Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: A Hierarchical clustering of differentially regulated transcripts from RNA‐seq between vehicle and TGFβ1‐treated AECs in the co‐culture ( P ‐adj < 0.05, t ‐test with Benjamini–Hochberg Correction). Significantly regulated profibrotic genes (marked by asterisk) and histone/DNA methyltransferase encoded genes (marked in blue) are listed on the right side. B Volcano plot representing logarithmic ratio of differentially secreted proteins from proteomics analysis of co‐culture medium upon apical TGFβ1 stimulation ( P ‐adj < 0.05, t ‐test), with examples of profibrotic secreted proteins (red dots indicating significantly upregulated proteins, blue dots indicating significantly downregulated proteins, and grey dots indicating no significant change in protein expression levels). C–E Gene Ontology (GO) analysis of differentially expressed genes/proteins ( P ‐adj < 0.05, hypergeometric test) that are enriched in (C) TGFβ1‐treated, (D) vehicle‐treated AECs and (E) differentially secreted proteins. F Gene set enrichment analysis (GSEA) shows enrichment of an IPF transcriptional and cellular phenotype in TGFβ1‐injured AECs/MCs co‐culture system (Kolmogorov–Smirnov test). Note, injured AECs displays an IPF transitional alveolar type 2 cells signature. G GSEA shows enrichment of genes defined as polycomb targets in injured AECs/MCs co‐culture (Kolmogorov–Smirnov test with Benjamini–Hochberg correction). H Representative H3K27me3 and EZH2 immunofluorescence images and box plots (minimum, first quartile, median, third quartile and maximum) showing decreased H3K27me3 levels but increased total EZH2 levels in TGFβ1‐injured AECs in co‐culture with MCs ( n = 5 biological replicates with > 50 cells per experiment, scale bars 50 µm, *P < 0.05, unpaired t ‐test). See also Appendix Fig S2.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: RNA Sequencing, Co-Culture Assay, Expressing, Immunofluorescence

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: A Representative simple western analysis (Peggy Sue) of ph‐EZH2 and quantification shows increased ph‐EZH2 levels on T311 in AECs subjected to apical TGFβ1 for 72 h compared to vehicle treatment (mean + s.d., n = 5 biological replicates, ** *P = 0.0008, unpaired t ‐test). B Representative simple western analysis (Peggy Sue) of SUZ12 immunoprecipitates shows co‐precipitation of EZH1 and EZH2 in AECs. TGFβ1‐induced injury leads to the EZ switch from SUZ12‐bound EZH2 to EZH1. Unspecific IgG binding was used as a negative control. A representative from 3 biological replicates is shown. C ChIP‐qPCR shows increased ph‐EZH2 occupancy at gene bodies of profibrotic genes in AECs subjected to TGFβ1 for 72 h. Note no changes in ph‐EZH2 levels at non‐target genes (mean + s.d., n = 3 biological replicates). Unspecific IgG was used as negative control. ChIP‐qPCR for non‐fibrotic genes is shown in Appendix Fig S3A. D ChIP‐qPCR shows increased EZH1 occupancy at promoters of non‐fibrotic genes in AECs subjected to apical TGFβ1 for 72 h (mean + s.d., n = 3 biological replicates). Unspecific IgG was used as negative control. E ChIP‐qPCR shows no changes in H3K27me3 at promoters of non‐fibrotic genes in AECs subjected to apical TGFβ1 for 72 h (mean + s.d., n = 3 biological replicates). Unspecific IgG was used as negative control. F Representative simple western analysis (Peggy Sue) and quantifications (right panels) for EZH2 and H3K27me levels from EZH2‐deleted AECs (sgEZH2) which were reintroduced empty vector (EV), T311 wildtype (WT), a phosphorylated‐deficient T311A or a phosphomimetic T311D form of EZH2. Quantifications show mean + s.d. ( n = 4 biological replicates, * P < 0.05, ** P < 0.01, Kruskal–Wallis/Dunn’s). G EZH2‐deleted AECs (sgEZH2) reintroducing empty vector (EV), T311 wildtype (WT), a phosphorylated‐deficient T311A or a phosphomimetic T311D form of EZH2 were quantified for the expression of profibrotic genes. Vehicle and TGFβ1‐treated AECs (sgNEG + vehicle/TGFβ1) were used as control. mRNA levels are normalised to HPRT1 expression. (mean + s.d., n = 4 biological replicates, *P < 0.05, ** P < 0.01, ** *P < 0.001, *** *P < 0.0001, Kruskal–Wallis/Dunn’s). See also Appendix Fig S3. Source data are available online for this figure.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Simple Western, Binding Assay, Negative Control, ChIP-qPCR, Plasmid Preparation, Expressing, Control

