Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: Single-cell RNA sequencing analysis of normal ileal and colonic tissues showing (A) SEPT9 expression in different types of intestinal cells and (B) its distribution in different IEC subtypes. (C) Immunofluorescence labeling of whole mount mouse small intestinal tissues with an anti-SEPT9 antibody (magenta) and F-actin probe phalloidin (green). Scale bar= 20 μm. (D and E) Localization of endogenous SEPT9 in the ileum and colon of SEPT9-mNG mice. Scale bars= 50 μm in upper and 10 μm in lower images. (F) Colocalization of endogenous SEPT9-NeonGreen with immunostained TJ (ZO-1) and AJ (β-catenin) markers in wholemount mouse ileal mucosa. Scale bar= 5 μm. (G) Fluorescent intensity plots of SEPT9 (cyan), ZO-1 (yellow) and β-catenin (magenta) at different Z-planes at the IEC apical junctions. (H) A schematic representation of SEPT9 localization at apical junctions in IEC.

Article Snippet: SEPT9-GFP protein was purified by coimmunoprecipitation, digested with trypsin, and desalted using ChromoTek iST GFP-Trap IP-MS sample preparation kit (Proteintech, Cat gtak-iST).

Techniques: RNA Sequencing Assay, Expressing, Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: The plots show correlation of SEPT9 transcript with the levels of SEPT2, SEPT7 and SEPT11transcripts in epithelial cell subpopulation in normal human ileum (A) and normal human colon (B) . Numbers of the top of the graphs are Pearson correlation between two genes. Cell cluster abbreviations are: DeepCrypt, deep crypt secretor; EC, enterocytes; EEC, enteroendocrine; GC, Goblet; SC, stem; TA, transit amplifying; Tuft, tuft cells.

Article Snippet: SEPT9-GFP protein was purified by coimmunoprecipitation, digested with trypsin, and desalted using ChromoTek iST GFP-Trap IP-MS sample preparation kit (Proteintech, Cat gtak-iST).

Techniques:

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: Validation of SEPT9 loss in SEPT9-KO mice. Representative immunoblots (A) and densitometric quantification (B) of SEPT9 expression in colonic scrapes, and immunolabeling for SEPT9 in ileal epithelia (C). Mean ± SEM, n= 4; *p<0.05. Scale bar= 10μm. (D and E) Gut-to-blood passage of 4 kDa FITC-dextran and 70 kDa Rhodamine-dextran in SEPT9-KO and control mice. Mean ± SEM, n= 6; ****p<0.0001. Representative en face images and quantification of immunolabeled junctional markers in wholemount colon of SEPT9-KO and control mice, including claudin3 (F and G), ZO-1 (H and I), β-catenin (J and K) and E-cadherin (L and M). Scale bars= 10 μm. White dash boxes indicate the zoomed areas. Arrowheads highlight accumulation of cytoplasmic TJ and AJ proteins in SEPT9-KO mice. (N-P) Measurements of junctional morphology in colonic mucosa of SEPT9-KO and control mice examined via measuring circularity (N), solidity (O), and the cell surface area (P); n= 5/group. Data represented as box-whisker plots with whiskers extending to the minimum and the maximum and mean at the middle of the box body. **p<0.01, ****p<0.0001.

Article Snippet: SEPT9-GFP protein was purified by coimmunoprecipitation, digested with trypsin, and desalted using ChromoTek iST GFP-Trap IP-MS sample preparation kit (Proteintech, Cat gtak-iST).

Techniques: Western Blot, Expressing, Immunolabeling, Control, Whisker Assay

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: (A) Full thickness sections of the distal colonic segments stained with H&E. (B and C) En face imaging of colonic epithelium labeled with F-actin probe, Alexa Fluor-tagged phalloidin (6 area/mouse, n= 6 mice/group). Representative images (B) and quantification of Goblet cell numbers (dark holes in the F-actin labeling) are shown (C). Data is shown as box whisker plots with whiskers extending to the minimum and the maximum with mean value at the middle of the box body. Student’s t-test. **p<0.01. Scale bar = 25μm (D) Dual fluorescence labeling of MUC2 and F-actin in colonic tissues of control and SEPT9-KO mice Scale bar = 10μm.

