2 expression levels were examined by qRT-PCR and western blot analyses. * p < 0.05, ** p < 0.01 vs mimic NC; ## p < 0.01 vs inhibitor NC.

Article Snippet: The EZH2 overexpressing plasmid was purchased by Ribobio (Guangzhou, China).

Techniques: Mutagenesis, Activity Assay, Luciferase, Expressing, Quantitative RT-PCR, Western Blot

Journal: Open Medicine

Article Title: miR-22-5p regulates the self-renewal of spermatogonial stem cells by targeting EZH2

doi: 10.1515/med-2022-0429

Figure Lengend Snippet: miR-22-5p regulates SSCs’ self-renewal by targeting EZH2. Human SSCs were co-transfected with miR-22-5p mimics and the EZH2 overexpression plasmid. (a and b) Cell proliferation was measured by EdU staining. (c and d) Cell apoptosis was analyzed by Annexin V/PI staining. (e and f) The expression of SSC markers (GDNF and DAZL) and apoptosis-related proteins (Caspase-3, Bax and Bcl-2) were detected by western blot. * p < 0.05, ** p < 0.01 vs mimic NC + vector; # p < 0.05, ## p < 0.01 vs miR-22-5p mimic + vector.

Article Snippet: The EZH2 overexpressing plasmid was purchased by Ribobio (Guangzhou, China).

