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. 2018 Jan 8;32(5):2644–2657. doi: 10.1096/fj.201700722R

Figure 7.

Figure 7.

TAZ-induced paracrine factors promote growth inhibition of normal kidney epithelial cells and trigger renal fibroblast growth. A) Conditioned media isolated from HK-2 cells stably expressing CMV-Con and CMV-TAZ were probed for CTGF and fibronectin expression by Western blot analysis. GAPDH expression of lysates of the cells provided an assessment of protein loading. B) Experimental design for assessing the potential influence of soluble factors in epithelial cell–cell crosstalk. Similarly confluent control shRNA-expressing HK-2 cells were treated with conditioned media isolated from CMV-Con– and CMV-TAZ–expressing HK-2 cells at a similar density for 3–5 d. C) Final cell densities were determined setting the number of cells in CM-CMV-Con treated HK-2 cells as 1 (means ± sd). *P < 0.01 vs. CM-CMV-Con. D) Crystal violet staining cell monolayers. Scale bars, 200 μm. E) Approach for investigating the potential paracrine role of CTGF in epithelial cell–cell communications. Conditioned media isolated from double-transgenic CMV-TAZ + Con shRNA­– and CMV-TAZ + CTGF shRNA–expressing HK-2 cells at equal densities were directly added to similarly seeded control shRNA transduced HK-2 cells. F) Relative cell counts 3 d after addition of conditioned media for triplicate studies. *P < 0.05 vs. CM-CMV-TAZ + Con shRNA cell count (arbitrarily set at 1). G) Control shRNA-expressing epithelial cells were stimulated with conditioned media isolated from CMV-Con– or CMV-TAZ–expressing HK-2 cells, and lysate extracts were probed for fibronectin, collagen-1, CTGF, and PAI-1 protein expression. H) Conditioned media isolated from HK-2 cells stably expressing Con-CMV or TAZ-CMV maintained at a similar density were directly added to NRK-49F fibroblasts (stably expressing Con- shRNA) seeded at similar initial densities. I) Cell counts for each condition were assessed 3 d later. The plot illustrates cell counts for triplicate cultures, arbitrarily setting the number of NRK-49F cells receiving conditioned media from vector-expressing HK-2 cultures at 1. Means ± sd (C, F, I). *P < 0.05, **P <0.01 vs. CMV-Con.