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: A Kinase enrichment analysis showing enrichment of TAK1 (encoded by MAP3K7 ) in injured AECs ( P ‐adj < 0.05, hypergeometric test with Benjamini–Hochberg correction). B Nuclear fractionation followed by simple western analysis (Peggy Sue) of AECs exposed to 72 h of TGFβ1 in the co‐culture system shows an increase in phosphorylated TAK1 and a parallel increase in ph‐EZH2. Quantifications (lower panels) show mean + s.d., n = 3 biological replicates (ns = non‐significant, ** *P < 0.001, Kruskal–Wallis/Dunn’s). C Simple western analysis (Peggy Sue) of EZH2 immunoprecipitates shows increased co‐precipitation of ph‐EZH2 (T311) and ph‐TAK1 in injured AECs. Unspecific IgG was used as negative control. A representative from three experiments is shown. D Simple western analysis (Peggy Sue) shows a TAK1‐dependent enrichment of ph‐EZH2 levels in injured AECs. Note: TAK1 inhibitor (5‐OZ) attenuates increased ph‐EZH2 levels in injured AECs. Quantifications (right panels) show mean + s.d., n = 5 biological replicates (ns = non‐significant, * *P = 0.0026, ** *P < 0.001, ANOVA/Tukey’s). E ChIP‐qPCR shows diminished POL2 occupancy on profibrotic genes in injured AECs subjected to 5‐OZ (mean + s.d., n = 3 biological replicates). Unspecific IgG was used as negative control. See also Appendix Fig S4. Source data are available online for this figure.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Fractionation, Simple Western, Co-Culture Assay, Negative Control, ChIP-qPCR

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: A Nuclear fractionation followed by simple western analysis (Peggy Sue) shows an increase in nuclear actin in AECs exposed to TGFβ1 for 48 h. Quantification (right panel) shows mean + s.d., n = 3 biological replicates, ns = non‐significant, *P = 0.014, ANOVA/Tukey’s). B Simple western analysis (Peggy Sue) of EZH2 co‐immunoprecipitates shows increased levels of EZH2‐bound POL2, ph‐EHZ2 and actin in injured AECs. Unspecific IgG binding was used as a negative control. A representative from 3 biological replicates is shown. C, D ChIP‐qPCR shows increased occupancy of (C) POL2‐S5p at promoters of profibrotic genes in AECs subjected to TGFβ1 for 24 h, whereas no enrichment of (D) POL2‐S2p at the gene bodies of these genes was detected. Negative IgG control is shown in Appendix Fig S5B (mean + s.d., n = 3 biological replicates). E, F ChIP‐qPCR shows increased occupancy of (E) POL2‐S5p at promoters and (F) POL2‐S2p at the gene bodies of profibrotic genes in AECs subjected to TGFβ1 for 48 h. Negative IgG control is shown in Appendix Fig S5C (mean + s.d., n = 3 biological replicates). See also Appendix Fig S5. Source data are available online for this figure.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Fractionation, Simple Western, Binding Assay, Negative Control, ChIP-qPCR, Control

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: Representative simple western analysis (Peggy Sue) shows histone fraction (upper panel) and non‐histone fraction (lower panel) from TGFβ1‐injured AECs and control. These cells were further treated with an EZH2 inhibitor GSK126. Note the loss of ph‐EZH2 in GSK126‐treated AECs. Quantifications (right panels) shows mean + s.d. ( n = 5 biological replicates, *P < 0.05, * *P = 0.01, ns = non‐significant, Friedman/Dunn’s test for H3K27me3, ANOVA /Sidak´s test for ph‐EZH2). Representative simple western analysis (Peggy Sue) shows increased POL2‐K7 methylation (K7m) levels in injured AECs. This increase is blocked by GSK126. Quantification (right panel) shows mean + s.d. ( n = 5 biological replicates, *P < 0.05, ANOVA/Tukey’s). Simple western analysis (Peggy Sue) of EZH2 co‐immunoprecipitates shows increased levels of EZH2‐bound POL2‐K7m and decreased levels of EZH2‐bound SUZ12 in injured AECs. Unspecific IgG binding was used as a negative control. A representative from 3 biological replicates is shown. ELISA of profibrotic markers shows that inhibition of EZH2 activity by GSK126 attenuates the profibrotic effect of injured AECs on MCs ( n = 3 biological replicates from 5 MCs donors, mean + s.d., *P < 0.05, * *P < 0.01, ** *P < 0.001, ANOVA/Tukey´s). Source data are available online for this figure.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Simple Western, Control, Methylation, Binding Assay, Negative Control, Enzyme-linked Immunosorbent Assay, Inhibition, Activity Assay

