Fig 2: Impact of NID1 conditioned media on cell migration. Representative images of toluidine blue stained cells on the bottom of collagen-coated transwell inserts for (A) parental RJ423, (B) RJ423con, (C) RJ423shNid1A and (D) RJ423shNid1D cells (magnification, ×4). (E) Quantification of the percent area occupied by parental RJ423, RJ423con, RJ423shNid1A and RJ423shNid1D cells. Migration and invasion of RJ423shNid1D cells when conditioned media from RJ423, RJ423con, RJ423shNid1A or RJ423shNid1D cells were placed (F and G) in the upper chamber of the well with the cells or (H and I) in the lower chamber. (F and H) Schematic indicating the location of the conditioned media and cells. (G and I) Quantitative data from these experiments. *P<0.05 vs. RJ423con cells (n=3). Nid1, nidogen 1; con, control; sh, short hairpin RNA; CM, conditioned media.

Fig 3: Expression levels of epithelial and mesenchymal genes following Nid1 knockdown. mRNA expression levels of (A) Cdh1, (B) Vim, (C) Snai1, (D) Snai2, (E) Twist1, (F) Twist2, (G) Zeb1 or (H) Zeb2 relative to Hprt. *P<0.05 vs. RJ423con cells (n=5). Nid1, nidogen 1; Hprt, hypoxanthine phosphoribosyltransferase; con, control; sh, short hairpin RNA.

Fig 4: Nid1 levels in cells stably expressing Nid1 shRNA. (A) Expression levels of Nid1 mRNA relative to Hprt in parental RJ423, RJ423con, RJ423Nid1A and RJ423shNid1D cells. The bars represent the mean value and the error bars represent the SEM. *Indicates significant difference (P<0.05) from RJ423con cells (n=6). (B) Western blot analysis of NID1 protein from cell lysates and conditioned media from RJ423, RJ423con, RJ423shNid1A and RJ423shNid1D cells. β-actin was used as a loading control for the cell lysates while amido black was used to ensure similar loading for the conditioned media samples. Nid1, nidogen 1; Hprt, hypoxanthine phosphoribosyltransferase; con, control; sh, short hairpin RNA.

Fig 5: FOXD1+ stromal progenitors differentiate abnormally in Zeb2-cKO mice.(A and B) Increased expression of stromal cell markers MEIS1/2/3 and CDKN1C in newborn Zeb2-cKO mice as compared with their wild-type littermate controls. (C) Immunostaining of stromal cell marker CSPG4 in 3-week-old (3W) Zeb2-cKO mouse kidney shows abnormal expression of CSPG4 in the kidney cortical region. (D) Basement membrane marker nidogen-1 staining shows a significant reduction in the interstitial space width (↔) in Zeb2-cKO mouse kidney as compared with their wild-type littermate controls. n = 3 per group. Scale bars: 10 μm (A, B, and D) and 25 μm (C). Data are expressed as mean ± SEM. A 2-tailed, unpaired Student’s t test was used for statistical significance. **P < 0.01.