en2  (Cusabio)

Journal: bioRxiv

Article Title: Lcn2 deficiency leads to social impairments independent of maternal immune activation

doi: 10.1101/2025.06.27.661499

Figure Lengend Snippet: A. Experimental timeline. Pregnant heterozygous mice were injected with LPS or saline at E16, E17, and E18. 4 hours after the last injection, the fetal forebrain was dissected for RNA-seq analysis. B, C. A volcano plot illustrating DEGs in the forebrain of E18. The red dots represent significantly upregulated genes, the blue dots represent significantly downregulated genes (|log2 FC| ≥ 1 and FDR < 0.05), and the black dots represent insignificant differentially expressed genes. (B) DEGs in the forebrain of Lcn2 KO animals at E18 (C) DEGs in the forebrain of WT MIA at E18, 4 hours after the last LPS injection. D, E. Gene ontology enrichment analysis using Metascape based on the genes deregulated (both up- and downregulated). A bar graph showing the top 5 clusters with the highest p-value. E. Heatmap of enriched terms across input differentially expressed gene lists. F. A Venn diagram of unique and overlapping gene expression between Lcn2 KO and MIA groups. G. The heat map of overlapping genes between Lcn2 KO and MIA. H, I. RT-qPCR confirmation of the results obtained with RNAseq analysis for Pax2 (H) and En2 (I) genes. mRNA expression levels in the forebrain were measured 4 hours after the final injection of LPS or saline in pregnant dams. (H) Two-way ANOVA: procedure: F (1, 33) = 4.616; p = 0.0391, genotype: F (1, 33) = 8.232; p = 0.0071, and interaction: F (1, 33) = 1.933; p = 0.1737, followed by Fisher’s LSD test. WT ctrl n = 7, WT MIA n = 11; Lcn2 KO n = 8, Lcn2 KO MIA n = 11. (I) Two-way ANOVA: procedure: F (1, 34) = 11.60; p = 0.0017, genotype: F (1, 34) = 4.019; p = 0.0530, and interaction: F (1, 34) = 7.669, p = 0.0090, followed by Fisher’s LSD post hoc test. WT ctrl n = 8, WT MIA n = 10; Lcn2 KO n = 9, Lcn2 KO MIA n = 11. Data are presented as mean ± SEM.

Article Snippet: RNA was reverse transcribed with SuperScript™ IV VILO (# 11766050, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. cDNA was amplified with TaqMan probes (Lcn2 Mm01324470_m1, Gapdh Mm99999915_g1, En2 Mm00438710_m1, Pax2: Mm01217939_m1, ThermoFisher, Waltham, MA, USA) that were specific for the mouse.

Techniques: Injection, Saline, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Expressing

Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: Heatmap ( a ) and volcano plot ( b ) displayed the expression profile of DEGs following LINC00973 knockdown or control cells. The mRNA ( c ) and protein ( d ) expression of EN2 were measured in Cal27 and HN6 cells with/without EN2 knockdown. e , f The correlations between EN2 and LINC00973 RNA expression were assessed in the public HNSCC datasets ( GSE41613 and GSE42743 ) and our in-house samples. Spearman’s correlation. The mRNA ( g ) and protein ( h ) abundance of EN2 were measured in Cal27 and HN6 cells under four conditions. Cell proliferation, migration and invasion were significantly reduced following LINC00973 knockdown but restored by ectopic EN2 overexpression as gauged by CCK-8 ( i ), wound healing ( j ) and Transwell invasion assays ( k ). Scale bar: 50 μm. l The migration/invasion-related markers were determined by western blot in Cal27 and HN6 cells with/without LINC00973 knockdown coupled with/without EN2 overexpression. m 1 × 10 5 HN6 cells with LINC00973 or/and EN2 manipulations were injected submucosally into the floor of the mouth. Fourteen days post-injection, tumor masses and cervical lymph nodes were harvested. Representative tumor masses images and estimated tumor volumes were showed. Scale bar: 1 cm. Representative IHC staining of Ki67 and EN2 in xenograft tumors ( n ) and their quantification data were shown ( o ). Scale bar: 50 μm. p Representative IHC staining of CK5/6 in lymph nodes (left panel) from orthotopic tumor-bearing mice among four indicated groups were displayed. The percentage of lymph nodes metastasis were shown and compared (right panel). Fisher’s exact test. Data were presented as mean ± SD, ** P < 0.01, Student’s t test.

