Figure 6.
Figure 6. Aligned matrix organization by CAFs is mediated by PDGFRα. (A) IF staining of PDGFRα in NFs and CAFs. Images are shown pseudocolor, with warmer colors indicating high intensity, and cooler colors indicating low intensity. Bar, 25 µm. (B) Quantification of mean fluorescent intensity of PDGFRα. More than 60 cells per condition from three independent experiments were analyzed. Error bars indicate SEM for three experiments. **, P < 0.01 as determined by Student’s t test. (C and E) WB analysis of PDGFRα (C) and pY762-PDGFRα (E) in NFs and CAFs. β-actin was used as a loading control. (D and F) Quantification of PDGFRα (D) and pY762-PDGFRα (E) mean intensity in NFs and CAFs, normalized to β-actin. Error bars represent the SEM from three independent experiments. *, P < 0.05 as determined by Student’s t test. (G) Collagen gel contraction of CAFs treated with either 10 µg/ml of control (Ctrl) antibody or PDGFRα neutralizing antibody (AF-307). Dashed lines circle the gels after 24 h treatment. (H) Quantification of collagen gel contraction at 24 h. Three gels from three independent experiments were analyzed. Error bars indicate SEM for three experiments. *, P < 0.05 as determined by Student’s t test. (I) Representative traction-force vector maps of CAFs treated with 10 µg/ml control IgG (left) or AF307 (right). Warmer colors indicate areas with high traction forces. (J) Scatter dot plot shows mean traction forces in control and AF307-treated CAFs. Line indicates means, whereas error bars indicate SEM. 17 control CAFs and 21 AF307-treated CAFs were analyzed in three independent experiments. ***, P < 0.001 as determined by Mann Whitney U test. (K) Fn staining of CAFs after 48 h treatment with 10 µg/ml control IgG or AF307. Bar, 25 µm. (L) Measurements of angles between Fn fibers in CAFs treated with control IgG or AF307. Greater than100 angles measured per condition from ≥12 images from three independent experiments. ***, P < 0.001 as analyzed by Mann-Whitney U test. The box plots range from the 25th to the 75th percentiles; the central line indicates the median, and the whiskers range from the 5th to the 95th percentiles. (M) Active α5 integrin staining of CAFs after 48 h treatment with 10 µg/ml control IgG or AF307. Bar, 25 µm. (N) Quantification of mean fluorescent intensity of active α5 integrin in control IgG or AF307-treated CAFs. Greater than 70 cells per condition were analyzed in three independent experiments. *, P < 0.05 as determined by Student’s t test.

Aligned matrix organization by CAFs is mediated by PDGFRα. (A) IF staining of PDGFRα in NFs and CAFs. Images are shown pseudocolor, with warmer colors indicating high intensity, and cooler colors indicating low intensity. Bar, 25 µm. (B) Quantification of mean fluorescent intensity of PDGFRα. More than 60 cells per condition from three independent experiments were analyzed. Error bars indicate SEM for three experiments. **, P < 0.01 as determined by Student’s t test. (C and E) WB analysis of PDGFRα (C) and pY762-PDGFRα (E) in NFs and CAFs. β-actin was used as a loading control. (D and F) Quantification of PDGFRα (D) and pY762-PDGFRα (E) mean intensity in NFs and CAFs, normalized to β-actin. Error bars represent the SEM from three independent experiments. *, P < 0.05 as determined by Student’s t test. (G) Collagen gel contraction of CAFs treated with either 10 µg/ml of control (Ctrl) antibody or PDGFRα neutralizing antibody (AF-307). Dashed lines circle the gels after 24 h treatment. (H) Quantification of collagen gel contraction at 24 h. Three gels from three independent experiments were analyzed. Error bars indicate SEM for three experiments. *, P < 0.05 as determined by Student’s t test. (I) Representative traction-force vector maps of CAFs treated with 10 µg/ml control IgG (left) or AF307 (right). Warmer colors indicate areas with high traction forces. (J) Scatter dot plot shows mean traction forces in control and AF307-treated CAFs. Line indicates means, whereas error bars indicate SEM. 17 control CAFs and 21 AF307-treated CAFs were analyzed in three independent experiments. ***, P < 0.001 as determined by Mann Whitney U test. (K) Fn staining of CAFs after 48 h treatment with 10 µg/ml control IgG or AF307. Bar, 25 µm. (L) Measurements of angles between Fn fibers in CAFs treated with control IgG or AF307. Greater than100 angles measured per condition from ≥12 images from three independent experiments. ***, P < 0.001 as analyzed by Mann-Whitney U test. The box plots range from the 25th to the 75th percentiles; the central line indicates the median, and the whiskers range from the 5th to the 95th percentiles. (M) Active α5 integrin staining of CAFs after 48 h treatment with 10 µg/ml control IgG or AF307. Bar, 25 µm. (N) Quantification of mean fluorescent intensity of active α5 integrin in control IgG or AF307-treated CAFs. Greater than 70 cells per condition were analyzed in three independent experiments. *, P < 0.05 as determined by Student’s t test.

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