Journal: Cell reports

Article Title: HCMV vCXCL1 Binds Several Chemokine Receptors and Preferentially Attracts Neutrophils over NK Cells by Interacting with CXCR2.

doi: 10.1016/j.celrep.2016.04.042

Figure Lengend Snippet: Figure 3. RvCXCL1 Induces Migration of Naive NK Cells (A) Transwell migration assays of freshly isolated NK cells toward increasing concentrations of rvCXCL1 (indicated on the x axis). RhFck and rhIL8 were used as positive controls. Percent of migrating cells was calculated out of total input cells. Data are presented as mean ± SEM (n = 4). (B) Freshly isolated NK cells were incubated for 1 hr with mAb against vCXCL1 or isotype-matched control. RvCXCL1 was placed in the bottom chamber, and migration was performed for 3 hr at 37C. The numbers of migrated cells was determined by FACS following 3 hr of incubation at 37C. The basal migration rate of NK cells toward medium that did not contain rvCXCL1 was set as 1 and the results presented as fold increase (FI). *p < 0.05. **p < 0.005.

Article Snippet: Filters were then plated in bottomwells containing 600 ml migrationmedium (RPMI 1640with 1% fetal calf serum [FCS]) supplemented with either rvCXCL1 (620-CM-025), rhIL8 (208-IL-050), rhFck (365-FR-025), or rhChemerin (2324-CM-025; obtained from R&D Systems), as indicated in each figure.

Techniques: Migration, Isolation, Incubation, Control

Journal: Cell reports

Article Title: HCMV vCXCL1 Binds Several Chemokine Receptors and Preferentially Attracts Neutrophils over NK Cells by Interacting with CXCR2.

doi: 10.1016/j.celrep.2016.04.042

Figure Lengend Snippet: Figure 4. RvCXCL1 Induces Naive NK Cell Migration via CX3CR1 and CXCR1 (A) Freshly isolated NK cells were incubated with and without 0.1 mg and 1 mg (indicated on the left side of the figure) of rhIL8 (red histograms, left), rhFck (blue histograms, middle), or rvCXCL1 (green histograms, right), at 37C for 1 hr. Next, cells were stained with anti-CX3CR1 (two upper rows) or with anti-CXCR1 (two lower rows). Open gray histograms show the staining of the chemokine receptors on untreated cells. Filled gray histograms show staining of the untreated NK cells with an isotype control. The backgrounds of the treated cells were similar to the untreated cells and are not shown in the figure. See also Table S2. (B) Binding of vCXCL1-Ig to 293T-CX3CR1 transfectant (black open histogram) or to the 293T parental cells (gray open histogram). Filled gray histogram is the staining of the control fusion protein (control-Ig) on the 293T-CX3CR1-transfected cells. Staining of the parental 293T cells with control-Ig was similar to the 293T- CX3CR1-transfected cells and is not shown in the figure. (C) Freshly isolated NK cells were incubated for 1 hr with and without the proteins indicated on the x axis. RvCXCL1 was placed in the bottom chamber, and migration was performed for 3 hr at 37C. Migrating cells were counted by FACS. NK cell migration toward rvCXCL1 without pre-blocking was set as 100%, and the results are presented as % of migration. *p < 0.05. NS, not significant. Figure shows one representative experiment out of three performed.

Article Snippet: Filters were then plated in bottomwells containing 600 ml migrationmedium (RPMI 1640with 1% fetal calf serum [FCS]) supplemented with either rvCXCL1 (620-CM-025), rhIL8 (208-IL-050), rhFck (365-FR-025), or rhChemerin (2324-CM-025; obtained from R&D Systems), as indicated in each figure.

Techniques: Migration, Isolation, Incubation, Staining, Control, Binding Assay, Transfection, Blocking Assay

Journal: Cell reports

Article Title: HCMV vCXCL1 Binds Several Chemokine Receptors and Preferentially Attracts Neutrophils over NK Cells by Interacting with CXCR2.

doi: 10.1016/j.celrep.2016.04.042

Figure Lengend Snippet: Figure 5. RvCXCL1 Induces Neutrophil Migration via CXCR1 and CXCR2 (A) Freshly isolated neutrophils were stained with mAb against CD16 and CEACAM1 (left dot plot). The double positive fraction (red square) was stained with specific antibodies against the chemokine receptors CXCR1, CXCR2, and CX3CR1. (B) Transwell migration assays were performed using freshly isolated neutrophils toward the recombinant proteins indicated on the x axis (rhFck, rhIL8, or rvCXCL1). The number of migrating cells was quantified by FACS, following a 30-min incubation period, at 37C. Percent of migrating cells out of total input cells was calculated. Data are presented as mean ± SEM (n = 6). (C) Freshly isolated neutrophils were incubated with and without 0.1 mg and 1 mg (indicated in the left of the figure) of rhIL8 (red histograms, left) or rvCXCL1 (green histograms, right) for 10 min at 37C, followed by staining with anti-CXCR1 (two upper rows) or anti-CXCR2 (two lower rows). Open gray histograms show chemokine receptors staining of the untreated cells. Filled gray histograms represent staining of the untreated cells with an isotype control. The backgrounds of the treated cells were similar to the untreated cells and are not shown in the figure. (D) Freshly isolated neutrophils were incubated at 37C for 10 min with or without the proteins indicated on the x axis. RvCXCL1 was placed in the bottom chamber, and the migrating neutrophils were counted using FACS following 30 min incubation at 37C. Neutrophil migration toward rvCXCL1 after pre-blocking with rhFck was set as 100%, and the results are presented as % of migration. *p < 0.05. **p < 0.005. Figure shows one representative experiment out of three performed. See also Table S3.

Article Snippet: Filters were then plated in bottomwells containing 600 ml migrationmedium (RPMI 1640with 1% fetal calf serum [FCS]) supplemented with either rvCXCL1 (620-CM-025), rhIL8 (208-IL-050), rhFck (365-FR-025), or rhChemerin (2324-CM-025; obtained from R&D Systems), as indicated in each figure.

Techniques: Migration, Isolation, Staining, Recombinant, Incubation, Control, Blocking Assay

Journal: Cell reports

Article Title: HCMV vCXCL1 Binds Several Chemokine Receptors and Preferentially Attracts Neutrophils over NK Cells by Interacting with CXCR2.

doi: 10.1016/j.celrep.2016.04.042

Figure Lengend Snippet: Figure 6. Neutrophils Migrate Faster and More Efficiently Than NK Cells in Response to rvCXCL1 (A) A transwell migration assay was performed using rvCXCL1, rhIL8, or rhFck as the chemoattractant with either freshly isolated neutrophils (gray triangles) or NK cells (black squares) placed in the upper chamber for 10, 20, and 30 min at 37C. Migration of untreated neutrophils and NK cells at the beginning of the experiment was set as 1, and the results are presented as FI. Figure shows one representative experiment out of two performed. (B) Diagram that describes competitive transwell migration assay in which NK cells and neutrophils were incubated together at the upper chamber and their ability to migrate toward rvCXCL1 (lower chamber) is determined. (C) Competitive transwell migration assays described in (B) was performed for 30 min (left panel) and 3 hr (right panel). Percent of migrating cells out of total input cells was calculated separately for neutrophils (gray) and NK cells (black). Data are presented as mean ± SEM (n = 3). *p < 0.05. **p < 0.005. ***p < 0.0005.

Article Snippet: Filters were then plated in bottomwells containing 600 ml migrationmedium (RPMI 1640with 1% fetal calf serum [FCS]) supplemented with either rvCXCL1 (620-CM-025), rhIL8 (208-IL-050), rhFck (365-FR-025), or rhChemerin (2324-CM-025; obtained from R&D Systems), as indicated in each figure.

Techniques: Transwell Migration Assay, Isolation, Migration, Incubation

Journal: Cell reports

Article Title: HCMV vCXCL1 Binds Several Chemokine Receptors and Preferentially Attracts Neutrophils over NK Cells by Interacting with CXCR2.

doi: 10.1016/j.celrep.2016.04.042

Figure Lengend Snippet: Figure 7. Reduced Neutrophil and NK Cell Migration in the Absence of UL146 during HCMV Infection (A) Freshly isolated neutrophils were incubated with increasing concentrations (indicated in the x axis) of rvCXCL1 (gray circles) for 30 min at 37C, followed by staining with anti-CXCR2. MFI of CXCR2 expression without blocking was set on 100%, and the residual CXCR2 expression was calculated. Estimated levels of vCXCL1 following infection of HFF cells with WT HCMV are shown as blue square on the graph. (B) HFFs were infected (MOI of 0.5) with WT HCMV (blue circles) or DUL146 (red circles), and supernatants containing progeny viruses were harvested at the indicated hours postinfection (x axis). The plaque-forming units (PFU) were determined using a standard plaque assay on HFF monolayers. (C and D) HFF cells were infected with WT HCMV or with HCMV DUL146 at a MOI of 1. Three days postinfection, cell supernatants were collected and used for transwell migration assays with either neutrophils (C) or NK cells (D). Neutrophils and NK cell migration toward supernatants from mock-infected HFF was set as 1, and the results are presented as FI. (E and F) Freshly isolated neutrophils were incubated at 37C for 30 min with or without the proteins indicated on the x axis. Transwell migration assays were performed with neutrophils (E) or NK cells (F) toward supernatant from mock-infected or WT-HCMV-infected HFFs. Neutrophils and NK cell migration toward supernatants from mock-infected cells was set as 1, and the results are presented as FI. *p < 0.05. **p < 0.005. ***p < 0.0005. (G) HCMV-infected endothelial cells secrete vCXCL1 (1) and recruit both neutrophils and NK cells to the infection site (2). Migration of NK cells is dependent on CXCR1 and CX3CR1 receptors, whereas neutrophil migration is dependent on CXCR2 and CXCR1. Neutrophils migrate faster and more efficiently in comparison to NK cells due to their CXCR2 receptor (3). Therefore, neutrophils reach the infection site early and can disseminate the virus while proceeding to travel throughout the body (4). This enables the virus to maintain a pool of HCMV-infected cells. NK cells that migrate toward vCXCL1 will get to the infection site at a later time point (5) and will be subverted by HCMV-immune evasion tactics.

Article Snippet: Filters were then plated in bottomwells containing 600 ml migrationmedium (RPMI 1640with 1% fetal calf serum [FCS]) supplemented with either rvCXCL1 (620-CM-025), rhIL8 (208-IL-050), rhFck (365-FR-025), or rhChemerin (2324-CM-025; obtained from R&D Systems), as indicated in each figure.

Techniques: Migration, Infection, Isolation, Incubation, Staining, Expressing, Blocking Assay, Plaque Assay, Comparison, Virus

Journal: The EMBO journal

Article Title: Pioneer activity distinguishes activating from non-activating SOX2 binding sites.

doi: 10.15252/embj.2022113150

Figure Lengend Snippet: Figure 1. Rapid depletion of SOX2 and OCT4 affects the accessibility landscape of thousands of sites.

Article Snippet: For the knock-in of the FKBP sequence at the genes of interest, we used previously described plasmids and approach (SOX2 fkbpdonor, Addgene # 175552; SOX2 sgRNA, Addgene #175553; NANOG donor, Addgene # 175554; NANOG sgRNA, Addgene # 175555) Briefly, cells were transfected with the plasmids containing the gRNA sequence and the donor plasmid designed to include the FKBP-2xHA-P2A-[GFP/mCherry] in between two homology arms for the gene of interest.

Techniques:

Journal: The EMBO journal

Article Title: Pioneer activity distinguishes activating from non-activating SOX2 binding sites.

doi: 10.15252/embj.2022113150

Figure Lengend Snippet: Figure 2. Loss of SOX2 and OCT4 effect accessibility at shared and independent regions.

Article Snippet: For the knock-in of the FKBP sequence at the genes of interest, we used previously described plasmids and approach (SOX2 fkbpdonor, Addgene # 175552; SOX2 sgRNA, Addgene #175553; NANOG donor, Addgene # 175554; NANOG sgRNA, Addgene # 175555) Briefly, cells were transfected with the plasmids containing the gRNA sequence and the donor plasmid designed to include the FKBP-2xHA-P2A-[GFP/mCherry] in between two homology arms for the gene of interest.

Techniques:

Journal: The EMBO journal

Article Title: Pioneer activity distinguishes activating from non-activating SOX2 binding sites.

doi: 10.15252/embj.2022113150

Figure Lengend Snippet: Figure 5. Open chromatin regions maintained by SOX2 are associated with transcription.

Article Snippet: For the knock-in of the FKBP sequence at the genes of interest, we used previously described plasmids and approach (SOX2 fkbpdonor, Addgene # 175552; SOX2 sgRNA, Addgene #175553; NANOG donor, Addgene # 175554; NANOG sgRNA, Addgene # 175555) Briefly, cells were transfected with the plasmids containing the gRNA sequence and the donor plasmid designed to include the FKBP-2xHA-P2A-[GFP/mCherry] in between two homology arms for the gene of interest.

Techniques:

Journal: Cell reports

Article Title: Reprogramming of nucleotide metabolism by interferon confers dependence on the replication stress response pathway in pancreatic cancer cells

doi: 10.1016/j.celrep.2021.110236

Figure Lengend Snippet: (A) STING transcript levels across TCGA tumor datasets relative to GTEx organ-matched normal tissues. (B) IHC analysis of STING expression in the UCLA PDAC tissue microarray (n = 138). (C) Immunoblot analysis of protein lysates prepared from STING WT and CRISPR-Cas9 KO HS766T or DANG subcutaneous xenograft tumors. (D) IncuCyte live-cell imaging analysis of SUIT2 TetR STINGR284M cells treated +50 ng/mL doxycycline (DOX) ± 500 nM VE-822 (ATR inhibitor [ATRi]) in anchorage-independent cultures (mean ± SD; n = 6; one-way ANOVA corrected for multiple comparisons by Bonferroni adjustment). Images are representative of respective experimental endpoints. (E) Immunoblot analysis of SUIT2 tumors from mice treated ± DOX diet ± AZD-6738 (25 mg/kg b.i.d.) for 3 days. (F) Experimental design to evaluate the impact of ATRi against PDAC tumors with active STING signaling. (G and H) NCG mice were inoculated with subcutaneous (s.c.; G; n = 8) or orthotopic (H; n = 4) SUIT2 TetR STINGR284M tumors. Mice were fed a DOX-supplemented diet starting 7 days after tumor inoculation and treated ±25 mg/kg ATRi AZD-6738 b.i.d. for 26 days (unpaired t test; mean ± SD). Tumor volumes were measured using micro-computed tomography (μCT). Mass of excised tumor tissue is indicated. *p < 0.05; ****p < 0.0001.

Article Snippet: pLENTI TetR BLAST mammalian expression plasmid , Addgene , Cat#17492.

Techniques: Expressing, Microarray, Western Blot, CRISPR, Live Cell Imaging, Micro-CT

Journal: Cell reports

Article Title: Reprogramming of nucleotide metabolism by interferon confers dependence on the replication stress response pathway in pancreatic cancer cells

doi: 10.1016/j.celrep.2021.110236

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pLENTI TetR BLAST mammalian expression plasmid , Addgene , Cat#17492.

Techniques: Virus, Microarray, Recombinant, Lysis, Protease Inhibitor, Membrane, Saline, Autoradiography, Plasmid Preparation, Control, BIA-KA, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Expressing, Software, Mass Spectrometry, Targeted Proteomics, Real-time Polymerase Chain Reaction, Live Cell Imaging

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) UMAP plot of integrated single-cell RNA sequencing (scRNAseq) datasets of mouse embryonic developing colon (hindgut), healthy adult colon and colon cancer (derived from AKPS organoid-based model). (B) Volcano plots of dual differential gene expression analysis comparing epithelial cell gene expression from scRNAseq data of embryonic hindgut and colon cancer to the healthy adult colon. Highlighted genes are examples of common genes in the embryonic and cancer cells. (log2FC (log2 fold change) > 0.6, p_adj.glb (p value adjusted global) < 0.05). Differential expression and p values were calculated via hierarchical permutation tests. (C) Immunofluorescent (IF) staining on embryonic hindgut, healthy adult colon and colon cancer for Tactsd2 and Marcksl1 (magenta). Blue DAPI. Scale bar: 50 μm. (D) Regulatory network inference using SCENIC analysis on the integrated epithelial scRNAseq data (from 1A). (E) Expression values of SoxC genes are shown as psupertime value learned for each cell (X-axis) and Z-scored log2 gene expression values (Y-axis) across three embryonic and one adult time-points. (F) Kaplan Meier plots showing the inverse correlation between SOXC family gene expression and CRC patients’ relapse-free survival. Hazard ratio (HR) and logarithmic ranked p Value (logrank P) were analyzed to infer the significance of the differences. Numbers below each graph represent number of patients at risk in any given time (months), black for low expression and red for high expression of each gene. The cut-off is automatically calculated based on the best performing threshold. (G) IF staining on embryonic hindgut, healthy adult colon and colon cancer for Sox11 (grey). Blue DAPI. Scale bar: 50 μm. (H) IF staining on human healthy and malignant colon for EPCAM (epithelial marker, green) and SOX11 protein (magenta). Blue DAPI. Scale bar: 50 μm.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: RNA Sequencing Assay, Derivative Assay, Expressing, Staining, Marker

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) Predicted functional gene interaction network of oncoembryonic genes derived from dual differential gene expression analysis (from ), showing significant interactions among key SoxC-regulated genes. (B) Summary of gene ontology (GO) terms represented in panel S3A, displayed as a pie chart, with numbers indicating the percentage of GO terms in each category. (C) Complete regulatory network inference using SCENIC analysis on integrated epithelial scRNAseq data (from ), highlighting Sox4 and Sox11 as master regulators in the oncoembryonic cells. (D) Quantitative PCR showing transcript levels of SoxC transcription factor targets in healthy, healthy-adjacent, and colon tumor tissues from mouse models. (E) RNA in situ hybridization (RNAscope) and quantification of signal intensity for Sox4 and Sox11 in mouse healthy and colon tumor tissues. Scale bar: 50 μm, DAPI (blue) counterstains nuclei. (F) Immunofluorescent staining of human healthy and cancerous colon tissues for E-CADHERIN (epithelial marker) and SOX11 protein. Scale bar: 50 μm.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Functional Assay, Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, RNA In Situ Hybridization, RNAscope, Staining, Marker

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) Ridge plot representing normalized counts of Sox11, Sox4 and Sox12 in epithelial cells at different stages of colon development. (B) Full R26-CreERT2; SoxC fl/fl or non-Cre littermate embryos at stage E14.5 or E18.5 after tamoxifen induction of Cre at E10.5. Arrows point at eye, back and digit phenotypes. (C) Length (in mm) of SoxC-KO or non-Cre littermate midguts at E18.5. (D) Quantification of mesenchymal nuclei present in the region below the epithelium and before the smooth muscle layer in SoxC-KO or non-Cre littermate E14.5 hindguts. (E) Marker genes were used for the annotation of the integrated WT and SoxC-KO E14.5 hindgut scRNAseq dataset. (F-H) Gene ontology and network analyses of top 200 genes enriched in clusters Epithelial 3 (F), Epithelial 4 (G) and Mesenchymal 4 (H) of the integrated WT and SoxC-KO E14.5 hindgut scRNAseq dataset.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Marker

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) Length of entire hindguts in SoxC-KO E14.5 embryos and non-Cre (unrecombined SoxC fl/fl) littermates. (B) Representative immunofluorescence (IF) staining of SoxC-KO and non-Cre littermates E14.5 hindguts (blue DAPI, green Phalloidin, grey E-Cadherin). Scale bar: 50 μm. (C) Measurement of stromal widths (defined as the distance between the lower epithelial border and the beginning of the smooth muscle layer) in multiple images similar to 2B. Each dot represents one measurement, between 3 to 5 measurements were taken from n=3 SoxC-KO and n=3 WT hindguts. (D) Schematics of the multi-omics experiments performed using E14.5 wild-type (WT) and SoxC-KO hindguts. (E) UMAP plot of SoxC-KO E14.5 hindgut scRNAseq integrated with previously published E14.5 WT hindgut scRNAseq. Dots represent single cells, colored by cell type as illustrated in . (F) Volcano plot of gene expression fold changes in scRNAseq of E14.5 hindguts in SoxC-KO or WT mice. Magenta dots represent genes significantly downregulated in SoxC-KO (log2FC (log2-fold change) < -0.6, p_adj.glb (p value adjusted global) < 0.05), blue dots represent genes significantly upregulated in SoxC-KO (log2FC > 0.6, p_adj.glb < 0.05). Selected genes are labeled. Differential expression and p values were calculated via hierarchical permutation tests. (G) Volcano plot of ATACseq peaks in SoxC-KO and WT E14.5 hindguts as identified via the ATACseq snakePipes pipeline. Magenta dots represent peaks significantly lost or reduced in SoxC-KO compared to WT (logFC < -1, FDR < 0.05), blue dots represent peaks significantly gained or increased in SoxC-KO compared to WT (logFC > 1, FDR < 0.05). (H) ChIPseq profiles in selected genomic regions bound by Sox4 and Sox11, showing two examples of positively regulated (top) and two negatively regulated (bottom) genes, also identified in scRNAseq and ATACseq (2F&G). (I) Number of genes with significant changes detected in scRNAseq, ATACseq or ChIPseq and schematics of overlaps between the three assays. Note that the Sankey plot shows only the genes shared between the datasets, while the total number of genes in each category exceeds the number of shared genes. (J) Representative IF staining of E14.5 SoxC-KO and SoxC fl/fl hindguts (blue DAPI, grey Sox11, magenta Klf4, green Phalloidin) and quantification of nuclear Klf4 measured as averaged intensity of all nuclei in the section. (K) qPCR analysis of Klf4 expression in WT and SoxC-KO E14.5 hindguts. (L) Representative IF staining of E14.5 SoxC-KO and SoxC fl/fl hindguts (blue DAPI, grey E-Cadherin, magenta Tead2, green Phalloidin) and quantification of nuclear Tead2 measured as averaged intensity of all nuclei in the section. (M) qPCR analysis of Tead2 expression in WT and SoxC-KO E14.5 hindguts. (N) Representative IF staining of E14.5 SoxC-KO and SoxC fl/fl hindguts (blue DAPI, magenta Mdk, green Phalloidin) and quantification of nuclear Mdk measured as averaged intensity of all nuclei in the section. (O) qPCR analysis of Mdk expression in WT and SoxC-KO E14.5 hindguts. Statistical analyses for 2B and 2J-O were performed using a two-tailed unpaired t-test. Scale bars 50 μm.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Immunofluorescence, Staining, Expressing, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A-C) Gene ontology analysis (biological processes) of all genes nearest to genomic regions with lost accessibility (A) or gained accessibility (B) in SoxC-KO E14.5 hindgut compared to WT or with significant ChIPseq peaks for Sox11 and or Sox4 (C). Top 20 gene ontologies are shown. (D) Representative immunofluorescent staining in SoxC-KO or non-Cre littermate E14.5 hindguts. Blue DAPI, magenta Klf5, green Phalloidin. Scalebar 50 μm. (E) qPCR analysis of Klf5 expression in WT and SoxC-KO E14.5 hindguts. Statistical analysis was performed using a two-tailed unpaired t-test.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Staining, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) Schematics of the generation of AKPS SoxC-KO organoids. (B) Primary tumor morphology of control (Cas12a intergenic targeting) and SoxC-KO (Cas12a targeting Sox4, Sox11 and Sox12 each with two specific gRNAs). Scalebar: 5 mm. (C) Bar plot of the tumor weights in the two groups. (D) Percentage of primary tumor and metastatic growth. (E) Quantitative PCR showing the transcript levels of SoxC TFs targets, Mdk and Tead2 . (F) Immunofluorescent staining of Mdk in Control and SoxC-KO colorectal cancer (CRC) tumors. Scalebars: 50 μm. Statistical analyses were performed using an unpaired t-test.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Control, Real-time Polymerase Chain Reaction, Staining

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) Immunofluorescent (IF) staining of Mdk (magenta) in E14.5 hindgut, healthy adult colon and colon cancer. Blue DAPI. (B) IF staining of Tead2 (magenta) in murine E14.5 hindgut, healthy adult colon and colon cancer. Blue DAPI. (C) Survival analysis of colorectal patients divided into high or low MDK expression. (D) Inferred interactions through Mdk signaling in E14.5 WT hindgut. Edge weights are proportional to the interaction strength. (E) IF staining and quantification of epithelial thickness of E13.5 hindgut explants cultured ex-vivo for 2 days in the presence of MDK inhibitor (iMDK, n=6) or control (DMSO, n=6). For each explant, 5-7 measurements were taken. Blue DAPI, grey E-cadherin, green Ki67. (F) Survival analysis of colorectal patients divided into high or low TEAD2 expression. (G) IF staining and quantification of epithelial thickness of E12.5 hindgut explants cultured ex-vivo for 2 days in the presence of TEAD inhibitor (iTEAD, n=4) or control (DMSO, n=3). For each explant, 5-7 measurements were taken. Blue DAPI, grey E-cadherin, green Ki67. (H) Activity of Tead2 promoter after Sox11 or Sox4 transfection assessed by luciferase assay in HEK293 cells. N=8 for Sox11, n=3 for Sox4. (I) IF staining of Klf4 and Sox11 in mouse colon cancer and adjacent tissue. Insets of representative regions with high nuclear Klf4 or Sox11 expression. On top: quantification of mean nuclear intensity along a left-to-right axis. Scale bars: 50 μm. Statistical analyses for (D), (G), (H) using two-tailed unpaired t-test.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Staining, Expressing, Cell Culture, Ex Vivo, Control, Activity Assay, Transfection, Luciferase, Two Tailed Test

Journal: bioRxiv

Article Title: An oncoembryology approach uncovers SoxC-driven regulation of colon development and cancer

doi: 10.1101/2024.10.08.617191

Figure Lengend Snippet: (A) Comparison of inferred information flow for all predicted cell-communication pathways in E14.5 hindgut or adult colon. Red-colored pathways are enriched in the E14.5 hindgut, blue-colored pathways are enriched in the adult. (B) Significant predicted Mdk ligand-receptor interactions from cell-cell communication prediction by CellChat in the scRNAseq of E14.5 WT including all cell types. Edge weights are proportional to the interaction strength. (C) Significant predicted Mdk ligand-receptor interactions originating from epithelial cells (Oncoembryonic/ISC-like, Enterocyte-like, Tuft-like, Secretory-like or Enteroendocrine-like). Inferred interactions are the result of cell-cell communication prediction by CellChat in the scRNAseq of cancer cells only (AKPS organoid-derived tumors) including all cell types. Edge weights are proportional to the interaction strength. (D) Inferred interactions through Mdk signaling from epithelial cells as in (B). (E) Quantification of nuclear Ki67 signal in and replicates (E13.5 hindgut explants cultured for 2 days ex vivo in the presence of DMSO or MDK inhibitor). (F) Quantification of nuclear Ki67 signal in and replicates (E12.5 hindgut explants cultured for 2 days ex vivo in the presence of DMSO or TEAD inhibitor). (G) Top: Activity of WT Tead2 promoter or with deletion of putative Sox11 binding site (BS) after Sox11 transfection assessed by luciferase assay in HEK293 cells. N=8 for WT, n=3 for BS mutation. Bottom: schematics of Tead2 promoter region cloned for the assay (−465 to +94 from the coding sequence start) and the mutated base (position +10 from the coding sequence start). Statistical analysis using two-way Anova with Bonferroni’s multiple comparison test. (H) Schematics of strategy to identify intermediate regulators of indirect targets of SoxC TFs. (I) Representative immunofluorescent staining of adult mouse colon (left, blue DAPI, green Pdgfra-GFP, magenta Klf4) or human healthy colon (right, blue DAPI, green E-CADHERIN, magenta KLF4). (J) Z-scored log2 expression values of Klf3, Klf4, Klf5, Klf6 with cell ordered along a pseudotime learned by pstupertime at different stages of colon development (x-axis). (K) Immunofluorescent staining from (mouse colon cancer) with nuclear mask applied on each separate channel. Top: grey, Klf4. Middle: magenta, Sox11. Bottom: blue DAPI, grey Klf4, magenta Sox11. If not otherwise stated, statistical analyses were performed using a two-tailed unpaired t-test. Scale bars 50 μm.

Article Snippet: For the Sox11 overexpression plasmid, the Sox11 coding sequence was amplified from pLenti-CMV-GFP-Sox11 (AddGene, 120387).

Techniques: Comparison, Derivative Assay, Cell Culture, Ex Vivo, Activity Assay, Binding Assay, Transfection, Luciferase, Mutagenesis, Clone Assay, Sequencing, Staining, Expressing, Two Tailed Test