Article Snippet: Next, membranes were blocked with 5% non-fat dry milk and incubated overnight at 4 °C with the indicated primary antibodies: EN2 (1:500, CSB-PA007660LA01HU, Cusabio, China), N-cadherin (1:2000, #22018-1-AP, Proteintech, China), E-cadherin (1:20000, #20874-1-AP, Proteintech, China), Vimentin (1:20000, #10366-1-AP, Proteintech, China), GAPDH (1:50000, #60004-1-Ig, Proteintech, China), FOSL1 (1:1000, #5281, Cell signaling, USA), NOTCH1 (1:1000, #20687-1-AP, Proteintech, China), BRD4 (1:1000, #13440, Cell signaling, USA), HEY1 (1:1000, #19929-1-AP, Proteintech, China), N1ICD (1:1000, #4147, Cell signaling, USA) or Rpb1 (1:1000, #14958, Cell signaling, USA).

Techniques: Expressing, Knockdown, Control, RNA Expression, Migration, Over Expression, CCK-8 Assay, Western Blot, Injection, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: a , b The half-life of EN2 mRNA were determined by qRT-PCR in LINC00973 knockdown or overexpressed Cal27 and HN6 cells with time-course Actinomycin D exposure. c Experimental design of AGO2-RIP was depicted by schematic diagram (left panel). Endogenous LINC00973 was assessed by qRT-PCR in cell lyses immunoprecipitated with either AGO2 antibody or mouse IgG (right panel). d , e The putative miRNA candidates with high EN2 mRNA binding affinity were computed by TargetScan webtool ( https://www.targetscan.org/vert_80/ ), while LINC00973 sponged miRNAs (top10) were predicted by DIANA tool kits ( http://diana.imis.athena-innovation.gr/DianaTools/index.php ). Six overlapped miRNA candidates were identified ( d ). Only miR-6756-3p expression were shown with significant upregulation followed by LINC00973 silencing in both Cal27 and HN6 cells ( e ). f The expression of miR-6756-3p in 50 HNSCC clinical samples and paired normal counterparts were determined by qRT-PCR. Paired Wilcoxon rank-sum test. g Kaplan-Meier analyses of OS and DSS stratified by median miR-6756-3p expression in 50 in-house samples Log-rank test. h , i Schematic diagram depicted the experimental design of MS2-RIP assays to detect the direct binding between LINC00973 and miR-6756-3p in HNSCC ( h ). The LINC00973-MS2-GFP complex was pulled down by anti-GFP antibody and the interacted miR-6756-3p were assessed by qRT-PCR in Cal27, HN6, and HEK293T cells ( i ). j The sequence alignment displayed the binding sequence between miR-6756-3p and LINC00973 [LINC00973 (WT)] or 3’UTR of EN2 [3’UTR EN2 (WT)]. The miR-6756-3p binding site mutated LINC00973 and EN2 transcripts [LINC00973 (MT) or 3’UTR EN2 (MT)] were showed below. k , l Cal27 and HN6 cells transfected with LINC00973/3’UTR EN2 wild type or miR-6756-3p binding site mutated pmirGLO luciferase reporter vectors and treated with miR-6756-3p or control mimics. The luciferase activated was measured. m The expression levels of EN2 were measured by qRT-PCR in Cal27 and HN6 treated with sh-LINC00973-1 lentivirus or in combination with miR-6756-3p inhibitor (inhibitor). n The correlations between EN2, LINC00973, and miR-6756-3p expression in HNSCC clinical samples were estimated by using Spearman’s correlation. o Schematic illustration of the regulatory model of LINC00973 decoys miR-6756-3p to stabilize EN2 mRNA. Data were presented as mean ± SD, # P ≥ 0.05, * P < 0.05, ** P < 0.01, Student’s t test.

Article Snippet: Next, membranes were blocked with 5% non-fat dry milk and incubated overnight at 4 °C with the indicated primary antibodies: EN2 (1:500, CSB-PA007660LA01HU, Cusabio, China), N-cadherin (1:2000, #22018-1-AP, Proteintech, China), E-cadherin (1:20000, #20874-1-AP, Proteintech, China), Vimentin (1:20000, #10366-1-AP, Proteintech, China), GAPDH (1:50000, #60004-1-Ig, Proteintech, China), FOSL1 (1:1000, #5281, Cell signaling, USA), NOTCH1 (1:1000, #20687-1-AP, Proteintech, China), BRD4 (1:1000, #13440, Cell signaling, USA), HEY1 (1:1000, #19929-1-AP, Proteintech, China), N1ICD (1:1000, #4147, Cell signaling, USA) or Rpb1 (1:1000, #14958, Cell signaling, USA).