Techniques: Transfection, Over Expression, Plasmid Preparation, Staining, Expressing, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: Ectopic expression of LINC00313 is associated with the poor prognosis of patients with osteosarcoma. A qRT-PCR analysis of LINC00313 in 64 pairs of osteosarcoma and adjacent normal tissues. The ratio of LINC00313 expression in osteosarcoma to adjacent normal tissues was calculated and ranked in a descending order. The ordinate showed the value of log2(2−ΔΔCt). B The relative LINC00313 expression levels across different osteosarcoma stages. The ordinate showed the value of ΔCt. (n = 13, 12, 19, and 20 in stage I, II, III and IV, respectively). C The relative LINC00313 expression levels at different lymph nodes spreading stages (n = 18, 22, and 24 in N0, N1, and N2, respectively) N0 indicates that there are no cancer cells in nearby lymph nodes. N1 and N2 indicate identification of lymph nodes containing cancer cells and a higher number after N associated with more cancer cell-positive nodes. The ordinate showed the value of ΔCt. D The relative LINC00313 expression levels in patients with or without distant metastasis (n = 24 in M0, n = 40 in M1) M0 and M1 refer to stage without and with distant metastasis, respectively. The ordinate showed the value of ΔCt. E The Kaplan–Meier overall survival analysis between patients with high (n = 32) and low (n = 32) LINC00313 expression levels. F qRT-PCR analysis of LINC00313 in osteoblast cell line hFOB 1.19 and four different osteosarcoma cell lines (n = 3). Error bars represented mean ± Standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: LINC00313 promotes the proliferation, migration, and invasion of osteosarcoma cells. A qRT-PCR assay confirmed the efficiency of LINC00313 overexpression or knockdown. B CCK-8 assay was performed to measure cell numbers at different time points after LINC00313 overexpression or knockdown. C and D EdU incorporation assay was applied to measure cell proliferation. E and F Transwell migration (upper) and invasion (lower) assay of cells with LINC00313 overexpression or knockdown. G and H Western blot assay to measure EMT-related proteins after LINC00313 overexpression or knockdown. n = 3. Error bars represented mean ± Standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: Migration, Quantitative RT-PCR, Over Expression, Knockdown, CCK-8 Assay, Western Blot, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: Depletion of LINC00313 inhibits tumor growth and metastasis in vivo. A Xenografted tumors were collected from mice subcutaneously inoculated with HOS cells stably transfected with NC (scramble) shRNA or LINC00313 shRNA. B The tumor volumes were measured every 7 days and compared between groups. C The tumors were weighed and compared between groups. D The relative LINC00313 expression between groups. E Ki-67 (proliferating marker) and EMT-related proteins were stained following IHC protocol and compared between groups. F Cancer cells were injected into tail veins and the number of pulmonary metastatic lesions were counted and compared between groups. n = 5. Error bars represented mean ± Standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: In Vivo, Stable Transfection, Transfection, shRNA, Expressing, Marker, Staining, Injection, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: PTEN manipulation affects LINC00313-mediated osteosarcoma progression. A qRT-PCR assay was conducted to measure the relative expression of PTEN in 64 pairs of osteosarcoma and adjacent normal tissues. B The relative expression of PTEN in osteoblast cell line hFOB 1.19 and four different osteosarcoma cell lines. C The expression levels of PTEN after LINC00313 overexpression or knockdown. D PTEN knockdown or forced expression rectified PTEN alterations by LINC00313 overexpression or knockdown. E CCK-8 assay was performed to assess the roles of PTEN in LINC00313-modualted cell growth. F and G Transwell assay was adopted to determine the roles of PTEN in LINC00313-modulated cell migration and invasion. H–K Western blot assay was employed to detect the alterations in EMT-related proteins (H and I) and AKT phosphorylation (J and K) after rectifying LINC00313-mediated PTEN changes. n = 3. Error bars represented mean ± Standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: Quantitative RT-PCR, Expressing, Over Expression, Knockdown, CCK-8 Assay, Transwell Assay, Migration, Western Blot, Phospho-proteomics, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: LINC00313 increases EZH2 mRNA stability by interacting with FUS. A, B qRT-PCR (A) and Western blot assay (B) confirmed LINC00313 overexpression or knockdown led to the alterations in mRNA and protein levels of EZH2. C Dual-luciferase reporter assay measured the roles of LINC00313 in EZH2 promoter activities. D RIP assay confirmed the physical interaction of FUS and LINC00313 and EZH2 mRNA. E LINC00313 overexpression or knockdown altered the binding capacity of EZH2 mRNA to FUS. F LINC00313 overexpression or knockdown-mediated changes of EZH2 mRNA levels could be rectified by modulating FUS levels. G mRNA degradation assay confirmed that LINC00313 enhanced EZH2 mRNA stability in FUS-dependent manner. n = 3. Error bars represented mean ± Standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: Quantitative RT-PCR, Western Blot, Over Expression, Knockdown, Luciferase, Reporter Assay, Binding Assay, Degradation Assay, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: EZH2 is crucial for LINC00313-mediated osteosarcoma initiation and progression. A Xenografted tumors were collected from mice subcutaneously inoculated with different HOS cells. B The tumor volumes were measured every 7 days and compared between groups. C The tumors were weighed and compared between groups. D and E The relative expression levels of PTEN and EZH2 mRNA (D) and protein (E) were determined by qRT-PCR and IHC, respectively. F Different HOS cells were injected into tail veins and the number of pulmonary metastatic lesions were counted. n = 5. Error bars represented mean ± Standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Injection, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: Illustrative diagram of the molecular mechanism underlying LINC00313-mediated osteosarcoma progression. LINC00313 contributs to carcinogenesis and metastasis of osteosarcoma by acting as a novel regulator of the EZH2/PTEN/AKT axis. LINC00313 promoted FUS-EZH2 interaction, resulting in the increased EZH2 mRNA stability and subsequent EZH2-mediated inhibition of the PTEN tumor suppressor gene. This in turn accelerated the proliferation and invasion of osteosarcoma cells, accompanied by an activation of AKT signaling pathway