Article Snippet: SEPT9-GFP protein was purified by coimmunoprecipitation, digested with trypsin, and desalted using ChromoTek iST GFP-Trap IP-MS sample preparation kit (Proteintech, Cat gtak-iST).

Techniques: Staining, Imaging, Labeling, Whisker Assay, Fluorescence, Control

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: B ) Representative images of en face intestinal epithelial segmentation in entire and detailed images. (A1) Representative image of junctional protein quantification. 0.6µm of wholemount colon tissue labeled with ZO-1 antibody was employed as an example (A2) Binary mask of cells generated from the original image with additional manual filtration. (A3) Overlay of the original image (green) with the binary mask (red). (A4) Binary mask of the junctions highlighting the intercellular borders. (A5) Overlay of the original image (green) with the junctional mask (red) in yellow. (B1) Representative image of junctional protein quantification. 0.6µm of wholemount colon tissue labeled with ZO-1 antibody was employed as an example (B2) Binary mask of the cell created from the original image. (B3) Binary mask of the junction of the cell of interest. (B4) Overlay of the original image (green) with the binary mask of the cell (red) and the binary mask of the junction (blue). (B5) Marked area (yellow ring) where junctional FI was measured using ImageJ. ( C and D ) Immunoblotting analysis of junctional protein expression in in colonic scrapes obtained from control SEPT9 Flox and SEPT9 cKO mice. Representative immunoblots (C) and densitometric quantification of protein expression (D) are shown (n= 4/group). The intensities of the bands for each sample were normalized to those for GAPDH, and the intensity of the bands in the controlled group was assigned a value of 1. Data are shown as mean ± SEM. * p<0.05.

Article Snippet: SEPT9-GFP protein was purified by coimmunoprecipitation, digested with trypsin, and desalted using ChromoTek iST GFP-Trap IP-MS sample preparation kit (Proteintech, Cat gtak-iST).

Techniques: Labeling, Generated, Filtration, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: (A) Immunoblotting analysis of SEPT9 expression in HT-29 cells with CRISPR/Cas9 mediated knockout of SEPT9 using two different sgRNAs. (B) Transepithelial electrical resistance (TEER) of control and SEPT9-KO HT-29 cells. (C) FITC-dextran flux in control and SEPT9-KO HT-29 cells. Mean ± SEM, n= 4; (D-K) Representative images and quantification of immunolabeled junctional markers in control and SEPT9-KO HT-29 cells, including claudin3 (D and E), ZO-1 (F and G), β-catenin (H and I), and E-cadherin (J and K). Scale bar= 10 μm. Data represented as box-whisker plots with whiskers extending to the minimum and the maximum and mean at the middle of the box body. **p<0.01, ****p<0.0001.

Article Snippet: SEPT9-GFP protein was purified by coimmunoprecipitation, digested with trypsin, and desalted using ChromoTek iST GFP-Trap IP-MS sample preparation kit (Proteintech, Cat gtak-iST).

Techniques: Western Blot, Expressing, CRISPR, Knock-Out, Control, Immunolabeling, Whisker Assay

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: (A) Immunoblotting analysis of junctional protein and myosin motor expression in control and SEPT9-KO HT-29 cells. Levels normalized to relative expression of GAPDH are shown in the boxes. (B) MTT assay and (C) cell number counting of control, and SEPT9-KO (sg4: brown; sg6: orange) HT-29 cells at different times after plating. Data shown as mean ± SEM (n =3).

Article Snippet: SEPT9-GFP protein was purified by coimmunoprecipitation, digested with trypsin, and desalted using ChromoTek iST GFP-Trap IP-MS sample preparation kit (Proteintech, Cat gtak-iST).

Techniques: Western Blot, Expressing, Control, MTT Assay

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: (A) Interactome analysis of the binding partners for SEPT9 in IECs in vitro . (B) Co-transfection of SEPT9 (green) and NMIIC (red) in COS-7 cell line. The white boxes indicated the zoomed area (Merged). Scale bar= 2 μm

Article Snippet: SEPT9-GFP protein was purified by coimmunoprecipitation, digested with trypsin, and desalted using ChromoTek iST GFP-Trap IP-MS sample preparation kit (Proteintech, Cat gtak-iST).

Techniques: Binding Assay, In Vitro, Cotransfection

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: (A) Super-resolution microscopy image of SEPT9 (green) and NMIIC (magenta) in DLD-1 human colonic epithelial cells. (B) Fluorescence intensities profiles show SEPT9 and NMIIC signal intercalation along the cell-cell junction highlighted by the white arrow. (C and D) Representative image and junction to cytoplasmic ratio of immunolabeled NMIIC in colonic mucosa of SEPT9-KO and control mice, (E and F) Representative image and junction to cytoplasmic ratio of immunolabeled NMIIC in control and SEPT9-KO HT-29 cells. Data presented as box-whisker plots with n= 5/group. **p<0.01, ****p<0.0001; Scale bars= 10 μm. ( G ) Immunoblotting analysis of NMIIC expression in Caco-2 cells with CRISPR/Cas9-mediated knockout of NMIIC. ( H ) TEER of control and NMIIC-KO cells. ( I ) FITC-dextran flux in control and NMIIC-KO Caco-2 cells. Mean ± SEM, n= 4; **p<0.01, **** p<0.001.

Article Snippet: SEPT9-GFP protein was purified by coimmunoprecipitation, digested with trypsin, and desalted using ChromoTek iST GFP-Trap IP-MS sample preparation kit (Proteintech, Cat gtak-iST).

Techniques: Super-Resolution Microscopy, Fluorescence, Immunolabeling, Control, Whisker Assay, Western Blot, Expressing, CRISPR, Knock-Out

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: ( A ) Principal Component Analysis (PCA) of gene expression profiles in colonic and ileal epithelial cells isolated from SEPT9-KO and control mice. Volcano plots comparing gene expression in ( B ) colonic and ( C ) ileal epithelium of SEPT9-KO and control mice. Significant differentially (p-value and log2FC cut-off) expressed genes are highlighted. Bubble plots representing pathway enrichment analysis of genes with statistically significant differences in expression between the SEPT9-KO and control mice in the ( D ) colonic and ( E ) ileal epithelium. Each bubble corresponds to a specific pathway, with size indicating the gene ratio and color representing the significance of the enrichment. The analysis was performed using isolated IEC from 5 mice/group.

Article Snippet: SEPT9-GFP protein was purified by coimmunoprecipitation, digested with trypsin, and desalted using ChromoTek iST GFP-Trap IP-MS sample preparation kit (Proteintech, Cat gtak-iST).

Techniques: Expressing, Isolation, Control

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: SEPT9-KO and control mice were exposed to 3% DSS in drinking water, or regular water for 7 days. ( A ) Body weight loss and ( B ) disease activity index, were recorded daily. ( C and D ) Intestinal permeability of DSS-treated SEPT9-KO and control mice. Gut-to-blood passage of 4 kDa FITC-dextran (C) and 70 kDa Rhodamine-dextran (D). ( E ) mRNA expression of inflammatory markers measured in colonic tissues of DSS-treated SEPT9-KO and control mice. (F-K) Immunolabeling and quantification of leukocyte infiltration (red) in the colonic mucosa of SEPT9-KO and control mice on day 7 of DSS or water exposure, including T-cells (F and G), monocytes/macrophages (H and I) and neutrophils (J and K), as well as TUNEL labeling of apoptotic cells (L and M). Nuclei are labeled with DAPI (blue). Scale bars= 20 μm. Mean ± SEM (n = 6); *p < 0.05, **p<0.01 *** p<0.001, ****p<0.0001. The scatter dots within the bars in C, D , and E represent individual mice. The scatter dots in the G, I, K and M graphs represent pooled cell numbers.

Article Snippet: SEPT9-GFP protein was purified by coimmunoprecipitation, digested with trypsin, and desalted using ChromoTek iST GFP-Trap IP-MS sample preparation kit (Proteintech, Cat gtak-iST).

Techniques: Control, Activity Assay, Permeability, Expressing, Immunolabeling, TUNEL Assay, Labeling

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: Mucosal injury and inflammation were examined in H&E-stained distal colonic sections of Control and SEPT9-KO mice on day 7 of DSS treatment. (A) Representative H&E images and (B) calculated tissue injury index are shown as mean ± SEM. (n= 5 in water-treated groups and n= 7 in DSS-treated groups)

Article Snippet: SEPT9-GFP protein was purified by coimmunoprecipitation, digested with trypsin, and desalted using ChromoTek iST GFP-Trap IP-MS sample preparation kit (Proteintech, Cat gtak-iST).

Techniques: Staining, Control

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: (A) Immunofluorescence labeling of SEPT9 (green) and E-cadherin (red) in cryosections of ileal and colonic tissue from IBD patients and non-IBD controls. Arrows point to SEPT9 localization at epithelial junctions in normal sample. Arrowheads indicate mislocalized and decreased SEPT9 labeling in CD and UC tissue samples. Scale bar= 50 μm ( B and C ) Quantification of the junction to cytoplasmic ratio of SEPT9 signal (B) and total SEPT9 signal intensity (C). Mean ± SEM (n= 6 for normal controls and CD patients, and 5 for UC patients). (D) Immunohistochemistry of SEPT9 labeling in paraffin sections of colonic tissue sections from control and IBD patients. (E) Quantification of total SEPT9 signal intensity. Mean ± SEM (n= 9 for normal controls, 5 for CD and 6 for UC patients). *p<0.05, **p<0.01, ***p<0.001

Article Snippet: SEPT9-GFP protein was purified by coimmunoprecipitation, digested with trypsin, and desalted using ChromoTek iST GFP-Trap IP-MS sample preparation kit (Proteintech, Cat gtak-iST).

Techniques: Immunofluorescence, Labeling, Immunohistochemistry, Control

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: Single-cell RNA sequencing analysis of normal ileal and colonic tissues showing (A) SEPT9 expression in different types of intestinal cells and (B) its distribution in different IEC subtypes. (C) Immunofluorescence labeling of whole mount mouse small intestinal tissues with an anti-SEPT9 antibody (magenta) and F-actin probe phalloidin (green). Scale bar= 20 μm. (D and E) Localization of endogenous SEPT9 in the ileum and colon of SEPT9-mNG mice. Scale bars= 50 μm in upper and 10 μm in lower images. (F) Colocalization of endogenous SEPT9-NeonGreen with immunostained TJ (ZO-1) and AJ (β-catenin) markers in wholemount mouse ileal mucosa. Scale bar= 5 μm. (G) Fluorescent intensity plots of SEPT9 (cyan), ZO-1 (yellow) and β-catenin (magenta) at different Z-planes at the IEC apical junctions. (H) A schematic representation of SEPT9 localization at apical junctions in IEC.

Article Snippet: DLD-1 cells were grown to sub-confluency and transfected with human SEPT9-GFP plasmid (OriGene, Rockville, MD; RG225682).

Techniques: RNA Sequencing Assay, Expressing, Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: The plots show correlation of SEPT9 transcript with the levels of SEPT2, SEPT7 and SEPT11transcripts in epithelial cell subpopulation in normal human ileum (A) and normal human colon (B) . Numbers of the top of the graphs are Pearson correlation between two genes. Cell cluster abbreviations are: DeepCrypt, deep crypt secretor; EC, enterocytes; EEC, enteroendocrine; GC, Goblet; SC, stem; TA, transit amplifying; Tuft, tuft cells.

Article Snippet: DLD-1 cells were grown to sub-confluency and transfected with human SEPT9-GFP plasmid (OriGene, Rockville, MD; RG225682).

Techniques:

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: Validation of SEPT9 loss in SEPT9-KO mice. Representative immunoblots (A) and densitometric quantification (B) of SEPT9 expression in colonic scrapes, and immunolabeling for SEPT9 in ileal epithelia (C). Mean ± SEM, n= 4; *p<0.05. Scale bar= 10μm. (D and E) Gut-to-blood passage of 4 kDa FITC-dextran and 70 kDa Rhodamine-dextran in SEPT9-KO and control mice. Mean ± SEM, n= 6; ****p<0.0001. Representative en face images and quantification of immunolabeled junctional markers in wholemount colon of SEPT9-KO and control mice, including claudin3 (F and G), ZO-1 (H and I), β-catenin (J and K) and E-cadherin (L and M). Scale bars= 10 μm. White dash boxes indicate the zoomed areas. Arrowheads highlight accumulation of cytoplasmic TJ and AJ proteins in SEPT9-KO mice. (N-P) Measurements of junctional morphology in colonic mucosa of SEPT9-KO and control mice examined via measuring circularity (N), solidity (O), and the cell surface area (P); n= 5/group. Data represented as box-whisker plots with whiskers extending to the minimum and the maximum and mean at the middle of the box body. **p<0.01, ****p<0.0001.

Article Snippet: DLD-1 cells were grown to sub-confluency and transfected with human SEPT9-GFP plasmid (OriGene, Rockville, MD; RG225682).

Techniques: Western Blot, Expressing, Immunolabeling, Control, Whisker Assay

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: (A) Full thickness sections of the distal colonic segments stained with H&E. (B and C) En face imaging of colonic epithelium labeled with F-actin probe, Alexa Fluor-tagged phalloidin (6 area/mouse, n= 6 mice/group). Representative images (B) and quantification of Goblet cell numbers (dark holes in the F-actin labeling) are shown (C). Data is shown as box whisker plots with whiskers extending to the minimum and the maximum with mean value at the middle of the box body. Student’s t-test. **p<0.01. Scale bar = 25μm (D) Dual fluorescence labeling of MUC2 and F-actin in colonic tissues of control and SEPT9-KO mice Scale bar = 10μm.

Article Snippet: DLD-1 cells were grown to sub-confluency and transfected with human SEPT9-GFP plasmid (OriGene, Rockville, MD; RG225682).

Techniques: Staining, Imaging, Labeling, Whisker Assay, Fluorescence, Control

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: B ) Representative images of en face intestinal epithelial segmentation in entire and detailed images. (A1) Representative image of junctional protein quantification. 0.6µm of wholemount colon tissue labeled with ZO-1 antibody was employed as an example (A2) Binary mask of cells generated from the original image with additional manual filtration. (A3) Overlay of the original image (green) with the binary mask (red). (A4) Binary mask of the junctions highlighting the intercellular borders. (A5) Overlay of the original image (green) with the junctional mask (red) in yellow. (B1) Representative image of junctional protein quantification. 0.6µm of wholemount colon tissue labeled with ZO-1 antibody was employed as an example (B2) Binary mask of the cell created from the original image. (B3) Binary mask of the junction of the cell of interest. (B4) Overlay of the original image (green) with the binary mask of the cell (red) and the binary mask of the junction (blue). (B5) Marked area (yellow ring) where junctional FI was measured using ImageJ. ( C and D ) Immunoblotting analysis of junctional protein expression in in colonic scrapes obtained from control SEPT9 Flox and SEPT9 cKO mice. Representative immunoblots (C) and densitometric quantification of protein expression (D) are shown (n= 4/group). The intensities of the bands for each sample were normalized to those for GAPDH, and the intensity of the bands in the controlled group was assigned a value of 1. Data are shown as mean ± SEM. * p<0.05.

Article Snippet: DLD-1 cells were grown to sub-confluency and transfected with human SEPT9-GFP plasmid (OriGene, Rockville, MD; RG225682).

Techniques: Labeling, Generated, Filtration, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: (A) Immunoblotting analysis of SEPT9 expression in HT-29 cells with CRISPR/Cas9 mediated knockout of SEPT9 using two different sgRNAs. (B) Transepithelial electrical resistance (TEER) of control and SEPT9-KO HT-29 cells. (C) FITC-dextran flux in control and SEPT9-KO HT-29 cells. Mean ± SEM, n= 4; (D-K) Representative images and quantification of immunolabeled junctional markers in control and SEPT9-KO HT-29 cells, including claudin3 (D and E), ZO-1 (F and G), β-catenin (H and I), and E-cadherin (J and K). Scale bar= 10 μm. Data represented as box-whisker plots with whiskers extending to the minimum and the maximum and mean at the middle of the box body. **p<0.01, ****p<0.0001.

Article Snippet: DLD-1 cells were grown to sub-confluency and transfected with human SEPT9-GFP plasmid (OriGene, Rockville, MD; RG225682).

Techniques: Western Blot, Expressing, CRISPR, Knock-Out, Control, Immunolabeling, Whisker Assay

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: (A) Immunoblotting analysis of junctional protein and myosin motor expression in control and SEPT9-KO HT-29 cells. Levels normalized to relative expression of GAPDH are shown in the boxes. (B) MTT assay and (C) cell number counting of control, and SEPT9-KO (sg4: brown; sg6: orange) HT-29 cells at different times after plating. Data shown as mean ± SEM (n =3).

Article Snippet: DLD-1 cells were grown to sub-confluency and transfected with human SEPT9-GFP plasmid (OriGene, Rockville, MD; RG225682).

Techniques: Western Blot, Expressing, Control, MTT Assay

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: (A) Interactome analysis of the binding partners for SEPT9 in IECs in vitro . (B) Co-transfection of SEPT9 (green) and NMIIC (red) in COS-7 cell line. The white boxes indicated the zoomed area (Merged). Scale bar= 2 μm

Article Snippet: DLD-1 cells were grown to sub-confluency and transfected with human SEPT9-GFP plasmid (OriGene, Rockville, MD; RG225682).

Techniques: Binding Assay, In Vitro, Cotransfection

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: (A) Super-resolution microscopy image of SEPT9 (green) and NMIIC (magenta) in DLD-1 human colonic epithelial cells. (B) Fluorescence intensities profiles show SEPT9 and NMIIC signal intercalation along the cell-cell junction highlighted by the white arrow. (C and D) Representative image and junction to cytoplasmic ratio of immunolabeled NMIIC in colonic mucosa of SEPT9-KO and control mice, (E and F) Representative image and junction to cytoplasmic ratio of immunolabeled NMIIC in control and SEPT9-KO HT-29 cells. Data presented as box-whisker plots with n= 5/group. **p<0.01, ****p<0.0001; Scale bars= 10 μm. ( G ) Immunoblotting analysis of NMIIC expression in Caco-2 cells with CRISPR/Cas9-mediated knockout of NMIIC. ( H ) TEER of control and NMIIC-KO cells. ( I ) FITC-dextran flux in control and NMIIC-KO Caco-2 cells. Mean ± SEM, n= 4; **p<0.01, **** p<0.001.

Article Snippet: DLD-1 cells were grown to sub-confluency and transfected with human SEPT9-GFP plasmid (OriGene, Rockville, MD; RG225682).

Techniques: Super-Resolution Microscopy, Fluorescence, Immunolabeling, Control, Whisker Assay, Western Blot, Expressing, CRISPR, Knock-Out

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: ( A ) Principal Component Analysis (PCA) of gene expression profiles in colonic and ileal epithelial cells isolated from SEPT9-KO and control mice. Volcano plots comparing gene expression in ( B ) colonic and ( C ) ileal epithelium of SEPT9-KO and control mice. Significant differentially (p-value and log2FC cut-off) expressed genes are highlighted. Bubble plots representing pathway enrichment analysis of genes with statistically significant differences in expression between the SEPT9-KO and control mice in the ( D ) colonic and ( E ) ileal epithelium. Each bubble corresponds to a specific pathway, with size indicating the gene ratio and color representing the significance of the enrichment. The analysis was performed using isolated IEC from 5 mice/group.

Article Snippet: DLD-1 cells were grown to sub-confluency and transfected with human SEPT9-GFP plasmid (OriGene, Rockville, MD; RG225682).

Techniques: Expressing, Isolation, Control

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: SEPT9-KO and control mice were exposed to 3% DSS in drinking water, or regular water for 7 days. ( A ) Body weight loss and ( B ) disease activity index, were recorded daily. ( C and D ) Intestinal permeability of DSS-treated SEPT9-KO and control mice. Gut-to-blood passage of 4 kDa FITC-dextran (C) and 70 kDa Rhodamine-dextran (D). ( E ) mRNA expression of inflammatory markers measured in colonic tissues of DSS-treated SEPT9-KO and control mice. (F-K) Immunolabeling and quantification of leukocyte infiltration (red) in the colonic mucosa of SEPT9-KO and control mice on day 7 of DSS or water exposure, including T-cells (F and G), monocytes/macrophages (H and I) and neutrophils (J and K), as well as TUNEL labeling of apoptotic cells (L and M). Nuclei are labeled with DAPI (blue). Scale bars= 20 μm. Mean ± SEM (n = 6); *p < 0.05, **p<0.01 *** p<0.001, ****p<0.0001. The scatter dots within the bars in C, D , and E represent individual mice. The scatter dots in the G, I, K and M graphs represent pooled cell numbers.

Article Snippet: DLD-1 cells were grown to sub-confluency and transfected with human SEPT9-GFP plasmid (OriGene, Rockville, MD; RG225682).

Techniques: Control, Activity Assay, Permeability, Expressing, Immunolabeling, TUNEL Assay, Labeling

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: Mucosal injury and inflammation were examined in H&E-stained distal colonic sections of Control and SEPT9-KO mice on day 7 of DSS treatment. (A) Representative H&E images and (B) calculated tissue injury index are shown as mean ± SEM. (n= 5 in water-treated groups and n= 7 in DSS-treated groups)

Article Snippet: DLD-1 cells were grown to sub-confluency and transfected with human SEPT9-GFP plasmid (OriGene, Rockville, MD; RG225682).

Techniques: Staining, Control

Journal: bioRxiv

Article Title: The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

doi: 10.1101/2024.12.20.629767

Figure Lengend Snippet: (A) Immunofluorescence labeling of SEPT9 (green) and E-cadherin (red) in cryosections of ileal and colonic tissue from IBD patients and non-IBD controls. Arrows point to SEPT9 localization at epithelial junctions in normal sample. Arrowheads indicate mislocalized and decreased SEPT9 labeling in CD and UC tissue samples. Scale bar= 50 μm ( B and C ) Quantification of the junction to cytoplasmic ratio of SEPT9 signal (B) and total SEPT9 signal intensity (C). Mean ± SEM (n= 6 for normal controls and CD patients, and 5 for UC patients). (D) Immunohistochemistry of SEPT9 labeling in paraffin sections of colonic tissue sections from control and IBD patients. (E) Quantification of total SEPT9 signal intensity. Mean ± SEM (n= 9 for normal controls, 5 for CD and 6 for UC patients). *p<0.05, **p<0.01, ***p<0.001

Article Snippet: DLD-1 cells were grown to sub-confluency and transfected with human SEPT9-GFP plasmid (OriGene, Rockville, MD; RG225682).

Techniques: Immunofluorescence, Labeling, Immunohistochemistry, Control

Journal: Cell Death & Disease

Article Title: Septin filament coalignment with microtubules depends on SEPT9_i1 and tubulin polyglutamylation, and is an early feature of acquired cell resistance to paclitaxel

doi: 10.1038/s41419-019-1318-6

Figure Lengend Snippet: a Subcellular localization of septin filaments in the paclitaxel-sensitive (Ts) and -resistant (Tr) MDA-MB 231 cell lines. Septin filaments associated with actin fibers in Ts cells mainly comprise SEPT9_i3, while SEPT9_i1 is the main long SEPT9 isoform recovered in septin filaments, which are associated with long-chain polyglutamylated MTs in paclitaxel-adapted Tr cells . Endogenous SEPT2 was used to reveal the localization of septin filaments in Ts and Tr cells. b Subcellular septin localization in Ts cells overexpressing various combinations of septins and TTLL enzymes alone or in association. SEPT2 labelling was used to detect endogenous septin filaments. With the exception of SEPT9_i1, which bears the V5 tag, all septins are GFP-tagged. TTLL enzymes are coexpressed with CFP. The bottom-left panels show examples of differential septin localization in transfected (t) versus non-transfected (nt) cells in the same field. The images shown are representative of at least three independent experiments. Scale bars = 10 µm

Article Snippet: Blots were probed with selected primary antibodies against: α-tubulin (clone DM1-A, Sigma), SEPT2 (Atlas, Ozyme, St Quentin en Yvelines, France), SEPT9 (Proteintech, Manchester, United Kingdom), EB1 (H70, Santa Cruz Biotechnology, Clinisciences, Nanterre, france), CLIP-170 (H300, Santa Cruz Biotechnology), MCAK (Abnova, VWR, Fontenay sous bois, France), GFP (Cell Signaling, Ozyme), V5 (Invitrogen, ThermoFischer).

Techniques: Transfection