Effects of TGFβ on differentiation, barrier function and scratch wound repair in NHU cell cultures.

<p>(a) NHU cells were differentiated with or without TZ/PD or ABS/Ca²⁺ in the presence or absence of TGFβ; UPK2 expression was assessed by RTqPCR after 4 days and normalised against GAPDH. Controls included SB431542, vehicle (0.1% DMSO), PD153035 and TZ alone. Bars represent means ± SD of triplicate PCR determinations. (b) