Article Figures & Data
Figures
- Figure 1.
Claudin-4 expression in HGSOC cells. A, CLDN4 gene expression levels in laser capture microdissected specimens of human high-grade serous ovarian tumors compared with isolated normal human ovarian surface epithelial cells (36). Western blot analysis of claudin-4 protein levels in human patient samples of normal fallopian tube and ovarian cancer (B) as well as high-grade serous ovarian tumor cell lines (C), using GAPDH as a loading control. Mean ± SEM, n = 10 HOSE, n = 10–53 HGSOC tumor specimens, n = 7 other EOC subtypes (*, P < 0.05; ***, P < 0.0001).
- Figure 2.
Disruption of claudin-4 activity with CMP enhances paclitaxel response. A, Representative images of nuclei (DAPI staining) and activated caspase-3 (fluorescent antibody directed to cleaved caspase-3) from OVCAR3 cells. Quantification of caspase-3 activation in OVCAR3 (B) and OVCAR8 (C) cells treated for 24 hours with vehicle control (Veh cont), 400 μmol/L inactive control peptide (ContP), 400 μmol/L CMP, 10 μmol/L Cisplatin (Cis), CMP + Cisplatin, 10 nmol/L paclitaxel (taxol), or CMP + paclitaxel. Percent of cell population positive for caspase-3 activation was calculated using SlideBook software (3i). Mean ± SEM; n = 3; NS, not significant; ***, P < 0.001 versus control/drug only.
- Figure 3.
Loss of claudin-4 expression enhances paclitaxel response. A, Western blot analysis of claudin-4 protein in OVCAR3 cells treated with control empty vector shRNA (shCTRL) or claudin-4–targeted shRNA (shCLDN4_1, shCLDN4_2, shCLDN4_3). GAPDH was used as a loading control. B, Immunofluorescence analysis of cells treated with 400 μmol/L inactive control peptide (ContP), 400 μmol/L CMP, 10 nmol/L paclitaxel (taxol), or CMP + paclitaxel for 24 hours. Cells were treated with fluorescent antibody directed to cleaved caspase-3 and DAPI (nuclei) and percent of cell population positive for caspase-3 was calculated using SlideBook software (3i). Mean ± SEM, n = 3 per treatment group; NS, not significant; *, P < 0.05, ***, P < 0.001 versus control/paclitaxel only.
- Figure 4.
Overexpression of claudin-4 reduces paclitaxel response. A, Western blot analysis of claudin-4 protein in OVCAR8 cells transduced with GFP only or claudin-4-GFP. GAPDH was used as a loading control. B, Immunofluorescence analysis of fixed OVCAR8 cells that were treated with 400 μmol/L inactive control peptide (ContP), 400 μmol/L CMP, 10 nmol/L paclitaxel (taxol), or CMP + paclitaxel for 24 hours. Cells were treated with fluorescent antibody directed to cleaved caspase-3 and DAPI (nuclei) and percent of cell population positive for caspase-3 was calculated using SlideBook software (3i). Mean ± SEM; n = 3 per treatment group; NS, not significant; **, P < 0.01; ***, P < 0.001 versus control/paclitaxel only.
- Figure 5.
Loss of claudin-4 leads to delayed mitotic progression. A, Representative images of DAPI (DNA) staining in fixed monolayers of control knockdown (shCTRL) and claudin-4 knockdown (shCLDN4_2) OVCAR3 cells. Yellow circles highlight mitotic figures. B, Visual counting of mitotic figures from DAPI images. C, Colorimetric measurement of phosphorylated H2B (mitotic marker) to quantify population of mitotic cells in shCTRL (white bars) and shCLDN4_2 (black bars) OVCAR3 cells. D, Proliferation rates of shCTRL (circles/solid line) and shCLDN4_2 (triangles/dotted line) OVCAR3 cells. E, Representative histograms of propidium iodide staining 6 hours after release from serum starvation synchronization in shCTRL and shCLDN4_2 cells. F, Quantification of the percent of the total cell population in the G2–M phase of the cell cycle using FlowJo software. Mean ± SEM; n = 3 (*, P < 0.05; ***, P < 0.001 versus control/paclitaxel only).
- Figure 6.
Claudin-4 interacts with tubulin. A, Proximity ligation assay using antibodies directed at claudin-4 and α-tubulin or β-tubulin. Red fluorescence indicates protein–protein interaction. Yellow outlined boxes are higher magnification of cell from image. Arrows point to sites of protein-protein interaction. B, IP of claudin-4 (cld-4 IP) and IgG (mouse IgG IP) from OVCAR3 lysates blotted for presence of α-tubulin and claudin-4. C, Immunofluorescence of claudin-4 (green) and β-tubulin (red), with DAPI (blue), in mitotic OVCAR3 cell. Yellow color indicates colocalization of claudin-4 and β-tubulin. D, Immunofluorescence of microtubules (β-tubulin; white/red) and nuclei (DAPI/blue) in cells expressing (shCTRL) and not expressing (shCLDN4_2) claudin-4.
- Figure 7.
Claudin-4 alters tubulin polymerization. A, Representative Western blot analysis of polymerized (P, pellet fraction) and nonpolymerized (S, supernatant fraction) tubulin levels in OVCAR3 cells expressing claudin-4 (shCTRL) and cells with silenced claudin-4 expression (shCLDN4_2). B, Quantification of the percent of the total tubulin that is polymerized. Mean ± SEM; n = 4 (**, P < 0.01).
Additional Files
Supplementary Data
- Supplementary Materials and Methods, Figure Legends
- Supplementary Figure 1 - Claudin-4 expression in patient tumors.
- Supplementary Figure 2 - Apoptotic response of cells with high and low claudin-4 expression.
- Supplementary Figure 3 - Annexin V binding in response to CMP and paclitaxel.
- Supplementary Figure 4 - Apoptotic response of cells with claudin-4 expression knocked down.
- Supplementary Figure 5 - Images of apoptotic response (loss of claudin-4 expression).
- Supplementary Figure 6 - Images of apoptotic response (claudin-4 overexpression).
- Supplementary Figure 7 - Apoptotic response to paclitaxel with claudin-4 overexpression.
Volume 17, Issue 3