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: A Representative immunofluorescence images of F‐actin (phalloidin) and DAPI show that treatment with ROCK inhibitor Y27632 but not depletion of EZH2 can prevent TGFβ1‐induced actomyosin remodelling in AECs (scale bars 200 µm). B Simple western analysis (Peggy Sue) of EZH2 immunoprecipitates shows abolition of TGFβ1‐induced profibrotic transcriptional complex of EZH2/POL2/actin upon the convergent treatment of TGFβ1 and Y27632. Unspecific IgG binding was used as a negative control. Representative from 3 biological replicates is shown. C, D Simple western analysis (Peggy Sue) of nuclear fractionation (C) shows an increase in nuclear actin, ph‐EZH2 and PO2‐S2p levels in injured AECs. RNAi‐mediated depletion of IPO9 (siIPO9) prevents injury‐induced nuclear actin and POL2‐S2p. Quantification (D) shows mean + s.d. ( n = 3 biological replicates, *P < 0.05, * *P < 0.01, ** *P < 0.001, ANOVA/Tukey’s). E qPCR analysis of profibrotic genes in MCs co‐culture with AECs shows that depletion of IPO9 in TGFβ1‐injured AECs blocks the fibrotic crosstalk with MCs. Data show mRNA levels of profibrotic genes normalised to S26 (mean + s.d., n = 3 biological replicates with 5 MCs donors, *P < 0.05, ** *P < 0.001, ANOVA/Tukey’s). See also Appendix Fig S6. Source data are available online for this figure.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Immunofluorescence, Simple Western, Binding Assay, Negative Control, Fractionation, Co-Culture Assay

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: A Simple western analysis (Peggy Sue) and quantifications (right panels) of mouse lung epithelial cells shows increased ph‐EZH2 (T311), ph‐TAK1, myosin activity (ph‐MLC2) and POL2‐K7m levels in AAV‐mediated TGFβ1 overexpression. Note, increased ph‐EZH2 and POL2‐K7m levels are attenuated by the EZH2 inhibitor GSK126, whereas ph‐TAK1 and ph‐MLC2 levels cannot be rescued by GSK126. Quantifications (right panels) show violin plots, *P < 0.05, * *P < 0.01, ** *P < 0.001, ns = non‐significant, Kruskal–Wallis/Dunn’s. B Representative of 3D computed tomography (CT) reconstruction of the lung from control, AAV‐TGFβ1 and GSK126‐treated AAV‐TGFβ1 mice (green: lung tissue, red: airways and region of interest (ROI): blue). Insets show µCT slices in the middle of the lung from respective mice. Note, GSK126 attenuates TGFβ1‐induced lung injury. Quantification (right panel) shows mean intensity of ROIs from the whole lung (violin plots, *P = 0.0385, * *P = 0.0012, ANOVA/Holm–Sidak’s). C qPCR analysis of differentiation genes in epithelial cells reveals that EZH2 is required for the effect of TGFβ1 on metaplastic differentiation gene expression. Data show mRNA levels of profibrotic genes normalised to S26 (violin plots, *P < 0.05, * *P < 0.01, ** *P < 0.001, ANOVA/Holm–Sidak’s). D Immunofluorescence analysis of KRT5 as a marker for alveolar metaplastic basal cells and ph‐EZH2 (scale bars 100 µm), pro‐SFTPC as a marker for alveolar type 2 epithelial cells (scale bars 50 µm) and quantifications (right panels) show percentage of KRT5 + pods area per 10X field and percentage of pro‐SFTPC + cells per 20X field ( *P < 0.05, unpaired t ‐test). Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12 AAV‐TGFβ1 and 13 GSK126‐treated AAV‐TGFβ1 mice. See also Appendix Fig S7. Source data are available online for this figure.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Simple Western, Activity Assay, Over Expression, Computed Tomography, Control, Gene Expression, Immunofluorescence, Marker