Article Snippet: Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: Inhibition, Activation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: Ectopic expression of LINC00313 is associated with the poor prognosis of patients with osteosarcoma. A qRT-PCR analysis of LINC00313 in 64 pairs of osteosarcoma and adjacent normal tissues. The ratio of LINC00313 expression in osteosarcoma to adjacent normal tissues was calculated and ranked in a descending order. The ordinate showed the value of log2(2−ΔΔCt). B The relative LINC00313 expression levels across different osteosarcoma stages. The ordinate showed the value of ΔCt. (n = 13, 12, 19, and 20 in stage I, II, III and IV, respectively). C The relative LINC00313 expression levels at different lymph nodes spreading stages (n = 18, 22, and 24 in N0, N1, and N2, respectively) N0 indicates that there are no cancer cells in nearby lymph nodes. N1 and N2 indicate identification of lymph nodes containing cancer cells and a higher number after N associated with more cancer cell-positive nodes. The ordinate showed the value of ΔCt. D The relative LINC00313 expression levels in patients with or without distant metastasis (n = 24 in M0, n = 40 in M1) M0 and M1 refer to stage without and with distant metastasis, respectively. The ordinate showed the value of ΔCt. E The Kaplan–Meier overall survival analysis between patients with high (n = 32) and low (n = 32) LINC00313 expression levels. F qRT-PCR analysis of LINC00313 in osteoblast cell line hFOB 1.19 and four different osteosarcoma cell lines (n = 3). Error bars represented mean ± Standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Cell transfection and generation of stable cell lines Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: LINC00313 promotes the proliferation, migration, and invasion of osteosarcoma cells. A qRT-PCR assay confirmed the efficiency of LINC00313 overexpression or knockdown. B CCK-8 assay was performed to measure cell numbers at different time points after LINC00313 overexpression or knockdown. C and D EdU incorporation assay was applied to measure cell proliferation. E and F Transwell migration (upper) and invasion (lower) assay of cells with LINC00313 overexpression or knockdown. G and H Western blot assay to measure EMT-related proteins after LINC00313 overexpression or knockdown. n = 3. Error bars represented mean ± Standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Cell transfection and generation of stable cell lines Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: Migration, Quantitative RT-PCR, Over Expression, Knockdown, CCK-8 Assay, Western Blot, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: Depletion of LINC00313 inhibits tumor growth and metastasis in vivo. A Xenografted tumors were collected from mice subcutaneously inoculated with HOS cells stably transfected with NC (scramble) shRNA or LINC00313 shRNA. B The tumor volumes were measured every 7 days and compared between groups. C The tumors were weighed and compared between groups. D The relative LINC00313 expression between groups. E Ki-67 (proliferating marker) and EMT-related proteins were stained following IHC protocol and compared between groups. F Cancer cells were injected into tail veins and the number of pulmonary metastatic lesions were counted and compared between groups. n = 5. Error bars represented mean ± Standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Cell transfection and generation of stable cell lines Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: In Vivo, Stable Transfection, Transfection, shRNA, Expressing, Marker, Staining, Injection, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: PTEN manipulation affects LINC00313-mediated osteosarcoma progression. A qRT-PCR assay was conducted to measure the relative expression of PTEN in 64 pairs of osteosarcoma and adjacent normal tissues. B The relative expression of PTEN in osteoblast cell line hFOB 1.19 and four different osteosarcoma cell lines. C The expression levels of PTEN after LINC00313 overexpression or knockdown. D PTEN knockdown or forced expression rectified PTEN alterations by LINC00313 overexpression or knockdown. E CCK-8 assay was performed to assess the roles of PTEN in LINC00313-modualted cell growth. F and G Transwell assay was adopted to determine the roles of PTEN in LINC00313-modulated cell migration and invasion. H–K Western blot assay was employed to detect the alterations in EMT-related proteins (H and I) and AKT phosphorylation (J and K) after rectifying LINC00313-mediated PTEN changes. n = 3. Error bars represented mean ± Standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Cell transfection and generation of stable cell lines Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: Quantitative RT-PCR, Expressing, Over Expression, Knockdown, CCK-8 Assay, Transwell Assay, Migration, Western Blot, Phospho-proteomics, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: LINC00313 increases EZH2 mRNA stability by interacting with FUS. A, B qRT-PCR (A) and Western blot assay (B) confirmed LINC00313 overexpression or knockdown led to the alterations in mRNA and protein levels of EZH2. C Dual-luciferase reporter assay measured the roles of LINC00313 in EZH2 promoter activities. D RIP assay confirmed the physical interaction of FUS and LINC00313 and EZH2 mRNA. E LINC00313 overexpression or knockdown altered the binding capacity of EZH2 mRNA to FUS. F LINC00313 overexpression or knockdown-mediated changes of EZH2 mRNA levels could be rectified by modulating FUS levels. G mRNA degradation assay confirmed that LINC00313 enhanced EZH2 mRNA stability in FUS-dependent manner. n = 3. Error bars represented mean ± Standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Cell transfection and generation of stable cell lines Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: Quantitative RT-PCR, Western Blot, Over Expression, Knockdown, Luciferase, Reporter Assay, Binding Assay, Degradation Assay, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: EZH2 is crucial for LINC00313-mediated osteosarcoma initiation and progression. A Xenografted tumors were collected from mice subcutaneously inoculated with different HOS cells. B The tumor volumes were measured every 7 days and compared between groups. C The tumors were weighed and compared between groups. D and E The relative expression levels of PTEN and EZH2 mRNA (D) and protein (E) were determined by qRT-PCR and IHC, respectively. F Different HOS cells were injected into tail veins and the number of pulmonary metastatic lesions were counted. n = 5. Error bars represented mean ± Standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Cell transfection and generation of stable cell lines Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Injection, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression

