Journal: bioRxiv

Article Title: In Vivo Multiplexed Modeling Reveals Diverse Roles of the TBX2 Subfamily and Egr1 in Ras -Driven Lung Adenocarcinoma

doi: 10.1101/2025.03.15.642187

Figure Lengend Snippet: A) Schematic representation of the molecular basis of TBX2-associated signaling pathways in cancer. TBX2 , TBX3 , and TBX5 contribute to the dysregulation of multiple cancer hallmarks, including cell proliferation, senescence, apoptosis, invasion, metastasis, and the pro-inflammatory response. EGR1 , TNFaip3 , ATF3 , Tbx4, and Chd2 are all modulated by TBX2 signaling in lung cancer cell lines and recurrently dysregulated in human lung cancers. While EGR1 , TNFAIP3, and ATF3 all directly modulate key carcinogenic pathways, the mechanisms by which TBX4 and CHD2 influence cancer remain largely unexplored( , , – ). AKT (AKT serine/threonine kinase), BAX (BCL2 associated X, apoptosis regulator), HDAC (Histone deacetylase), MDM2 (MDM2 proto-oncogene, E3 ubiquitin protein ligase), MMP9 (Matrix metallopeptidase 9), NDRG1 (N-myc downstream-regulated gene 1), p14ARF (Cyclin-dependent kinase inhibitor 2A, isoform p14ARF), p 21CIP1 (Cyclin-dependent kinase inhibitor 1A), p53 (Tumor protein p53), PAsrp (Phosphatidylserine receptor protein), PRC2 (Polycomb repressive complex 2), SNCG (Synuclein gamma), and TNFa (Tumor necrosis factor alpha). B) Experimental schematic of the Tuba-seq approach used to investigate combinatorial inactivation of potential LUAD regulators in vivo. Tumor initiation was achieved through intratracheal intubation with Lenti-sgTS-Pool/Cre in Kras LSL-G12D ,Rosa26 CAG-LSL-Cas9-GFP mice. The Lenti-sgTS-Pool contained two inert sgRNA vectors along with a positive control (sg Rb1 ) and a negative control (sg Pcna ). Each of the eight genes studied was targeted using two distinct sgRNAs. Each sgRNA vector included a unique sgID and a random barcode, allowing quantification of individual tumor sizes via deep sequencing. The genotype, time points and lentiviral titers are as indicated. C) Representative images of lung lobes from mice at 6- and 20-weeks post-tumor initiation. Images include fluorescence dissecting scope views, H&E-stained sections, and TTF1-stained (lung epithelial marker) sections of lung lobes. Scale bars are indicated on each image. D) Quantification of the percent tumor area in representative mice revealed a significant increase at 20 weeks compared to 6 weeks (Wilcoxon test, p<0.05). Each dot represents an individual mouse, with horizontal bars indicating the mean tumor area.

Article Snippet: Tumor-bearing lung lobes from mice intubated with Lenti-sg Egr1 /Cre or Lenti-sg Inert /Cre pools were mixed with TRIzol reagent (Invitrogen Life Technologies) and homogenized using a gentleMACS Dissociator (Miltenyi Biotec).

Techniques: Protein-Protein interactions, Histone Deacetylase Assay, Ubiquitin Proteomics, In Vivo, Positive Control, Negative Control, Plasmid Preparation, Sequencing, Fluorescence, Staining, Marker

