Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: MED19 localizes to the nucleolus. ( A ) Protein sequence of MED19. The predicted NoLS is highlighted in blue with lysine (K) residues included marked in red. ( B ) The NoLS prediction of MED19 protein using the NOD web tool. The x -axis indicates the amino acid position along the MED19 protein sequence, while the y -axis shows the nucleolar localization score assigned by the NOD for each amino acid. Each blue dot represents an individual amino acid. The pink region above 0.8 represents potential NoLSs. ( C ) Immunofluorescence staining of MED19 using specific antibodies in various cell lines. FBL serves as a commonly used nucleolar marker, and DAPI stains nuclear DNA. Scale bar: 10 μm. ( D ) Immunofluorescence images of the MED23, CDK8, MED4, and MED8 subunits of the Mediator complex in HuH7 cells, using respective antibodies. Scale bar: 10 μm. ( E ) Schematic representation of exogenously expressed full-length MED19 and its truncated or mutated variants. HA-MED19-FL represents full-length MED19. HA-MED19-NoLS represents MED19 with the NoLS. HA-MED19-ΔNoLS represents MED19 with the NoLS deleted. HA-MED19-K-A represents MED19 with all lysine residues (K) in the NoLS mutated to alanines (A). ( F ) Immunofluorescence images of exogenously expressed full-length MED19 and its truncated or mutated variants in HuH7 cells, detected using an anti-HA antibody. Scale bar: 10 μm. ( G ) Schematic illustration of the GFP localization experiment. GFP-WT, a plasmid expressing wild-type GFP protein. GFP-19NoLS, a plasmid expressing a GFP fusion protein with the NoLS of MED19 fused to its C-terminus. ( H ) Co-localization results of GFP-WT and GFP-19NoLS proteins. ( I ) Immunofluorescence was performed using an HA-tag antibody. Three representative cells were selected for display. The HA tag was knocked into the N-terminus of the MED19 protein via gene editing technology in the HuH7 hepatocellular carcinoma cell line.​

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: Sequencing, Immunofluorescence, Staining, Marker, Plasmid Preparation, Expressing

Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: MED19 directly binds rRNA via its NoLS. ( A ) Schematic illustration of the silica enrichment assay. RNA-binding proteins can be enriched by silica material under UV cross-linking and denaturing conditions. ( B – F ) Western blot analysis of silica-enriched fractions probed with antibodies against FBL, NPM1, tubulin, actin, and MED19. Input: total cell lysates. UV: samples subjected to UV cross-linking. ( G ) Western blot analysis of silica-enriched fractions using an anti-HA antibody. HA-19-FL: full-length HA-tagged MED19. HA-ΔNoLS: HA-tagged MED19 lacking the NoLS. ( H ) Western blot analysis of silica-enriched fractions using an anti-GFP antibody. GFP-19-FL: full-length GFP-tagged MED19. GFP-NoLS: the NoLS of MED19 was fused to the C-terminus of GFP. ( I ) Western blot analysis of silica-enriched fractions using an anti-HA antibody. HA-19-K-A: HA-tagged MED19 with all lysine residues in the NoLS mutated to alanine. ( J ) Schematic diagram of identifying the RNA-binding sites on the corresponding protein by UV cross-linking mass spectrometry. ( K ) Distribution of RNA-binding sites across the MED19 protein. The x -axis represents the amino acid sequence positions; the y -axis represents the summed quantitative scores of the RNA-amino acid cross-linking sites. ( L ) Peaks distribution of MED19 CLIP-seq. IGS, Intergenic Spacer; 5′ETS, 5′ External Transcribed Spacer; 3′ETS, 3′ External Transcribed Spacer; ITS1, Internal Transcribed Spacer 1; ITS2, Internal Transcribed Spacer 2. ( M ) Reads frequency distribution of MED19-binding RNAs. ( N ) Reads frequency distribution of MED19-binding snoRNAs. ( O ) Denaturing UV-RIP-qPCR of MED19. Three kinds of denaturing wash buffer (10% guanidine hydrochloride, 8 M urea, 10% SDS) were used in the “wash” step of the UV-RIP-qPCR of HaloTag-MED19 and HaloTag-GFP. The Ct value of the target proteins was normalized to the input sample represented as the percentage of the corresponding RNA in the input. ( P ) EMSA blot of the purified MED19 proteins with rRNA probes. The rRNA probe without biotin was used as a competitive probe and was added at a molar amount 10 times that of the biotin-labeled probe. The signal on the membrane was detected using SA-HRP luminescence system.

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: RNA Binding Assay, Western Blot, Structural Proteomics, Sequencing, Binding Assay, Purification, Labeling, Membrane

Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: MED19 interacts with the BoxC/D-snoRNP. ( A ) Intersection analysis of MED19 interacting proteins identified by the MED19 IP-MS with nucleolar proteins annotated in the GO database. ( B ) Enrichment analysis of MED19 interactome based on the GO database at the molecular function level. ( C ) Enrichment analysis of annotated nucleolar proteins in the MED19 interactome by the GO database at the molecular function level. ( D ) Enrichment analysis of MED19 interactome by the Reactome database. ( E ) Enrichment analysis of annotated nucleolar proteins in the MED19 interactome by the Reactome database. ( F ) Identification of MED19 and four core Box C/D snoRNP subunit proteins by MED19 IP-MS. ( G ) Western blot analysis of MED19 immunoprecipitation (IP-WB) following RNase A treatment. ( H ) Western blot of FBL immunoprecipitation. ( I ) Western blot analysis of MED19 immunoprecipitation using exogenously expressed full-length and mutant forms of MED19. HA-MED19-FL: HA-tagged full-length MED19; HA-MED19-K-A: HA-tagged MED19 with all lysines in the NoLS mutated to alanines; HA-MED19-NoLS: HA-tagged NoLS domain of MED19. ( J ) Western blot of MED19 IP by exogenously expressed full-length and different truncation forms of MED19. HA-MED19-FL, HA-tagged MED19 full-length form; HA-MED19-ΔN, HA-tagged MED19 with the N-terminal region deleted (1–72); HA-MED19-ΔM, HA-tagged MED19 with the middle-region deleted (73–158); HA-MED19-ΔC, HA-tagged MED19 with the C-terminal region deleted (159–244). ( K ) IP experiments were performed using endogenously HA-tagged HA-MED23 and HA-MED19 by HA-magnetic beads. The results demonstrate that MED19 specifically associates with snoRNP proteins.