doi: 10.1007/s00018-022-04376-1

Figure Lengend Snippet: Illustrative diagram of the molecular mechanism underlying LINC00313-mediated osteosarcoma progression. LINC00313 contributs to carcinogenesis and metastasis of osteosarcoma by acting as a novel regulator of the EZH2/PTEN/AKT axis. LINC00313 promoted FUS-EZH2 interaction, resulting in the increased EZH2 mRNA stability and subsequent EZH2-mediated inhibition of the PTEN tumor suppressor gene. This in turn accelerated the proliferation and invasion of osteosarcoma cells, accompanied by an activation of AKT signaling pathway

Article Snippet: Cell transfection and generation of stable cell lines Plasmids for LINC00313 overexpression (pcDNA3.1-LINC00313), LINC00313 knockdown (shRNA-LINC00313 in pGPU6/Neo), PTEN overexpression (pcDNA3.1-PTEN), PTEN knockdown (siPTEN, siRNA oligonucleotides), EZH2 overexpression (pcDNA3.1-EZH2), EZH2 knockdown (shRNA-EZH2 in pGPU6/Pac backbone), FUS overexpression (pcDNA3.1-FUS) and FUS knockdown (siFUS, siRNA oligonucleotides) were ordered from GenePharma (Shanghai, China).

Techniques: Inhibition, Activation Assay

Journal: PLOS Biology

Article Title: Mutant EZH2 alters the epigenetic network and increases epigenetic heterogeneity in B cell lymphoma

doi: 10.1371/journal.pbio.3003191

Figure Lengend Snippet: A. Histogram of BCL6 levels in OCI-Ly7 WT, EZH2 Y646N cells, and the double mutant of EZH2 Y646N and KMT2D −/− , as measured by CyTOF. B. Normalized coverage values of H3K27me3 on the promoter of BCL6 gene, as measured by Cut&Run, in OCI-Ly7 and EZH2 Y646N. Chr3:187742009-187750134. C – D. BCL6 mean levels in cells binned according to H3K27me3 levels. Red hue represents standard error of the mean. Number of cells in each bin is shown. C. OCI-Ly7 cells with WT EZH2. D. OCI-Ly7 cells with mutant-EZH2 (isogenic cell line). For this analysis measurements from four CyTOF experiments were pooled. H3K27me3 and BCL6 show a non-linear relationship. E. Model for the non-linear ‘bell-shaped’ relationship between H3K27me3 and BCL6. For low H3K27me3 levels, as in most OCI-Ly7 cells, it is positively correlated with BCL6. For high H3K27me3 levels, as in most EZH2-mutant cells, it is negatively correlated with BCL6. Thus, inhibiting EZH2 in the context of WT or mutant-EZH2 generates opposite effects on BCL6 levels. Generated with Biorender.com . F. Expression levels of H3K27me3 and BCL6, as measured by CyTOF, in the isogenic OCI-Ly7 WT and EZH2-mutant cells treated with EZH2 inhibitor for 48 h at a concentration of 10 µM. In OCI-Ly7 with low H3K27me3 levels, EZH2 inhibition downregulated BCL6. In the same cells expressing mutant-EZH2 and high H3K27me3, EZH2 inhibition led to upregulation of BCL6, validating a non-linear relationship. P values were calculated by Welch’s t test. *** p value < 0.001. G. Heatmap showing mean values of OCI-Ly7 control and BCL6 overexpressing cells, for the indicated histone modifications. BCL6 overexpression results in an increase in H3K27me3 as well as additional marks of heterochromatin, and a decrease in histone acetylation levels. H. H3K27me3 mean levels in cells binned according to BCL6 levels. For this analysis measurements from four CyTOF experiments were pooled. Red hue represents standard error of the mean. Number of cells in each bin is shown. I–J. Quantitative RT-PCR analysis of EHZ2 expression. ΔΔ C T values relative to OCI-Ly7 ± s.d ( n = 3) are shown. I. OCI-Ly7 cells were treated with 50 µM of the BCL6 inhibitor (BCL6i) FX1 for the indicated time points. J. OCI-Ly7 control and BCL6 over expression. Both treatments show an increase in EZH2 expression. The data underlying this figure can be found in Raw data 1, 6 at 10.17605/OSF.IO/NTGUX , under CyTOF and RT-PCR folders.