Techniques: Quantitative RT-PCR, Knockdown, Immunoprecipitation, Binding Assay, Expressing, Sequencing, Transfection, Luciferase, Control

Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: a SEs were calculated and ranked by H3K27ac signal using ROSE algorithm. And a proximal SE within LINC00973 gene locus were identified in 3 HNSCC cell lines (Cal27, HN12, and HSC3). b Genomic tracks plot displayed the H3K27ac enrichment (including 6 cell lines, 2 primary HNSCC and 1 human normal oral mucosa (NOM) tissues), BRD4, and EP300 binding and chromatin accessibility at the LINC00973-SE region. Four individual enhancer fragments within LINC00973-SE were observed and referred as E1-E4. c The H3K27ac, BRD4 and EP300 enrichment on E1-E4 or E-NC were measured by ChIP-qPCR in Cal27 and HN6 cells. d , e The expression changes of LINC00973 were measured by qRT-PCR and the binding changes of H3K27ac, BRD4, and EP300 on E1-E4 were determined by ChIP-qPCR in Cal27 and HN6 cells treated with JQ1 (1 μM, 12 h) or NEO2734 (1 μM, 12 h). f Representative FISH staining of LINC00973 and IHC staining of EN2 in PDX samples treated with vehicle, JQ1 or NEO2734. g Schematic diagram showing the procedure of E1-E4 repression by dCas9-KRAB-MeCP2 system. h LINC00973 expression in Cal27-dCas9 and HN6-dCas9 cells with repressed LINC00973 enhancer activity were assessed by qRT-PCR. Cell proliferation, migration and invasion were significantly suppressed following sgE2 treatment as gauged by CCK-8 ( i ), wound healing ( j ) and Transwell invasion assays ( k ). Scale bar: 50 μm. l De novo motif discovery at LINC00973-SE regions via HOMER algorithm. m LINC00973 expression was detected by qRT-PCR in Cal27 and HN6 cell with/without FOSL1 silencing. n , o co-IP assays revealed the endogenous FOSL1 protein interacted with Rpb1 (RNA pol II subunit) and BRD4 in both Cal27 and HN6 cells ( i ), while FOSL1-depletion disrupted these interactions ( j ). p The FOSL1 binding enrichments at E2 region in SCC1 with/without FOSL1 knockdown were displayed by track plot. q FOSL1 binding on E1-E4 or E-NC regions in Cal27 and HN6 transfected with si-FOSL1 were measured by ChIP-qPCR. r The core DNA sequences of E2 or E-NC were subcloned into pGL3 vectors and the luciferase activities were detected in Cal27 and HN6 cells transfected with si-FOSL1 or control siRNAs. s Schematic model of LINC00973 transcription regulation: cis -activation by LINC00973-SE and trans -regulation by AP-1/FOSL1. Data were presented as mean ± SD, # P ≥ 0.05,* P < 0.05, ** P < 0.01, Student’s t test.

Article Snippet: Next, membranes were blocked with 5% non-fat dry milk and incubated overnight at 4 °C with the indicated primary antibodies: EN2 (1:500, CSB-PA007660LA01HU, Cusabio, China), N-cadherin (1:2000, #22018-1-AP, Proteintech, China), E-cadherin (1:20000, #20874-1-AP, Proteintech, China), Vimentin (1:20000, #10366-1-AP, Proteintech, China), GAPDH (1:50000, #60004-1-Ig, Proteintech, China), FOSL1 (1:1000, #5281, Cell signaling, USA), NOTCH1 (1:1000, #20687-1-AP, Proteintech, China), BRD4 (1:1000, #13440, Cell signaling, USA), HEY1 (1:1000, #19929-1-AP, Proteintech, China), N1ICD (1:1000, #4147, Cell signaling, USA) or Rpb1 (1:1000, #14958, Cell signaling, USA).