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: Protein-Protein interactions, Western Blot, Immunoprecipitation, Mutagenesis, Magnetic Beads

Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: Depletion of MED19 attenuates the 2′-O-methylation level of rRNA. ( A ) Mass spectrometry assay of 2′-O-methylation level of adenosine and guanosine in the total RNA. sh19, stable knockdown of MED19 using a retrovirus with shRNA sequence designed against the 3′UTR. Re-OE, re-expression of MED19 using its coding sequence (CDS). Statistical differences were calculated using a two-way unpaired Student’s t -test with experiments repeated three times. *Represents P < .05; **represents P < .01; ***represents P < .001. ns represents no significance. ( B ) Schematic diagram of the RTL-Q assay. RT, reverse transcription primer. R, reverse PCR primer located downstream of the 2′-O-Me site. Fu, forward PCR primer located upstream of the 2′-O-Me site. Fd, forward PCR primer located downstream of the 2′-O-Me site. ( C – E ) Changes of the 2′-O-methylation ratio of selected 2′-O-methylation sites after knockdown of MED19 (sh19) and re-expression (Re-OE) in 293T cells using RTL-Q method. ( F ) Schematic illustration of the Ribometh-seq principle. ( G – I ) Differentially changed 2′-O-methylated sites on rRNAs after MED19 knockdown in the HuH7 cell line. The x -axis represents the positions and corresponding nucleotides of the 2′-O-methylated sites on their respective rRNAs. The y -axis indicates the methylation modification ratio, where a value of 1 signifies 100% modification at that site. ( J ) Western blot of the MED19 and other BoxC/D-snRNP proteins after knockdown of MED19 in 293T cells. ( K, L ) CLIP-qPCR of FBL under MED19 knockdown (sh19) and re-overexpression (Re-OE). The pre-rRNA bound to FBL was measured by real-time qPCR both in 293T and HuH7 cell lines.

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: Methylation, Mass Spectrometry, Knockdown, shRNA, Sequencing, Expressing, Reverse Transcription, Modification, Western Blot, Over Expression

Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: Phase separation of MED19 with FBL. ( A ) IDR profiling of FBL and MED19 proteins. The x -axis represents the amino acid sequence of the protein, while the y -axis represents the scores of disorder propensity calculated by the PONDA software . Scores >50 indicate potential IDR regions. ( B ) Coomassie blue staining of the purified mEGFP-MED19 and mCherry-FBL. ( C ) Droplet formation of mEGFP-MED19 protein (6 μM) under 1% PEG8000 condition. DIC, differential interference contrast microscopy. ( D ) Droplet formation of equimolar mixtures of mEGFP-MED19 and mCherry-FBL (6 μM) under 1% PEG8000 condition. ( E ) FRAP analysis mEGFP-MED19 in transfected HuH7 cell line. The MED19 plaque was bleached and gradually recovered within minutes. ( F ) The bleach and recovery profile of the bleached region.

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: Sequencing, Software, Staining, Purification, Microscopy, Transfection

Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: MED19 promotes the IRES-dependent translation. ( A ) Schematic illustration of cap-dependent translation and IRES-dependent translation (cap-independent translation). ( B ) Schematic diagram of the bicistronic reporter system for measuring the IRES element translational activity. ( C ) Schematic diagram of the c-Myc mRNA composition with the IRES element located in the 5′UTR. ( D ) Predicted secondary structure of the c-Myc IRES sequence. ( E ) Measurement of the EMCV-IRES translation activity dynamics by the bicistronic reporter system. Left: overexpression of MED19 at increasing dosages. Right: knockdown of MED19 (sh19) and re-overexpression of MED19 (Re-OE). EMCV: Encephalomyocarditis virus. Statistical differences were calculated using a two-way unpaired Student’s t -test with experiments repeated three times. *Represents P < .05, ****represents P < .0001. ns represents no significance. ( F ) Measurement of the c-Myc-IRES translation activity by the bicistronic reporter system under knockdown of MED19 (sh19) and re-overexpression of MED19 (Re-OE). Left: in 293T cells. Right: in HuH7 cells. ( G ) Western blot of the c-Myc and tubulin proteins expression levels under knockdown of MED19 (sh19) and re-overexpression of MED19 (Re-OE). ( H ) RT-qPCR analysis of c-Myc mRNA expression levels under knockdown of MED19 (sh19) and re-overexpression of MED19 (Re-OE). Actin was used for normalization. Left: in 293T cells. Right: in HuH7 cells.

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: Activity Assay, Sequencing, Over Expression, Knockdown, Virus, Western Blot, Expressing, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: MED19 promotes cancer development and correlates with c-Myc at the protein level. ( A ) Expression profiling of MED19 in 33 types cancer clinical samples based on the TCGA database. The x -axis represents the sample names and quantities, while the y -axis represents the mRNA expression level of MED19. Red color indicates cancer samples, while blue color represents adjacent normal samples. ( B ) Cell proliferation disturbance profiling of various cancer cell lines under MED19 knockout. The x -axis values represent the cell proliferation changes compared to the control cell lines. A value >0 indicates that MED19 knockout promotes cell proliferation, while a value <0 indicates that MED19 knockout suppresses cell proliferation. The corresponding lineages of the cancer cell lines are plotted in the y -axis. The source data are from the DepMap database. ( C, D ) Effects of MED19 knockdown and re-expression on cell proliferation determined by CCK8 assay. ( E ) The mRNA expression correlation of MED19 and c-Myc across 1749 cancer cell lines. The source data are from the DepMap database. ( F ) The protein expression correlation of MED19 and c-Myc across 225 cancer cell lines. The source data are from the DepMap database. ( G ) The mRNA expression correlation of MED19 and c-Myc across 3850 tumor samples. The source data are from the TCGA database. ( H ) The protein expression correlation of MED19 and c-Myc across 203 tumor samples. The source data are from the CPTAC database . ( I, J ) Survival analysis of MED19 in multiple cancer types. LIHC: liver hepatocellular carcinoma. ACC: adrenocortical carcinoma. ( K ) Schematic model illustrating that MED19 enhances IRES-mediated translation of c-Myc and other genes through its role in promoting rRNA 2′-O-methylation in the nucleolus.