Article Snippet: BCL6 or EZH2 overexpressing cells were obtained by nucleofecting OCI-Ly7 with BCL6 overexpression plasmid pCXN2-BCL6 (Addgene: #40346) or EZH2 WT overexpression plasmid (Addgene: #173717), and compared to blank controls.

Techniques: Mutagenesis, Generated, Expressing, Concentration Assay, Inhibition, Control, Over Expression, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: EZH2 negatively regulates PD-L1 expression in hepatocellular carcinoma

doi: 10.1186/s40425-019-0784-9

Figure Lengend Snippet: IRF1 is a potential transcription factor involved in the negative regulation of PD-L1 by EZH2. a Schematic diagram of a series of CD274 (PD-L1) gene promoter luciferase reporter plasmids. b After transfection with EZH2 -targeted or NC siRNA overnight, Huh7 cells were co-transfected with pGL3-basic vector or the indicated CD274 promoter luciferase reporter gene plasmid and the pRL-TK plasmid for 48 h, and then treated with IFNγ for an additional 24-h. Luciferase activity was determined and normalized using the dual luciferase reporter system. (Mean ± S.E.M.; n = 3; the asterisk represents a comparison between the siEZH2 group and the corresponding control group; * P < 0.05, ** P < 0.01, *** P < 0.001, Wilcoxon test). c TFs that could potentially bind to the P1 truncated promoter were predicted using the PROMO bioinformatics software (pink circle). Genes showing the absolute values of both the Pearson and Spearman expression correlation coefficient (positively or negatively correlated) of more than 0.3 with CD274 (PD-L1 gene) in HCC tissues (TCGA, Provisional) were analyzed on the cBioportal website (blue circle). Venn diagram showing that IRF-1 was the only candidate gene in both gene sets. d Scatter gram showing the mRNA expression correlation of CD274 and IRF1 from TCGA (HCC, Provisional). Pearson and Spearman correlation coefficients and P values are shown. e Huh7 and Hep3B cells were transfected with NC or EZH2 -targeted, IRF1 -targeted, or both, siRNA overnight, and then co-transfected with pGL3-basic vector or the P1 luciferase reporter gene plasmid and pRL-TK plasmid for 48 h. The cells were then treated with IFNγ for an additional 24 h. Luciferase activity was determined and normalized using the dual luciferase reporter system (Mean ± S.E.M.; n = 4; * P < 0.05, ** P < 0.01, Wilcoxon test). f After transfection with NC or EZH2 siRNA targeting 3′-UTR, Huh7 and Hep3B cells were transfected with the indicated plasmids for 48 h, and then treated with IFNγ for 24 h. Luciferase activity was determined and normalized using the dual luciferase reporter system (Mean ± S.E.M.; n = 3; * P < 0.05, ** P < 0.01, Wilcoxon test). pEZH2 and pIRF-1 represent ectopic expression of EZH2 and IRF-1 respectively, and the corresponding control groups were transfected with NC siRNA and/or vector plasmids. g After transfection with NC or the indicated siRNA targeting 3′-UTR, Huh7 and Hep3B cells were transfected with the indicated plasmids for 48 h, and then treated with IFNγ for 24 h. Luciferase activity was determined and normalized using the dual luciferase reporter system (Mean ± S.E.M.; n = 3; * P < 0.05, ** P < 0.01, Wilcoxon test). pIRF-1 represent ectopic expression of IRF-1, and the corresponding control groups were transfected with NC siRNA and/or vector plasmids. h Sequence logo of IRF1 binding site frequency matrix of Homo sapiens predicted using the online software JASPAR. i Schematic representation of IRF1 binding sites in the CD274 P1 promoter region, as predicted by JASPAR. IRE, IRF1 response element. j Huh7 and Hep3B cells were transfected with NC or EZH2 -targeted siRNA overnight, and then co-transfected with pGL3-basic vector or the indicated P1 with or without IREs sequence deletion luciferase reporter gene plasmid and pRL-TK plasmid for 48 h. The cells were then treated with IFNγ for an additional 24 h. Luciferase activity was determined and normalized using the dual luciferase reporter system (Mean ± S.E.M.; n = 4; NS, no significant difference; * P < 0.05, ** P < 0.01, *** P < 0.001, Wilcoxon test)

