Disorganization of collagen IV-rich PFHR9 matrix under low-Cl conditions. (A–D) Transmission electron microscopy of the BM-like matrix from PFHR9 cells under standard (A, Cl “+”) or low-Cl (B, Cl “−”) conditions. Standard conditions yielded organized matrices (A), whereas low-Cl conditions yielded disorganized matrices with patches of accumulated granular material (B). (C and D) Transmission electron microscopy of matrix deposited in alternating standard and low-Cl conditions. (C) Cells grown in standard media (Cl “+”) for 5 d followed by 5 d in low-Cl media. An abrupt density change occurred approximately in the middle of the deposited matrix (bracketed area). (D) Cells grown in low-Cl media followed by standard conditions (5 d each) displayed a disorganized patchy matrix nearest the support membrane with more ordered structure nearest the cells (bracketed area). (E–G) Confocal microscopy reveals that low-Cl conditions stimulate the enhanced localization of laminin and PXDN to collagen IV in PFHR9 matrix. (E) Maximum intensity projection of immunofluorescent confocal z-stacks (7.2-µm projections) of matrix after 5 d in standard media or low-Cl media; collagen IV (red), laminin (green), or PXDN (green), merge where colocalization is yellow/orange, and nuclei/Hoechst stain (blue). Secondary antibody only negative control inset. Bars, 15 µm. (F and G) Collagen IV (F) and laminin or collagen IV and PXDN (G) colocalization analysis by Pearson correlation coefficient (1.0 = perfect colocalization) and ratio of green/red signal per field-of-view. B, n = 8; C, n = 4. *, P < 0.001.