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: Expressing, Knock-Out, Control, Knockdown, CCK-8 Assay, Methylation

Journal: Bioactive Materials

Article Title: AntagomiR-192-5p-engineered exosomes encapsulated in MXene-modified GelMA hydrogel facilitated epithelization of burn wounds by targeting OLFM4

doi: 10.1016/j.bioactmat.2025.06.013

Figure Lengend Snippet: Screening and validation of downstream target genes of miR-192-5p. A. Volcano plot of differentially expressed genes. B. Heatmap of differentially expressed genes with P < 0.05, |log 2 FC| > 1, and FPKM >1. C. Venn diagram showing the intersection of differentially expressed genes and predicted miR-192-5p target genes from the miRWalk database. D. Predicted binding sites of wild-type and mutated OLFM4 with miR-192-5p. E. Luciferase reporter assays in HEK-293 T cells after cotransfection with wild-type (WT) or mutant (MUT) CDS OLFM4 plasmids and miRNA mimics. (n = 3). F. RT-qPCR detection of OLFM4 expression changes in the HaCaTs oxidative stress model (n = 3). G-H. RT-qPCR analysis of OLFM4 mRNA expression changes in HaCaTs after overexpression and knockdown of miR-192-5p (n = 3). I-L. Immunofluorescence detection of OLFM4 protein expression changes in HaCaTs after overexpression and knockdown of miR-192-5p (scale bar: 50 μm, n = 3). M-P. Western blot analysis of OLFM4 protein expression changes in HaCaTs after overexpression and knockdown of miR-192-5p. Data are shown as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, and ns, no significance, by two-tailed unpaired Student's t -test (F-H, J, L, N and P), and by one-way ANOVA followed by the Tukey-Kramer's post hoc test (E).

Article Snippet: Similarly, The overexpression plasmids of OLFM4 (OLFM4 OE) and NC (OEC) (Shanghai Genechem Co.,Ltd.) as well as siRNA targeting OLFM4 (siOLFM4) and siNC (Shanghai Hanbio Co., Ltd.) were transfected into HaCaTs using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., USA) according to the manufacturer's instructions. showed the sequences.

Techniques: Biomarker Discovery, Binding Assay, Luciferase, Cotransfection, Mutagenesis, Quantitative RT-PCR, Expressing, Over Expression, Knockdown, Immunofluorescence, Western Blot, Two Tailed Test

Journal: Bioactive Materials

Article Title: AntagomiR-192-5p-engineered exosomes encapsulated in MXene-modified GelMA hydrogel facilitated epithelization of burn wounds by targeting OLFM4

doi: 10.1016/j.bioactmat.2025.06.013

Figure Lengend Snippet: Overexpression of OLFM4 protected HaCaTs from dysfunction induced by miR-192-5p overexpression. A-C. RT-qPCR and immunofluorescence validation of the transfection efficiency of the OLFM4 overexpression plasmid (scale bar: 50 μm, n = 3). D. CCK8 assay to assess the effect of miR-192-5p and OLFM4 overexpression on HaCaTs cell viability (n = 3). E and F. Scratch assay to evaluate the effect of miR-192-5p and OLFM4 overexpression on HaCaTs migration ability (scale bar: 500 μm, n = 3). G and H. Flow cytometry analysis of the effect of miR-192-5p and OLFM4 overexpression on HaCaTs apoptosis (n = 3). I. ROS staining fluorescence images after overexpressing miR-192-5p and OLFM4(scale bar: 50 μm). J-K. Flow cytometry assessment of ROS levels in HaCaTs after overexpressing miR-192-5p and OLFM4 (n = 3). Data are shown as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001, and ns, no significance, by two-tailed unpaired Student's t -test (A and C), and by one-way ANOVA followed by the Tukey-Kramer's post hoc test (D, F, H and K).

Article Snippet: Similarly, The overexpression plasmids of OLFM4 (OLFM4 OE) and NC (OEC) (Shanghai Genechem Co.,Ltd.) as well as siRNA targeting OLFM4 (siOLFM4) and siNC (Shanghai Hanbio Co., Ltd.) were transfected into HaCaTs using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., USA) according to the manufacturer's instructions. showed the sequences.

Techniques: Over Expression, Quantitative RT-PCR, Immunofluorescence, Biomarker Discovery, Transfection, Plasmid Preparation, CCK-8 Assay, Wound Healing Assay, Migration, Flow Cytometry, Staining, Fluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: AntagomiR-192-5p-engineered exosomes encapsulated in MXene-modified GelMA hydrogel facilitated epithelization of burn wounds by targeting OLFM4

doi: 10.1016/j.bioactmat.2025.06.013

Figure Lengend Snippet: Knockdown of OLFM4 reversed the protective effect of miR-192-5p knockdown on HaCaTs function under oxidative stress model. A-C. RT-qPCR and immunofluorescence validation of the transfection efficiency of OLFM4 small interfering RNA (siRNA) (scale bar: 50 μm, n = 3). D. CCK8 assay to assess the effect of miR-192-5p and OLFM4 knockdown on HaCaTs cell viability (n = 3). E and F. Scratch assay to evaluate the effect of miR-192-5p and OLFM4 knockdown on HaCaTs migration ability (scale bar: 500 μm, n = 3). G and H. Flow cytometry analysis of the effect of miR-192-5p and OLFM4 knockdown on HaCaTs apoptosis (n = 3). I. ROS staining fluorescence images after miR-192-5p and OLFM4 knockdown (scale bar: 50 μm). J-K. Flow cytometry assessment of ROS levels in HaCaTs after knockdown of miR-192-5p and OLFM4 (n = 3). Data are shown as the mean ± SD. ∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, and ns, no significance, by two-tailed unpaired Student's t -test (A and C), and by one-way ANOVA followed by the Tukey-Kramer's post hoc test (D, F, H and K).

Article Snippet: Similarly, The overexpression plasmids of OLFM4 (OLFM4 OE) and NC (OEC) (Shanghai Genechem Co.,Ltd.) as well as siRNA targeting OLFM4 (siOLFM4) and siNC (Shanghai Hanbio Co., Ltd.) were transfected into HaCaTs using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., USA) according to the manufacturer's instructions. showed the sequences.

Techniques: Knockdown, Quantitative RT-PCR, Immunofluorescence, Biomarker Discovery, Transfection, Small Interfering RNA, CCK-8 Assay, Wound Healing Assay, Migration, Flow Cytometry, Staining, Fluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: AntagomiR-192-5p-engineered exosomes encapsulated in MXene-modified GelMA hydrogel facilitated epithelization of burn wounds by targeting OLFM4

doi: 10.1016/j.bioactmat.2025.06.013

Figure Lengend Snippet: Mechanistic exploration of the effect of OLFM4 on HaCaTs proliferation and apoptosis. A. Volcano plot of differentially expressed genes in HaCaTs overexpressing OLFM4. B. KEGG pathway enrichment analysis. C. Violin Plot of selected genes in the "Cell growth and death" pathway. D. Heatmap of enriched genes in the "Cell growth and death" pathway. E. RT-qPCR validation of the expression changes of genes enriched in the "Cell growth and death" pathway (n = 3). Data are shown as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, by two-tailed unpaired Student's t -test (E).

Article Snippet: Similarly, The overexpression plasmids of OLFM4 (OLFM4 OE) and NC (OEC) (Shanghai Genechem Co.,Ltd.) as well as siRNA targeting OLFM4 (siOLFM4) and siNC (Shanghai Hanbio Co., Ltd.) were transfected into HaCaTs using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., USA) according to the manufacturer's instructions. showed the sequences.