Techniques: Binding Assay, ChIP-qPCR, Expressing, Quantitative RT-PCR, Staining, Immunohistochemistry, Activity Assay, Migration, CCK-8 Assay, Co-Immunoprecipitation Assay, Knockdown, Transfection, Luciferase, Control, Activation Assay

Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: a Schematic diagram outlining the experimental design to identify and characterize downstream signaling pathways modulated by EN2 in HNSCC. b Differential analyses of KEGG pathways were performed in Cal27 and HN6 cells with/without EN2 silencing and the results were represented as scatter plot (left panel) and heatmap (right panel). KEGG pathway scores were calculated by GSVA method. c Correlation analyses between LINC00973 expression and KEGG_NOTCH_SIGNALING_PATHWAY scores (GSVA method) in TCGA-HNSC, GSE186775 and GSE65858 datasets. d The transcription activity of NOTCH1, the core member of NOTCH pathway, was measured by NOTCH1 luciferase reporter assays in Cal27 and HN6 with EN2 manipulations. e The protein abundance of NOTCH1, N1ICD and HEY1 were determined by western blot in Cal27 and HN6 cells with EN2 knockdown. f The endogenous NOTCH1, N1ICD and HEY1 protein expression was detected in Cal27 and HN6 cells followed by LINC00973 siRNAs or HN30 cells with LINC00973 overexpression, respectively. g NOTCH1, N1ICD and HEY1 were determined by western blot in Cal27 and HN6 cells with/without LINC00973 knockdown coupled with/withoutEN2 overexpression. Cell proliferation, migration and invasion were significantly increased following LINC00973 overexpression but reduced by DAPT treatment as gauged by CCK-8 ( h ), wound healing ( i ) and Transwell invasion assays ( j ).Scale bar: 50 μm. k Representative H&E, IHC staining of FOSL1, EN2, and NOTCH1 as well as FISH staining of LINC00973, in serial sections of the same HNSCC clinical samples were shown. Scale bar: 50 μm. l The correlation among FOSL1, EN2 and NOTCH1 protein expression (IHC quantifications data) and LINC00973 RNA abundance (FISH signal intensity) were analyzed by using Spearman’s correlation. Data were presented as mean ± SD, ** P < 0.01, Student’s t test.

Article Snippet: Next, membranes were blocked with 5% non-fat dry milk and incubated overnight at 4 °C with the indicated primary antibodies: EN2 (1:500, CSB-PA007660LA01HU, Cusabio, China), N-cadherin (1:2000, #22018-1-AP, Proteintech, China), E-cadherin (1:20000, #20874-1-AP, Proteintech, China), Vimentin (1:20000, #10366-1-AP, Proteintech, China), GAPDH (1:50000, #60004-1-Ig, Proteintech, China), FOSL1 (1:1000, #5281, Cell signaling, USA), NOTCH1 (1:1000, #20687-1-AP, Proteintech, China), BRD4 (1:1000, #13440, Cell signaling, USA), HEY1 (1:1000, #19929-1-AP, Proteintech, China), N1ICD (1:1000, #4147, Cell signaling, USA) or Rpb1 (1:1000, #14958, Cell signaling, USA).

Techniques: Protein-Protein interactions, Expressing, Activity Assay, Luciferase, Quantitative Proteomics, Western Blot, Knockdown, Over Expression, Migration, CCK-8 Assay, Immunohistochemistry, Staining

Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: The proximal SE in LINC00973 locus cis -activates LINC00973 transcription via recruiting AP-1/FOSL1, BRD4 and EP300; the cytoplasmically localized LINC00973 sponges miR-6756-3p to increase the mRNA half-life of EN2; EN2 activates downstream NOTCH signaling to modulate the aggressiveness of HNSCC.

Article Snippet: Next, membranes were blocked with 5% non-fat dry milk and incubated overnight at 4 °C with the indicated primary antibodies: EN2 (1:500, CSB-PA007660LA01HU, Cusabio, China), N-cadherin (1:2000, #22018-1-AP, Proteintech, China), E-cadherin (1:20000, #20874-1-AP, Proteintech, China), Vimentin (1:20000, #10366-1-AP, Proteintech, China), GAPDH (1:50000, #60004-1-Ig, Proteintech, China), FOSL1 (1:1000, #5281, Cell signaling, USA), NOTCH1 (1:1000, #20687-1-AP, Proteintech, China), BRD4 (1:1000, #13440, Cell signaling, USA), HEY1 (1:1000, #19929-1-AP, Proteintech, China), N1ICD (1:1000, #4147, Cell signaling, USA) or Rpb1 (1:1000, #14958, Cell signaling, USA).

Techniques: