Volume 54, Issue 2, Pages 407-415 (August 1998) Fibronectin synthesis by human tubular epithelial cells in culture: Effects of PDGF and TGF-β on synthesis and splicing  Antje Bürger, Christof Wagner, Christiane Viedt, Bettina Reis, Friederike Hug, Gertrud Maria Hänsch  Kidney International  Volume 54, Issue 2, Pages 407-415 (August 1998) DOI: 10.1046/j.1523-1755.1998.00009.x Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 1 Identification of fibronectin by biosynthetic labeling with [35S]-methionine and SDS-PAGE and Western blotting: TEC were cultivated in the presence of [35S]-methionine for 24hours. The cell supernatant (lane 3) and the cell lysates (lane 2) were absorbed to gelatin sepharose and after elution subjected to SDS-PAGE followed by autoradiography. The deposited extracellular matrix (lane 1) was solubilized with urea and then separated by SDS-PAGE. In parallel, a Western blot was performed with a polyclonal antibody to human FN (left lane). Kidney International 1998 54, 407-415DOI: (10.1046/j.1523-1755.1998.00009.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 2 TGF-β and PDGF in the supernatant of cultivated TEC. (A) TEC were cultivated for various times with (grey bar) or without (hollow bar) PDGF (1ng/ml). Then PDGF (lower panel) or TGF-β (upper panel) was measured in the cell supernatant by ELISA. The values represent the mean of triplicates and are expressed as pg FN per ml supernatant. (B) TEC were cultivated for various times with TGF-β 2ng (dark grey bar), TGF-β 0.2ng (grey bar) or without TGFβ (hollow bar). Again, PDGF (lower panel) or TGF-β (upper panel) were measured as described above. Note that the two experiments were done with different TEC, which might explain the differences in the spontaneous cytokine release. Kidney International 1998 54, 407-415DOI: (10.1046/j.1523-1755.1998.00009.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 3 Expression of FN mRNA in TEC after stimulation with PDGF. TEC were incubated for various times with (+) or without (-) PDGF (1ng/ml). Then total RNA was isolated and Northern blots were performed with FN specific cDNA probe (upper panel), a cDNA specific for EDA (middle panel) and for comparison with GAPDH (lower panel). Kidney International 1998 54, 407-415DOI: (10.1046/j.1523-1755.1998.00009.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 4 Fibronectin synthesis by TEC after stimulation with TGF-β or with PDGF. TEC were cultivated in the absence (hollow bar) or presence of either TGF-β (0.2ng/ml; grey bar) or PDGF (2ng/ml; hatched bar) for various times. Then FN was measured in the cell supernatant (right panel) or in the solubilized deposited matrix proteins (left panel) by ELISA. The data are the mean of five replicates. Kidney International 1998 54, 407-415DOI: (10.1046/j.1523-1755.1998.00009.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 5 Determination of the EDA splice variant in TEC. TEC were cultivated in medium with TGF-β (24 hr) or PDGF (2 hr), and then RNA was isolated and RT-PCR was performed with primers flanking the EDA coding region. The PCR products are separated on agarose gels and visualized by ethidium bromide staining (M size marker, - unstimulated TEC). Kidney International 1998 54, 407-415DOI: (10.1046/j.1523-1755.1998.00009.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 6 Determination of the EDB splice variant in TEC. TEC were cultivated in medium, with TGF-β (24 hr) or PDGF (2 hr); then RNA was isolated and RT-PCR was performed with [32P]dCTP and primers flanking the EDB coding region. The PCR products are separated on polyacrylamide gels and visualized by autoradiography (- unstimulated TEC). Kidney International 1998 54, 407-415DOI: (10.1046/j.1523-1755.1998.00009.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 7 Analysis of the IIICS splice variants in TEC. The IIICS region contains three potential splice sites (schematically shown in A) giving rise to five different mRNA (shown as shaded bars). With use of primers flanking the IIICS region (PCS2 and PCS1) the five PCR products were obtained (B shows the autoradiography of 2 independent RT-PCR reactions). The relative percentage of the different PCR was calculated. V95 and V89 were separated using the two other primer pairs (PCS3/PCS1or PCS2/PCS4) (B right panel; ethidium bromide gels are shown). On lane M the DNA size markers were separated (from top: 2176, 1756, 1230, 1033, 653, 517, 453, 394, 298, 234, 154bp). With use of the flanking primers (PCS2/PCS1) the band pattern of TEC, stimulated with PDGF (C), or TGF-β (D) for various times was analyzed (ethidium bromide agarose gels are shown). Kidney International 1998 54, 407-415DOI: (10.1046/j.1523-1755.1998.00009.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Kidney International 1998 54, 407-415DOI: (10. 1046/j. 1523-1755. 1998 Kidney International 1998 54, 407-415DOI: (10.1046/j.1523-1755.1998.00009.x) Copyright © 1998 International Society of Nephrology Terms and Conditions