Journal: bioRxiv

Article Title: In Vivo Multiplexed Modeling Reveals Diverse Roles of the TBX2 Subfamily and Egr1 in Ras -Driven Lung Adenocarcinoma

doi: 10.1101/2025.03.15.642187

Figure Lengend Snippet: A) Schematic illustration of the experimental design for Egr1 knockout versus control. KC mice were administered a pool of two sgRNAs targeting Egr1 (n=3) or a pool of control sg Inerts (n=3). (B) Representative images of lung lobes from mice 20 weeks post-tumor initiation via Lenti-sg Egr1 -pool/Cre or Lenti-sg Inerts . Images include fluorescence views under a dissecting microscope, H&E-stained sections, TTF1-stained sections to confirm lung adenomatous origin, and Egr1 staining to confirm knockout efficiency. C) Bulk RNA-seq analysis was conducted on lung tissues 20 weeks post-tumor initiation in Egr1 knockout and control mice. The left panel displays a heatmap of 421 differentially expressed genes (DEGs) identified with an FDR q < 0.05, illustrating two distinct expression clusters. The right panel highlights a heatmap of the top 50 most deregulated genes, showing clear differences between the two groups. D) Gene Ontology analysis was performed using Metascape on significantly upregulated genes between Egr1 knockout and control mice. Statistically enriched terms were identified based on cumulative hypergeometric p-values and enrichment scores. The top 20 most significant terms, predominantly associated with immune processes, are visualized.

Article Snippet: Tumor-bearing lung lobes from mice intubated with Lenti-sg Egr1 /Cre or Lenti-sg Inert /Cre pools were mixed with TRIzol reagent (Invitrogen Life Technologies) and homogenized using a gentleMACS Dissociator (Miltenyi Biotec).

Techniques: Knock-Out, Control, Fluorescence, Microscopy, Staining, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: In Vivo Multiplexed Modeling Reveals Diverse Roles of the TBX2 Subfamily and Egr1 in Ras -Driven Lung Adenocarcinoma

doi: 10.1101/2025.03.15.642187

Figure Lengend Snippet: A) The immune network and top 12 hub genes are visualized. From the top 30 most significantly upregulated pathway clusters, 18 immunity-related terms and their associated genes were selected for Maximal Clique Centrality analysis using the CytoHubba plugin in Cytoscape. Gene interactions within the network indicate their participation in shared pathways. The background color of each gene box represents its centrality ranking: four genes co-ranked 1st (red), five genes co-ranked 5th (orange), and three genes co-ranked 10th (yellow). The 16 pathways associated with these 12 hub genes were further consolidated into 8 primary categories, represented by blue rectangular boxes. (B) Analysis of the DepMap CRISPR–Cas9 cancer dataset evaluating the effect of Egr1 knockout in 342 human cancer cell lines, including independent assessments in KRAS-driven lung cancer lines, showed no significant impact on cell proliferation in either case. (C) Immunohistochemical staining of Cd4, Cd8, Cd3, Cd19, and F4/80 in Egr1 -deficient lung tumors compared to control tumors. Scale bars are provided for each image. (D) A heatmap illustrating the expression patterns of 55 curated T-cell exhaustion markers (CellMarker 2.0 and 10X Genomics) in control and Egr1 -deficient tumors. Differential expression analysis reveals that most exhaustion markers are upregulated in Egr1 -deficient samples. The color scale represents relative expression levels, ranging from red (high expression) to blue (low expression).

Article Snippet: Tumor-bearing lung lobes from mice intubated with Lenti-sg Egr1 /Cre or Lenti-sg Inert /Cre pools were mixed with TRIzol reagent (Invitrogen Life Technologies) and homogenized using a gentleMACS Dissociator (Miltenyi Biotec).

Techniques: CRISPR, Knock-Out, Immunohistochemical staining, Staining, Control, Expressing, Quantitative Proteomics

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A) Total cell lysates from MRC5 and other indicated NSCLC cell lines were immunoblotted with anti-EGR1. β-actin was used as loading control. (B) Representative of H1299 cells with or without overexpressed EGR1 immunofluorescence-stained with antibodies against EGR1 (red color). The nuclei were counterstained with DAPI (blue color). (C) The growth of H1299 cells infected with CD513B-1-EGR1 or -dnEGR1 was analyzed by CCK8 assay. Up panel: western blot for EGR1 expression levels in H1299 cells transfected with EGR1 or dnEGR1. (D) Histogram showing the percentages of apoptotic cells by flow cytometric staining with Annexin V-PE/7-AAD. (E) Western blot for apoptotic caspase-3 and -7 in H1299 cells with increasing quantities of EGR1 compared with control. Numbers presented the fold change compared with the vector. Relative values ± SD were obtained from three independent assays. The full-length blots of Fig. 1A, 1C and 1E were presented in .

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Immunofluorescence, Staining, Infection, CCK-8 Assay, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A) The effect of EGR1 on cell migration was determined by wound healing assay. The spreading speed of EGR1-transfected cells along the wound edge was slower than that of vector- or dnEGR1-transfected cells over a period of 72 h. (C) Representative images showing the transfected cells that migrated through the cell. The number of migrated tumor cells was quantified and is shown in the right panel. Columns, mean of triplicate experiments; *P < 0.05.

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Migration, Wound Healing Assay, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A and B) Hierarchical clustering of 100 genes that exhibited significantly altered expression in EGR1-transfected H1299 cells as compared with vector-transfected H1299 cells. The color bar indicates the fold change (log 2 ). (C) IPA launch of bio-function enrichment profiles, plotted by relative statistical significance. P value, Fisher's exact test. (D) The expression of 15 upregulated genes (ARC, GDF15, CDKN1C, TGM2, HES1, EFNA1, NR4A1, TRIB1, IGFBP6, ITGB8, PDGFA, KRT18, TIMP1, and ID2) was analyzed by qPCR. Relative values ± SD were obtained from three independent assays.

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: Genes differentially expressed between EGR1-overexpressed H1299 cells and control cells

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Binding Assay, Dominant Negative Mutation, Expressing

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A) Luciferase reporter assays of sequential deletions of the KRT18 promoter, cotransfected with increasing quantities of EGR1. The predicted positions of the EBS (black boxes) were indicated on the schematic. Nucleotides with red colors presented consensus EBS. Values are expressed as mean fold-activation to the promoter cotransfected with empty vector. (B) Luciferase reporter assays of point mutant of the KRT18 promoter. The consensus EBS in pGL3-KRT18 was mutated, creating pGL3-KRT18-mut. Relative reporter activity in response to EGR1 transfection was compared as in (A). (C) EGR1 and KRT18 protein levels after treatment with different si-EGR1 doses or NC (negative control). β-actin was used as loading control. Values are presented as fold change relative to NC. The full-length blot of Fig. 4C is presented in .

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Luciferase, Activation Assay, Plasmid Preparation, Mutagenesis, Activity Assay, Transfection, Negative Control

Journal: Scientific Reports

Article Title: EGR1 decreases the malignancy of human non-small cell lung carcinoma by regulating KRT18 expression

doi: 10.1038/srep05416

Figure Lengend Snippet: (A) The growth of H1299 and A549 cells infected with CD513B-1-KRT18 was analyzed by CCK-8 assay. The results are expressed as mean ± SD of three independent experiments. (B) The effect of EGR1 on cell migration was determined by wound healing assay. (C) Representative images showing the transfected cells that migrated through the cell. The number of migrated tumor cells was quantified as in . (D) Western blot for apoptotic caspase-3 and -7 in H1299 cells with different KRT18 transfected dose as compared with control cells. β-actin was used as loading control. The full-length blot of Figure 5D is presented in . (E) Expression level of EGR1 and KRT18 in 2 primary NSCLC cases. (F) Correlation of the EGR1 (+) expression with age, lymph node metastasis and KRT18 in 36 NSCLC cases. (G) Correlation of the KRT18 (+) expression with lymph node metastasis in 36 NSCLC cases.

Article Snippet: Sections were incubated with rabbit anti-human EGR1 polyclonal antibody (Abgent, USA) at a dilution of 1∶500 at 4°C overnight.

Techniques: Infection, CCK-8 Assay, Migration, Wound Healing Assay, Transfection, Western Blot, Expressing