Article Snippet: The EZH2 and IRF1 overexpression plasmids were purchased from VectorBuilder company ( https://www.vectorbuilder.cn/ ).

Techniques: Luciferase, Transfection, Plasmid Preparation, Activity Assay, Comparison, Control, Software, Expressing, Sequencing, Binding Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: EZH2 negatively regulates PD-L1 expression in hepatocellular carcinoma

doi: 10.1186/s40425-019-0784-9

Figure Lengend Snippet: EZH2 inhibits PD-L1 transcription by inhibiting transcription factor IRF1. a Huh7 cells were transfected with NC or EZH2 -targeted siRNAs for 72 h, and then treated with IFNγ for the indicated times. Immunoblotting analysis was performed to detect the levels of EZH2, IRF1, and PD-L1. GAPDH was used as a loading control. b Huh7 and Hep3B cells were transfected with NC or EZH2 -targeted siRNAs, with or without IRF1 -targeted siRNAs for 48 h, and then treated with IFNγ for 24 h. Immunoblotting was performed to detect the levels of EZH2 and PD-L1. c After transfection with EZH2 siRNA targeting 3′-UTR, Huh7 and Hep3B cells were transfected with the indicated plasmids for 48 h, and then treated with IFNγ for 24 h. Immunoblotting was performed to detect the levels of EZH2, IRF-1 and PD-L1. d After transfection with the indicated siRNA targeting 3′-UTR, Huh7 and Hep3B cells were transfected with the indicated plasmids for 48 h, and then treated with IFNγ for 24 h. Immunoblotting was performed to detect the levels of EZH2, IRF-1 and PD-L1. In c and d , the corresponding control groups were transfected with NC siRNA or vector plasmids. pEZH2 and pIRF-1 represent ectopic expression of EZH2 and IRF-1, respectively. e Huh7 and PLC/PRF/5 cells were pretreated with GSK126, DZNep, or DMSO for 48 h, and then treated with IFNγ for an additional 12 h. GAPDH and H3 were used as loading controls for EZH2 and H3K27me3, respectively. f An H3K27me3 ChIP assay was performed in shEZH2 Hep3B and vector control cells. H3K27me3 levels on the IRF1 gene promoter were normalized to the input. TSS, transcription start site; − 0.5 kb, − 1.0 kb, − 1.5 kb indicate the corresponding upstream locus in the IRF1 gene TSS. CXCL10 was used as positive control (Mean ± S.E.M.; n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001, Wilcoxon test)

Article Snippet: The EZH2 and IRF1 overexpression plasmids were purchased from VectorBuilder company ( https://www.vectorbuilder.cn/ ).

Techniques: Transfection, Western Blot, Control, Plasmid Preparation, Expressing, Positive Control