Techniques: Quantitative RT-PCR, Biomarker Discovery, Expressing, Two Tailed Test

Journal: Genes & Diseases

Article Title: Dysfunction of PDE4DIP contributes to LVNC development by regulating cell polarity, skeleton, and energy metabolism via Rho-ROCK pathway

doi: 10.1016/j.gendis.2025.101568

Figure Lengend Snippet: Changes of PDE4DIP expression after differentiation of hiPSCs into hiPSC-CMs. (A) Quantitative reverse transcription PCR analysis of PDE4DIP relative mRNA expression on days 0, 5, 10, 15, 20, 25, and 30 during hiPSC-CM differentiation. (B, C) Protein expression of PDE4DIP on the 30th day of inducement, and the analysis of the PDE4DIP protein expression ( n = 3 samples per group). (D, E) Immunostaining of hiPSC-CMs for PDE4DIP (scale bar = 10 μm) among the NC-hiPSC-CMs and LVNC-hiPSC-CMs on the 30th day of inducement, and the analysis of PDE4DIP protein expression in 30th hiPS-CMs ( n = 80–120 cells per group). (F–H) The schematic diagram shows the location of PDE4DIP in the Golgi apparatus (F), the supposed 3D structure map of PDE4DIP protein (G), and the interaction of PDE4DIP with other cytoskeleton-related genes in red boxes (H). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 versus the NC group.

Article Snippet: The PDE4DIP overexpression plasmid (P-PDE4DIP) and its negative control (P-NC) were purchased from GeneChem Co., China ( ; ).

Techniques: Expressing, Reverse Transcription, Immunostaining

Journal: Genes & Diseases

Article Title: Dysfunction of PDE4DIP contributes to LVNC development by regulating cell polarity, skeleton, and energy metabolism via Rho-ROCK pathway

doi: 10.1016/j.gendis.2025.101568

Figure Lengend Snippet: P-PDE4DIP has abnormal skeleton, polarity, and mitochondria in H9C2 cells compared with the P-NC. (A) Representative transmission electron microscope images of H9C2 cells between the P-NC group and P-PDE4DIP group. Myofibrils, yellow arrow; endoplasmic reticulum, blue arrow; mitochondria, green arrow. (B) The analysis of the proportion of the vacuolated mitochondria ( n = 3 samples per group). (C, D) Representative confocal microscope images of mitochondrial morphology stained by MitoTracker-Green (scale bar = 10 μm) in H9C2 cells transfected with plasmids, and the analysis of the fluorescence intensity level ( n = 80–120 cells per group). (E) Skeletonization of the mitochondrial network from MitoTracker staining by NIS analysis software, and ATP content (F) (nmol per mg protein) in H9C2 among the P-NC and P-PDE4DIP groups ( n = 6). (G – M) Immunostaining of H9C2 transfected with plasmids for vinculin, F-actin (G–I), Par6 (J, K), and α/β-tubulin (L, M), and the analysis of the fluorescence intensity level ( n = 80–120 cells per group). (N, O) Protein expression of H9C2 transfected with plasmids of Scribble, Crb2, α/β-tubulin, and vinculin, and the analysis of the protein expression ( n = 3 samples per group). (P) Quantitative reverse transcription PCR was employed in H9C2 cells to assess the relative mRNA expression levels of cell polarity and cytoskeletal genes, including Crb2, Myh6, Par6b, α-actin4, and α-tubulin, in H9C2 cells, comparing the P-NC group with the P-PDE4DIP group. ( n = 4 samples per group). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 versus the NC group.

Article Snippet: The PDE4DIP overexpression plasmid (P-PDE4DIP) and its negative control (P-NC) were purchased from GeneChem Co., China ( ; ).

Techniques: Transmission Assay, Microscopy, Staining, Transfection, Fluorescence, Software, Immunostaining, Expressing, Reverse Transcription

Journal: Genes & Diseases

Article Title: Dysfunction of PDE4DIP contributes to LVNC development by regulating cell polarity, skeleton, and energy metabolism via Rho-ROCK pathway

doi: 10.1016/j.gendis.2025.101568

Figure Lengend Snippet: P-PDE4DIP has abnormal skeleton, polarity, and mitochondria in primary cardiomyocytes of neonatal Sprague–Dawley rats within 3 days of birth (PC), compared with the P-NC. (A) Representative transmission electron microscope images of primary cardiomyocytes between the P-NC group and P-PDE4DIP group. Myofibrils, yellow arrow; endoplasmic reticulum, blue arrow; mitochondria, green arrow. (B) The analysis of the proportion of the vacuolated mitochondria ( n = 3 samples per group). (C, D) Representative confocal microscope images of mitochondrial morphology stained by MitoTracker-Green (scale bar = 10 μm) in PC transfected with plasmids, and the analysis of the fluorescence intensity level ( n = 80–120 cells per group). (E, F) Skeletonization of the mitochondrial network from MitoTracker staining by NIS analysis software (E), and ATP content (nmol per mg protein) in PC among the P-NC and P-PDE4DIP groups ( n = 6) (F). (G – M) Immunostaining of PC transfected with plasmids of vinculin, F-actin (G–I), Par6 (J, K), and α/β-tubulin (L, M), and the analysis of the fluorescence intensity level ( n = 80–120 cells/group). (N, O) Protein expression of Scribble, vinculin, and α/β-tubulin, and the analysis of the protein expression ( n = 3 samples/group). (P) Quantitative reverse transcription PCR was employed in PC to assess the relative mRNA expression levels of cell polarity and cytoskeletal genes, including Crb2, Myh6, Par6b, α-actin1, and α-tubulin, in H9C2 cells, comparing the P-NC group with the P-PDE4DIP group ( n = 4 samples per group). ∗∗∗∗ p < 0.0001, ∗∗ p < 0.01, and ∗ p < 0.05 versus the NC group.

Article Snippet: The PDE4DIP overexpression plasmid (P-PDE4DIP) and its negative control (P-NC) were purchased from GeneChem Co., China ( ; ).

Techniques: Transmission Assay, Microscopy, Staining, Transfection, Fluorescence, Software, Immunostaining, Expressing, Reverse Transcription

Journal: Genes & Diseases

Article Title: Dysfunction of PDE4DIP contributes to LVNC development by regulating cell polarity, skeleton, and energy metabolism via Rho-ROCK pathway

doi: 10.1016/j.gendis.2025.101568

Figure Lengend Snippet: Changes in skeleton, polarity, and mitochondria after transfection of siRNA-PDE4DIP in H9C2 cells. (A) Representative transmission electron microscope images of H9C2 cells between the si-NC group and si-PDE4DIP group. Myofibrils, yellow arrow; endoplasmic reticulum, blue arrow; mitochondria, green arrow. (B) The analysis of the proportion of the vacuolated mitochondrial ( n = 3 samples per group). (C, D) Representative confocal microscope images of mitochondrial morphology stained by MitoTracker-Green (scale bar = 10 μm) in H9C2 cells transfected with siRNA, and the analysis of the fluorescence intensity level ( n = 80–120 cells per group). (E, F) Skeletonization of the mitochondrial network from MitoTracker staining by NIS analysis software (F), and ATP content (nmol per mg protein) in H9C2 among the si-NC and si-PDE4DIP groups ( n = 6) (F). (G – M) Immunostaining of H9C2 transfected with plasmids of vinculin, F-actin (G–I), Par6 (J, K), and α/β-tubulin (L, M), and the analysis of the fluorescence intensity level ( n = 80–120 cells per group). (N, O) Protein expression of PC transfected with siRNA of Scribble, Crb2, vinculin, and α/β-tubulin, and the analysis of the protein expression ( n = 3 samples per group). (P) Quantitative reverse transcription PCR was employed in H9C2 cells to assess the relative mRNA expression levels of cell polarity and cytoskeletal genes, including Crb2, Myh6, Par6b, α-actin4, and α-tubulin, comparing the si-NC group and si-PDE4DIP group ( n = 4 samples per group). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 versus the NC group.

Article Snippet: The PDE4DIP overexpression plasmid (P-PDE4DIP) and its negative control (P-NC) were purchased from GeneChem Co., China ( ; ).

Techniques: Transfection, Transmission Assay, Microscopy, Staining, Fluorescence, Software, Immunostaining, Expressing, Reverse Transcription

Journal: Genes & Diseases

Article Title: Dysfunction of PDE4DIP contributes to LVNC development by regulating cell polarity, skeleton, and energy metabolism via Rho-ROCK pathway

doi: 10.1016/j.gendis.2025.101568

Figure Lengend Snippet: Changes in skeleton, polarity, and mitochondria after transfection of siRNA-PDE4DIP in the primary cardiomyocyte (PC) of neonatal Sprague–Dawley rats within 3 days of birth. (A) Representative transmission electron microscope images of primary cardiomyocytes between the si-NC group and si-PDE4DIP group. Myofibrils, yellow arrow; Z-disks, red arrow; endoplasmic reticulum, blue arrow; mitochondria, green arrow. (B) The analysis of the proportion of the vacuolated mitochondria ( n = 3 samples per group). (C, D) Representative confocal microscope images of mitochondrial morphology stained by MitoTracker-Green (scale bar = 10 μm) in PC transfected with siRNA, and the analysis of the fluorescence intensity level ( n = 80–120 cells per group). (E, F) Skeletonization of the mitochondrial network from MitoTracker staining by NIS analysis software (E), and ATP content (nmol per mg protein) in PC among the si-NC and si-PDE4DIP groups ( n = 6) (F). (G – M) Immunostaining of PC transfected with siRNA for vinculin, F-actin (G–I), Par6 (J, K), and α/β-tubulin (L, M), and the analysis of the fluorescence intensity level ( n = 80–120 cells per group). (N, O) Protein expression of PC transfected with siRNA of Scribble and vinculin, and the analysis of the protein expression ( n = 3 samples per group). (P) Quantitative reverse transcription PCR was employed to assess the relative mRNA expression levels of cell polarity and cytoskeletal genes, including Crb2, Myh6, Par6b, α-actin1, and α-tubulin between the P-NC group and P-PDE4DIP group ( n = 4 samples per group). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 versus the NC group.

Article Snippet: The PDE4DIP overexpression plasmid (P-PDE4DIP) and its negative control (P-NC) were purchased from GeneChem Co., China ( ; ).

Techniques: Transfection, Transmission Assay, Microscopy, Staining, Fluorescence, Software, Immunostaining, Expressing, Reverse Transcription

Journal: Genes & Diseases

Article Title: Dysfunction of PDE4DIP contributes to LVNC development by regulating cell polarity, skeleton, and energy metabolism via Rho-ROCK pathway

doi: 10.1016/j.gendis.2025.101568

Figure Lengend Snippet: Changes of H9C2 cell migration and proliferation after transfection of plasmid-PDE4DIP and siRNA-PDE4DIP. (A) H9C2 cell migration in scratch wound experiment. (B) The analysis of the proportion of migration area (%) ( n = 5 samples per group). (C, D) The H9C2 cells transfected with plasmid-NC, plasmid-PDE4DIP, siRNA-NC, and siRNA-PDE4DIP were investigated by the CCK-8 assay ( n = 5). ∗∗∗∗ p < 0.0001, ∗∗ p < 0.01, and ∗ p < 0.05 versus the NC group.

Article Snippet: The PDE4DIP overexpression plasmid (P-PDE4DIP) and its negative control (P-NC) were purchased from GeneChem Co., China ( ; ).

Techniques: Migration, Transfection, Plasmid Preparation, CCK-8 Assay

Journal: Genes & Diseases

Article Title: Dysfunction of PDE4DIP contributes to LVNC development by regulating cell polarity, skeleton, and energy metabolism via Rho-ROCK pathway

doi: 10.1016/j.gendis.2025.101568

Figure Lengend Snippet: PDE4DIP plays a role in cell polarity and skeleton through the RhoA-ROCK pathway. (A – C) Protein expression in H9C2 after transfection of plasmid-PDE4DIP, including CDC42 and RhoA, analysis of the protein expression ( n = 3 samples per group), and quantitative reverse transcription PCR analysis was employed to assess the relative mRNA expression levels of RhoA-ROCK pathway genes, including CDC42, RhoA, and Rac1, between the P-NC group and P-PDE4DIP group ( n = 4 samples per group). (D – F) Protein expression in PC after transfection of plasmid-PDE4DIP, including CDC42 and RhoA, analysis of the protein expression ( n = 3 samples per group), and quantitative reverse transcription PCR analysis was employed to assess the relative mRNA expression levels of RhoA-ROCK pathway genes, including CDC42, RhoA, and Rac1, between the P-NC group and P-PDE4DIP group ( n = 4 samples per group). (G – I) Protein expression in H9C2 after transfection of siRNA-PDE4DIP, including CDC42 and RhoA, analysis of the protein expression ( n = 3 samples/group), and quantitative reverse transcription PCR analysis of was employed to assess the relative mRNA expression levels of RhoA-ROCK pathway genes, including CDC42, RhoA, and Rac1, between the si-NC group and si-PDE4DIP group ( n = 4 samples per group). (J – L) Protein expression in PC after transfection of siRNA-PDE4DIP, including CDC42 and RhoA, analysis of the protein expression ( n = 3 samples per group), and quantitative reverse transcription PCR analysis was employed to assess the relative mRNA expression levels of RhoA-ROCK pathway regenes, including CDC42, RhoA, and Rac1, between the si-NC group and si-PDE4DIP group ( n = 4 samples per group). ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 versus the NC group.

Article Snippet: The PDE4DIP overexpression plasmid (P-PDE4DIP) and its negative control (P-NC) were purchased from GeneChem Co., China ( ; ).

Techniques: Expressing, Transfection, Plasmid Preparation, Reverse Transcription

Journal: Genes & Diseases

Article Title: Dysfunction of PDE4DIP contributes to LVNC development by regulating cell polarity, skeleton, and energy metabolism via Rho-ROCK pathway

doi: 10.1016/j.gendis.2025.101568

Figure Lengend Snippet: Possible mechanism of PDE4DIP causing LVNC through Rho-ROCK pathway. (A) PDE4DIP is located in the Golgi apparatus and regulates a series of physiological activities of cells through the Rho-ROCK pathway. (B) Simplified mechanism of PDE4DIP possibly causing LVNC.

Article Snippet: The PDE4DIP overexpression plasmid (P-PDE4DIP) and its negative control (P-NC) were purchased from GeneChem Co., China ( ; ).

Techniques:

Journal: Science Advances

Article Title: Inhibition of craniosynostosis and premature suture fusion in Twist1 mutant mice with RNA nanoparticle gene therapy

doi: 10.1126/sciadv.adx9763

Figure Lengend Snippet: ( A ) Procedure for injecting the PEG-peptide plasmid DNA expressing pre-miR-200a , under the scalp of mice. microCT, micro–computed tomography. ( B ) WT mice were injected at P4 and harvested at P16, the scalp was removed, and GFP fluorescence was visualized in the metopic (M.) and coronal (C.) sutures. ( C ) WT mice were injected at P4 with either PBS (control) or plasmid miR-200a–GFP and harvested at P10, and heads were fixed and processed for 4′,6-diamidino-2-phenylindole (DAPI) and GFP staining. The left panel is PBS controls showing DAPI staining but no GFP. The right panel is plasmid miR-200a–GFP treatment. ( D ) miR-200a expression in isolated P10 coronal suture cells from WT and Twist1 +/− mice with and without miR-200a treatment. Transcripts were measured in three independent mice; fold change ( n = 3, * P < 0.05).

Article Snippet: PEI transfections were used to transfect BMSCs with plasmid DNA overexpressing miR-200a or EV, and RNA was isolated from the cells using TRIzol reagent (Thermo Fisher Scientific) and subjected to quantitative polymerase chain reaction primers for BGLAP , TGFB1 , TGFBR1 , and Wnt5a .

Techniques: Plasmid Preparation, Expressing, Micro-CT, Injection, Fluorescence, Control, Staining, Isolation

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: A Schema of BMI1-induced SREs (iSRE) establishment and culture from PBMCs. B Lentiviral transduction leads to an approximately four-fold increase in BMI1 transcript levels in iSREs compared to compared to untransduced (UT) SREs and empty vector transduced (EV) SREs at day 13 of expansion, which have, as expected, similar levels of BMI1. 4 independent iSRE cultures derived from 3 donors, mean ± SEM. p -value was calculated based on paired two-tailed Student's t -test. ***p < 0.0001. Source data are provided as a Source Data file. C Lentiviral transduction leads to an approximately four-fold increase in BMI1 protein levels compared to compared to UT and EV SREs at day 13 of expansion. 4 independent iSRE cultures derived from 3 donors, mean ± SEM. p -value was calculated based on paired two-tailed Student's t -test **p < 0.01. Source data are provided as a Source Data file. D iSREs have approximately a 10 4 -fold expansion, compared to untransduced (UT) SREs and empty vector transduced (EV) SREs, which have similar limited (200-300-fold) expansion. 4 independent iSRE cultures derived from 3 donors, mean ± SEM. E FACS-based analysis of iSRE cultures reveals 3 subpopulations of cells. Population 1 is CD36+CD235 lo and consist of cells with an immature erythroblast morphology. Population 2 is CD36 + CD235+ and consists of cells with a slightly more mature morphology. Population 3 is CD36 lo CD235+ and consist of smaller hemoglobinizing cells with condensed nuclei. Cells are colored based on GFP expression, which is highest in Population 1 and lowest in Population 3. Size bar = 10um. A representative example of three independent experiments from three donors is shown. F Enrichment of immature erythroblasts by weekly fluorescence-activated cell sorting (FACS) leads to a 10 12 -fold expansion of iSREs, compared to untransduced (UT) and empty vector transduced (EV) control SREs. 3 independent iSRE cultures derived from 3 donors.

Article Snippet: The pReceiver-Lv165 human BMI1 overexpression plasmid (EX-B0015-Lv165, GeneCopoeia) and the pReceiver-Lv165 empty vector plasmid (EX- NEG-Lv165, GeneCopoeia) contain an EEF1A1 promoter with an IRES2 followed by an eGFP fluorescent marker.

Techniques: Transduction, Plasmid Preparation, Derivative Assay, Two Tailed Test, Expressing, Fluorescence, FACS, Control

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: A Morphology of self-renewing iSREs at expansion day 34. Size bar = 10 um. Representative example of 6 independent cultures derived from 3 donors. B Surface expression of CD71, CD44, CD49d, and CD34 on iSREs at expansion day 30. Representative plots from one of three independent iSRE cultures. C Consistency of cell and nuclear area of iSREs on days 15, 30, and 55 of culture, as analyzed by imaging flow cytometry. Data from 3 independent cultures derived from 3 donors, mean ± SEM. D Karyotype of iSREs at day 45 of culture. Data from 1 of 3 independent cultures are shown (see Supplementary Fig. for additional two donors). E Principal Component Analysis (PCA) of global transcriptomes of CD34-derived erythroid populations (GEO GSE53983 and GSE128268 ) , and of iSREs (Supplementary Fig. ) indicate that iSREs are closest in gene expression identity to proerythroblasts (ProE). HSPC = hematopoietic stem and progenitor cells, BFU-E = burst forming units erythroid, CFU-E = colony forming units erythroid, EBasoE = early basophilic erythroblasts, LBasoE = late basophilic erythroblasts, PolyE = polychromatophilic erythroblasts, and OrthoE = orthochromatic erythroblasts. F BMI1 and EEF1A1 transcript levels during the differentiation trajectory of CD34+ HSPCs to OrthoE (GEO GSE53983 and GSE128268 ) , .

Article Snippet: The pReceiver-Lv165 human BMI1 overexpression plasmid (EX-B0015-Lv165, GeneCopoeia) and the pReceiver-Lv165 empty vector plasmid (EX- NEG-Lv165, GeneCopoeia) contain an EEF1A1 promoter with an IRES2 followed by an eGFP fluorescent marker.

Techniques: Derivative Assay, Expressing, Imaging, Flow Cytometry, Gene Expression

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: A Morphology of terminally maturing iSREs after 8 days of maturation culture. Representative example of 6 independent cultures derived from 3 donors. B Flow-based assay of enucleation of iSREs after 8 days of maturation. Representative example of 4 independent iSRE cultures derived from 3 donors. Percent gated is the mean ± SEM of the replicates. C Globin gene expression of iSREs matured for 3 days and quantified by qPCR. 4 independent iSRE cultures derived from 3 donors, mean ± SEM. Source data are provided as a Source Data file. D BMI1 transcript levels rapidly decrease during in vitro maturation of iSREs. 4 independent iSRE cultures derived from 3 donors. Paired two-tailed Student's t -test * p = 0.027. Source data are provided as a Source Data file. E Flow-based surface expression of CD235a, Band3, Kell, Glycophorin C (GPC), and Rh on day 8 matured iSREs compared to FMO (fluorescence minus one) controls. F Gel column assay of 1.5 million terminally maturing iSREs after 7 days of maturation culture . Absence of antibody served as a negative control. G Gel column assay of expression of Rh antigens on D + C + c + E-e+ terminally maturing iSREs. H Gel column assay of Glycophorin B (S+, s-), Kell (K-, k+), and Duffy Fy(a-b+) blood groups on terminally maturing iSREs. I Gel column assay of type A terminally maturing iSREs using plasma from a type O and a type A donor. J Ficin-treated terminally maturing iSREs agglutinated with plasma from type A patients containing anti-D, anti-C, or anti-e, respectively.

Article Snippet: The pReceiver-Lv165 human BMI1 overexpression plasmid (EX-B0015-Lv165, GeneCopoeia) and the pReceiver-Lv165 empty vector plasmid (EX- NEG-Lv165, GeneCopoeia) contain an EEF1A1 promoter with an IRES2 followed by an eGFP fluorescent marker.

Techniques: Derivative Assay, Gene Expression, In Vitro, Two Tailed Test, Expressing, Fluorescence, Negative Control, Clinical Proteomics

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: A shRNA knockdown of BMI1 decreases BMI1 transcript levels compared to control luciferase shRNA culture. 3 independent iSRE cultures derived from 3 donors, mean ± SEM. p -value was calculated based on one-tailed Student's t -test. ** p < 0.01. B shRNA knockdown of BMI1 leads to rapid collapse of iSRE cultures. Representative example shown, 3 independent iSRE cultures derived from 3 donors. C PTC-209 inhibition of BMI1 for 24 hours decreases BMI1 transcript levels compared to vehicle-treated controls. 3 independent iSRE cultures derived from 3 donors, mean ± SEM. p -value was calculated based on a one-tailed Student's t -test. ** p = 0.010. *** p = 7.8e-6. D PTC-209 treatment leads to a dose-dependent inhibition of iSRE self-renewal. Representative example shown of 3 independent iSRE cultures, derived from 3 donors. E iSRE self-renewal remains dependent on the continued presence of exogenous erythropoietin (EPO), stem cell factor (SCF), and Dexamethasone (Dex). 6 independent iSRE cultures from 3 donors, mean ± SEM. All experiments were conducted with iSREs between expansion days 25 and 35. Source data are provided as a Source Data file.

Article Snippet: The pReceiver-Lv165 human BMI1 overexpression plasmid (EX-B0015-Lv165, GeneCopoeia) and the pReceiver-Lv165 empty vector plasmid (EX- NEG-Lv165, GeneCopoeia) contain an EEF1A1 promoter with an IRES2 followed by an eGFP fluorescent marker.

Techniques: shRNA, Knockdown, Control, Luciferase, Derivative Assay, One-tailed Test, Inhibition

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: See Supplementary Fig. for samples used in CUT& RUN. A shRNA knockdown of RING1B leads to rapid collapse of iSRE cultures. p -value was calculated based on two-way ANOVA of all RING1B shRNA vs Luciferase. 2 independent iSRE cultures from 1 donor, mean ± range, expansion day 30. B Heatmaps of BMI1 enrichment in untransduced (UT) SREs and iSREs over union peaks (±2 kb). The color scale represents the RPKM of each sample using merged replicates. Ranked based on mean RPKMs. C Volcano plot showing differential occupancy of BMI1 in iSRE versus untransduced SREs. Significantly increased and decreased peaks are shown in red and blue, respectively, with FDR < 0.05. D Heatmaps of BMI1 occupancy, RING1B occupancy, H2A119ub, and H3K27me3 over BMI1 differentially increased (top) and decreased (bottom) peaks in untransduced SREs and in iSREs. The color scale represents RPKM of each sample using merged replicates. Ranking was sorted based on BMI1 binding in iSREs. Blue line- increased BMI1 peaks. Orange line- decreased BMI1 peaks. E Heatmaps of BMI1, RING1B, H2A119ub, and H3K27me3 over 2 clusters of BMI1 differentially increased peaks (kmeans=2, clustering based on H3K27me3 in iSREs) in untransduced SREs and in iSREs. The color scale represents RPKM of each sample using merged replicates. Ranking was based on BMI1 binding in iSREs. Blue line- Cluster 1. Orange line- Cluster 2. F Table of selected published gene sets that contain significant overlap with CUT&RUN gene sets (Enrichr). See Supplementary Data for details.

Article Snippet: The pReceiver-Lv165 human BMI1 overexpression plasmid (EX-B0015-Lv165, GeneCopoeia) and the pReceiver-Lv165 empty vector plasmid (EX- NEG-Lv165, GeneCopoeia) contain an EEF1A1 promoter with an IRES2 followed by an eGFP fluorescent marker.

Techniques: shRNA, Knockdown, Luciferase, Binding Assay

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: See Supplementary Fig. for samples used in CUT&RUN and Supplementary Fig. for samples used in RNA-Seq. A Genomic annotations of BMI1 differentially increased peaks (left) and decreased peaks (right). B Heatmaps of BMI1, RING1B, H2A119ub, and H3K27me3 over genes (scaled TSS to TES ± 2 kb) associated with iSRE BMI1 peaks in untransduced SREs and iSREs. The color scale represents RPKM of each sample using merged replicates ranked based on the binding of BMI1 in iSREs. C Heatmaps of BMI1, RING1B, H2A119ub, and H3K27me3 over promoters (TSS ± 2 kb) of differentially expressed, upregulated (top) and downregulated (bottom), genes in untransduced SREs and in iSREs as determined by DeSeq2 analysis of RNA-Seq. The color scale represents RPKM of each sample using merged replicates ranked based on BMI1 binding in iSREs. Blue line- upregulated genes. Orange line- downregulated genes. D Quantitation of log2-fold change (iSRE/SRE) for BMI1, RING1B, H2A119ub, and H3K27me3 over promoters (TSS ± 500 bp) of the same differentially expressed genes shown in Fig. 6C. Box is Q1-Q3 with bar at the median and whiskers represent Q1-1.5x IQR and Q3 + 1.5x IQR. Statistics for log2 -old change is not equal to 0 based on a two-tailed Wilcoxon signed rank test with continuity correction. **** p < 2.2e−16, ns not significant. E Table of published gene sets with significant overlap with differentially upregulated and downregulated genes that have BMI1 associated with their promoters indicated on the left (Enrichr). See Supplementary Data for details.

Article Snippet: The pReceiver-Lv165 human BMI1 overexpression plasmid (EX-B0015-Lv165, GeneCopoeia) and the pReceiver-Lv165 empty vector plasmid (EX- NEG-Lv165, GeneCopoeia) contain an EEF1A1 promoter with an IRES2 followed by an eGFP fluorescent marker.

Techniques: RNA Sequencing, Binding Assay, Quantitation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: A Occupancy of indicated factors and presence of CpG islands (purple) in SRE (black) and iSRE (green) at the INK/ARF locus. Data represent merged replicates for all CUT&RUN studies. B The INK/ARF genes are expressed at lower levels in iSREs compared to SREs at day 13 of expansion. 6 independent cultures derived from 3 donors. C Relative expression of INK/ARF genes in day 25-35 iSRE following 24 hours of PTC-209 treatment relative to DMSO (Veh) control. N = 3 for p16. N = 5 for CDKN1B and CDKN2B, each derived from 2 donors. D Expression of BMI1 and p16 relative to luciferase (Luc) at 24 hours after siRNA treatment of expansion day 41-46 CD34-derived iSREs. N = 7 for Luc and BMI1-1 and N = 4 for BMI1-2, each derived from 2 donors. N = 3 for p16 with iSREs derived from 2 donors. E PTC-209 for 12 hours decreases the percentage of iSREs in S-phase in a dose-dependent manner compared to vehicle (Veh)-treated controls. 4 independent iSRE cultures expanded for 25-35 days, derived from 2 donors. F PTC-209 treatment of iSREs for 48 hours leads to dose-dependent increases of G0 cells. 4 independent iSRE cultures expanded for 25-35 days, derived from 2 donors. G PTC-209 treatment of iSRE for 12 hours leads to dose-dependent increases in early apoptotic cells. 4 independent iSRE cultures expanded for 25-35 days, derived from 2 donors. H Venn Diagram showing overlap in BMI1 occupied ribosomal protein genes (yellow) and ribosomal protein genes differentially upregulated in iSREs vs. SREs (blue). I Occupancy of indicated factors in SRE (black) and iSRE (green), and CpG islands (purple) at the Ribosomal Protein S19 ( RPS19 ) locus. Data represent merged replicates for all CUT&RUN studies. J Occupancy of indicated factors in SRE (black) and iSRE (green) and compared with RNA-seq reads at the BMI1 locus. Boxed region is not present in the viral overexpression construct and thus represents endogenous gene expression. All graphs plot mean ± SEM. p values were calculated using a two-tailed Student's t -test. * p < 0.05 ** p < 0.01 *** p < 0.001. Source data are provided for as a Source Data file.

Article Snippet: The pReceiver-Lv165 human BMI1 overexpression plasmid (EX-B0015-Lv165, GeneCopoeia) and the pReceiver-Lv165 empty vector plasmid (EX- NEG-Lv165, GeneCopoeia) contain an EEF1A1 promoter with an IRES2 followed by an eGFP fluorescent marker.

Techniques: Derivative Assay, Expressing, Control, Luciferase, RNA Sequencing, Over Expression, Construct, Gene Expression, Two Tailed Test

Journal: Nature Communications

Article Title: BMI1 regulates human erythroid self-renewal through both gene repression and gene activation

doi: 10.1038/s41467-025-62993-3

Figure Lengend Snippet: A Cholesterol homeostasis depends on cholesterol synthesis, import, export and storage. B CUT&RUN studies reveal BMI1 and RING1B bind the HMGCR locus at higher levels in iSRE than SRE. Data represent merged replicates for all CUT&RUN studies. C The BMI1 inhibitor PTC-209 decreases the expression of BMI1, HMGCR, and HMGCS1 in iSREs at 25 to 35 days of expansion. In contrast, PTC-209 treatment increases expression of p16. 6 independent iSRE cultures expanded for 25 to 35 day and derived from 2 donors, mean. ± SEM. p -value was calculated using a two-tailed Student's t -test. * p < 0.05, ** p < 0.01. Source data are provided as a Source Data file. D iSREs contain cytoplasmic droplets that stain with filipin (cholesterol). Representative example of one of two cultures shown. E iSREs contain cytoplasmic droplets that stain with Nile red (lipids). Representative example of one of two cultures shown. F iSREs contain higher levels of cholesterol (filipin stain) compared to primary human proerythroblast (ProE). Staining of both cell populations normalized to primary orthochromatic erythroblasts (OrthoE). Human marrow samples derived from 3 donors. 4 independent iSRE cultures expanded for 25 to 35 day and derived from 2 donors, mean ± SEM. Two-tailed Student's t -test. ** p < 0.01. Source data are provided as a Source Data file. G iSREs contain higher levels of lipid rafts (CT-B staining) compared to primary human ProE. Staining of both cell populations normalized to primary OrthoE. Human marrow samples derived from 3 donors. 2 independent iSRE cultures expanded for 25 to 35 day and derived from 2 donors. Paired two-tailed Student's t -test. * p < 0.05 mean ± range. Source data are provided as a Source Data file. H While short-term removal of exogenous lipids does not alter iSRE proliferation, concomitant block of cholesterol synthesis with Ro 48 obviates iSRE proliferation, which can be partially rescued by addition of exogenous lipids. 3 independent iSRE cultures expanded for 25 to 35 day and derived from 2 donors. p -value was calculated based on two-tailed Student's t -test, mean ± SEM. ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: The pReceiver-Lv165 human BMI1 overexpression plasmid (EX-B0015-Lv165, GeneCopoeia) and the pReceiver-Lv165 empty vector plasmid (EX- NEG-Lv165, GeneCopoeia) contain an EEF1A1 promoter with an IRES2 followed by an eGFP fluorescent marker.

Techniques: Expressing, Derivative Assay, Two Tailed Test, Staining, Blocking Assay