Table 1.
Primer sequences.
Fig 1.
Pten knockout increases collagen fiber formation.
(A) Second harmonic generation (SHG) and picrosirius red staining surrounding ducts in murine mammary tissue sections. SHG images are shown above their corresponding picrosirius red images. (B) Quantification of the number of collagen fibers in SHG images, shown as mean+SD with individual values plotted, n = 32 ducts imaged across 7 WT mice, 23 ducts imaged across 5 Pten-/- mice. (C) Histogram of collagen fiber length in SHG images. (D) Histogram of collagen fiber width in SHG images. ***p<0.001.
Fig 2.
Pten knockout does not change collagen expression.
(A) Normalized collagen mRNA expression, n = 5+SD. (B) Western blot of collagen I, with GAPDH used as a loading control for cell lysates. Individual bands likely represent procollagen, processing intermediates, and alpha subunit chains. (C) Quantification of collagen in cell lysates, normalized to WT. n = 3+SD. (D) Quantification of collagen in culture medium, normalized to WT. n = 3+SD. *p<0.05.
Fig 3.
Pten knockout alters collagen shuttling and fibrillogenesis.
(A) Immunofluorescence images of WT and Pten-/- MMF plated for 24 hours and cultured with or without 50 μg/mL ascorbic acid for 1 hour, then fixed and stained for collagen, GM130, and nuclei. (B) Quantification of collagen colocalization with GM130, determined by Manders’ correlation coefficient and normalized to the WT condition. n = 5+SD. *p<0.05. (C) Immunofluorescence images of cells incubated with fluorescently labeled collagen and fibronectin for 24 hours, then fixed and stained for actin and nuclei. (D) Quantification of the area of each image covered by collagen fibers normalized to number of nuclei in each image. (E) Quantification of the area of each image covered by fibronectin fibers normalized to number of nuclei in each image. n = 3+SD. *p<0.05 relative to WT.
Fig 4.
Pten loss upregulates SPARC expression.
(A) mRNA expression of collagen-processing proteins. n = 5+SD. (B) Western blotting for SPARC expression, with GAPDH as a loading control. (C) Quantification of SPARC expression in cell lysate and culture medium, normalized to WT. n = 3+SD. (D) mRNA expression of transcription factors known to affect SPARC. n = 3+SD. *p<0.05, ****p<0.0001
Fig 5.
SPARC knockdown does not affect collagen shuttling.
(A) Western blot verifying SPARC knockdown in both the cell lysate and the culture medium, with GAPDH as a loading control. (B) Western blot for collagen I in the cell lysate and culture medium with SPARC knockdown. (C) Quantification of collagen expression in the culture medium with SPARC knockdown, normalized to Pten-/- MMF. n = 3+SD, with no significant difference observed between conditions. (D) Immunofluorescence images of MMF plated for 24 hours and cultured with 50 μg/mL ascorbic acid for 1 hour, then fixed and stained for collagen, GM130, and nuclei. (E) Quantification of collagen colocalization with GM130, determined by Manders’ correlation coefficient and normalized to the Pten-/- parental condition. n = 3+SD. **p<0.01, ***p<0.001, ****p<0.0001 compared to the same cell line without ascorbic acid treatment.
Fig 6.
SPARC expression induced by Pten knockout promotes collagen and fibronectin fiber formation.
(A) Immunofluorescence images of cells incubated with fluorescently labeled collagen and fibronectin for 24 hours, then fixed and stained for actin and nuclei. (B) Quantification of the area of each image covered by collagen fibers normalized to number of nuclei in each image. (C) Quantification of the area of each image covered by fibronectin fibers normalized to number of nuclei in each image. n = 3+SD. *p<0.05 relative to Pten-/-. **p<0.01 relative to Pten-/-.
Fig 7.
SPARC expression induced by Pten knockout promotes fibrillar adhesion formation.
(A) Top row: Immunofluorescence images of cells incubated with fluorescently labeled fibronectin for 24 hours, then fixed and stained for tensin and nuclei. Inset boxes indicate areas of interest, magnified in bottom row. (B) Quantification of the number of fibrillar adhesions (FAs) per field of view (FOV) relative to the total amount of tensin signal present. n = 3+SD. #p<0.05 relative to Pten-/-.
Fig 8.
SPARC knockdown decreases extracellular matrix alignment.
(A) Fibroblast-derived extracellular matrices, color-coded by fiber orientation. (B) Quantification of fibroblast-derived matrix alignment, n = 3+SD.
Fig 9.
SPARC expression is upregulated in the tumor microenvironment.
Stromal gene expression of SPARC in normal or breast cancer patients from (A) Finak et al, (B) Karnoub et al, or (C) Ma et al. *p<0.05, ***p<0.001, ****p<0.0001.