Journal: EMBO Reports

Article Title: An EZH2‐dependent transcriptional complex promotes aberrant epithelial remodelling after injury

doi: 10.15252/embr.202152785

Figure Lengend Snippet: TGFβ1‐injured epithelium activates TAK1 and actomyosin remodelling, which subsequently induces nuclear translocation of TAK1 and actin. Nuclear TAK1 mediates the phosphorylation of EZH2 on T311 and facilitates the release of EZH2 from PRC2. The liberation of EZH2 is accompanied by an EZ switch to EZH1‐PRC2, which is required to maintain H3K27me3 at TGFβ1 non‐target genes. Simultaneously, EZH2 establishes the fibrotic transcriptional complex with POL2 and nuclear actin to promote the metaplastic differentiation of AECs and triggers the fibrotic crosstalk with MCs. Perturbing this fibrotic complex blocks the fibrotic cascade, reinforces tissue repair and restores homeostasis.

Article Snippet: In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 μM ATP (Cell Signaling, 9804) for 60 min at 30°C.

Techniques: Translocation Assay, Phospho-proteomics

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: DUXAP9 promoted xenograft tumor growth and metastasis of OSCC cells. A,B) The volumes and weights A) and the growth curves B) of tumors derived from CAL27 cells transfected with SS‐NC or SS‐DUXAP9 ASO were measured, and representative tumor images were taken. n = 6/group. C,D) The volumes and weights C) and the growth curves D) of tumors derived from CAL27 cells transfected with vector or DUXAP9 plasmids were measured, and representative tumor images were taken. n = 6/group. E) Immunohistochemical staining of Ki67, PCNA, EZH2, and E‐cadherin in tumors derived from CAL27 cells transfected with SS‐NC and SS‐DUXAP9 ASO or control‐ and DUXAP9‐expressing vectors, respectively, Scale bars, 100 µm (left), 25 µm (right). F–I) The expression of Ki67 F), PCNA G), EZH2 H), and E‐cadherin I) was determined by the IHC score in the indicated groups. J) Representative bioluminescence images of lung metastasis in mice injected with CAL27 cells stably expressing vector or DUXAP9 via the tail vein. K) Representative images of H&E staining and GFP fluorescence in the lungs of nude mice injected with CAL27 cells overexpressing vector or DUXAP9. Scale bars, 100 µm. Data in (B) and (D) are presented as the mean ± SEM, and data in (A), (C), and (F–I) are presented as the mean ± SD from three independent experiments. Data in (A–D) and (F–I) were calculated by two‐tailed unpaired Student's t ‐test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Derivative Assay, Transfection, Plasmid Preparation, Immunohistochemical staining, Staining, Control, Expressing, Injection, Stable Transfection, Fluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: Physical interaction between DUXAP9 and EZH2 in OSCC cells. A) DUXAP9‐interacting proteins were separated by SDS‐PAGE followed by an endogenous DUXAP9 RNA pull‐down assay and manifested by Coomassie Brilliant Blue staining. The distinct protein bands in the gel were excised, dissolved, and subjected to mass spectrometry. The red asterisk denotes the location of EZH2. B) Venn diagram shows a group of nucleoplasm‐located proteins identified by proteomic analysis. The five proteins listed are considered candidates for DUXAP9 binding proteins. C) RIP‐qPCR assay using IgG or anti‐EZH2 antibody shows the enrichment of DUXAP9 expression in the precipitated EZH2 binding complex. D) RNA pull‐down assays with biotin‐labeled DUXAP9 probes show the interaction between DUXAP9 and EZH2 in CAL27 cells. E) Confocal FISH and IF images showing the colocalization of EZH2 (green) and DUXAP9 (red) in CAL27 and HN6 cells. Scale bars, 5 µm. F) Predicted DUXAP9 interaction region using catRAPID. G) qRT‐PCR analysis of DUXAP9 enrichment by RIP assay using anti‐EZH2 in CAL27 cells. Thirteen specific primers for DUXAP9 were used to detect the binding region of DUXAP9. H) Predicted secondary structure of DUXAP9 using Mfold software. I) Western blot followed by RNA pull‐down assay of DUXAP9‐ or mutant DUXAP9 (mut‐DUXAP9)‐transfected cells. The construction of the mutant DUXAP9 vector is shown above, and the expression level of DUXAP9‐associated EZH2 protein is shown below. J) In vitro reconstituted RIP‐qPCR assay using IgG or EZH2 antibody shows the enrichment of DUXAP9 expression in the precipitated EZH2 binding complex. Data are presented as the mean ± SD from three independent experiments. Data in (C), (G), and (J) were calculated by two‐tailed unpaired Student's t ‐test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: SDS Page, Pull Down Assay, Staining, Mass Spectrometry, Binding Assay, Expressing, Labeling, Quantitative RT-PCR, Software, Western Blot, Mutagenesis, Transfection, Plasmid Preparation, In Vitro, Two Tailed Test

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: DUXAP9 increases EZH2 protein expression via inhibition of its proteasomal degradation. A) The mRNA expression of EZH2 was measured by qRT‐PCR in CAL27 and HN6 cells transfected with SS‐NC or SS‐DUXAP9 ASO (left) or control‐ or DUXAP9‐overexpressing vectors (right). B) The protein expression of EZH2 was detected by western blot in CAL27 and HN6 cells transfected with SS‐NC or SS‐DUXAP9 ASO (left) or control or DUXAP9 overexpressing vectors (right). C–E) Western blot shows EZH2 protein in CAL27 and HN6 cells transfected with SS‐NC or SS‐DUXAP9 ASO C) or control‐ or wild type DUXAP9‐ D) or mutant DUXAP9‐ E) overexpressing vectors and treated with CHX (20 µg mL −1 ) for the indicated time (left). The quantification of the EZH2 degradation rate was measured by grayscale analysis (right). F) Western blot showing EZH2 protein in CAL27 and HN6 cells transfected with SS‐NC or SS‐DUXAP9 and treated with MG132 (20 µ m for 6 h). G, H) Ubiquitination of EZH2 in DUXAP9‐silenced G) and DUXAP9‐overexpressing H) CAL27 cells after MG132 treatment (20 µ m for 6 h) was detected by western blot analysis. I) Ubiquitination of EZH2 in 293T cells transfected with EZH2 and DUXAP9 after MG132 treatment (20 µ m for 6 h) was detected by western blot analysis. Data are presented as the mean ± SD from three independent experiments. Data in (A) and (C–E) were calculated by two‐tailed unpaired Student's t ‐test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Expressing, Inhibition, Quantitative RT-PCR, Transfection, Control, Western Blot, Mutagenesis, Two Tailed Test

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: DUXAP9 suppresses EZH2 degradation via inhibition of the phosphorylation (Thr345/Thr487) of EZH2. A,B) Western blot showing the levels of p‐EZH2 (T345), p‐EZH2 (T487), and CDK1 in DUXAP9‐silenced A) and DUXAP9‐overexpressing B) CAL27 and HN6 cells treated with MG132 (20 µ m for 6 h). C,D) Western blot showing the interaction between EZH2 and CDK1 in control and DUXAP9‐overexpressing CAL27 C) and 293T D) cells treated with MG132 (20 µ m for 6 h) in a coimmunoprecipitation assay using an anti‐EZH2 antibody. E,F) The binding of DUXAP9 to mutant EZH2 or wild‐type EZH2 was detected by RNA pull‐down assay E) and RIP‐qPCR assay F). The diagram shows the mutation site of EZH2. G,H) Western blot showing the levels of p‐EZH2 (T345), p‐EZH2 (T487), and CDK1 in the different combination of transfection of DUXAP9‐ and CDK1 overexpressing vectors in CAL27 G) and 293T H) cells transfected with different combinations of DUXAP9‐ and CDK1‐overexpressing vectors and treated with MG132 (20 µ m for 6 h). I,J) Western blot showing the ubiquitination of EZH2 in the different combination of transfection of DUXAP9‐ and CDK1 overexpressing vectors in CAL27 I) and 293T J) cells transfected with different combinations of DUXAP9‐ and CDK1‐overexpressing vectors and treated with MG132 (20 µ m for 6 h). K) Western blot shows the ubiquitination of mutant EZH2 in the different combination of transfection of DUXAP9‐ and CDK1 overexpressing vectors in 293T cells transfected with different combinations of DUXAP9‐ and CDK1‐overexpressing vectors and treated with MG132 (20 µ m for 6 h). L) Western blot showing the levels of p‐EZH2 (T345), p‐EZH2 (T487), and CDK1 in the different combinations of mutant DUXAP9‐ and CDK1‐overexpressing vectors treated with MG132 (20 µ m for 6 h). M) Western blot analysis of the ubiquitination of EZH2 in 293T cells transfected with different combinations of mutant DUXAP9‐ and CDK1‐overexpressing vectors and treated with MG132 (20 µ m for 6 h). Data are presented as the mean ± SD from three independent experiments. Data in (F) were calculated by two‐tailed unpaired Student's t ‐test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Inhibition, Western Blot, Control, Co-Immunoprecipitation Assay, Binding Assay, Mutagenesis, Pull Down Assay, Transfection, Two Tailed Test

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: DUXAP9 suppresses EZH2 degradation via nuclear‐to‐cytoplasmic translocation. A–C) Western blot showing the levels of nuclear and cytoplasmic EZH2 in CDK1‐ A) and DUXAP9‐ B) overexpressing and DUXAP9 knockdown C) CAL27 and HN6 cells. Quantification of the EZH2 nucleus/cytoplasm ratio by grayscale analysis is shown on the right. D) Western blot shows the levels of nuclear and cytoplasmic mutant EZH2 in vector‐ or CDK1‐overexpressing 293T cells. E) Western blot shows the levels of nuclear and cytoplasmic wild‐type EZH2 and mutant EZH2 in CDK1‐overexpressing 293T cells. F) Western blot shows the levels of nuclear and cytoplasmic EZH2 in 293T cells transfected with control or mutant DUXAP9‐overexpressing vectors in combination with CDK1‐overexpressing vector. Data are presented as the mean ± SD from three independent experiments. Data in (A–F) were calculated by two‐tailed unpaired Student's t ‐test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Translocation Assay, Western Blot, Knockdown, Mutagenesis, Plasmid Preparation, Transfection, Control, Two Tailed Test

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: DUXAP9 promotes the proliferation and invasion of OSCC cells by mediating EZH2 expression and function. A) The mRNA expression of 9 known EZH2 target genes was analyzed by qRT‐PCR assays in CAL27 and HN6 cells transfected with vector, DUXAP9, and EZH2. B) ChIP‐qPCR assay of EZH2 or IgG occupancy at the CDKN1A, DAB2IP, and RUNX2 loci in CAL27 cells transfected with SS‐NC or SS‐DUXAP9. C) The volumes, weights and growth curves of tumors derived from CAL27 cells transfected with control‐ or DUXAP9‐expressing vectors and siRNAs targeting NC or EZH2 were measured and imaged; D,E) The expression of Ki67, PCNA, and EZH2 was determined by the IHC score in the indicated groups. n = 5/group, Scale bars, 100 µm (left), 25 µm (right). Data in (C) are presented as the mean ± SEM, and data in (A,B) and (D) are presented as the mean ± SD from three independent experiments. Data were calculated by two‐tailed unpaired Student's t ‐test.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Derivative Assay, Control, Two Tailed Test

Journal: Advanced Science

Article Title: Yin Yang 1‐Induced Long Noncoding RNA DUXAP9 Drives the Progression of Oral Squamous Cell Carcinoma by Blocking CDK1‐Mediated EZH2 Degradation

doi: 10.1002/advs.202207549

Figure Lengend Snippet: Schematic depicting YY1‐induced DUXAP9 drives OSCC by blocking CDK1‐mediated EZH2 degradation. DUXAP9 orchestrates a different biological function of CDK1‐mediated phosphorylation of the T345 and T487 sites of EZH2 in controlling the protein stability of EZH2 via nuclear to cytoplasmic translocation, suggesting the importance of lncRNA regulation at the posttranslational level in OSCC progression.

Article Snippet: An in vitro reconstituted RIP assay was conducted with human recombinant EZH2 protein (#TP302054, Origene, US) and DUXAP9 transcribed by a T7 High Yield RNA Transcription Kit (Beyotime, China).

Techniques: Blocking Assay, Translocation Assay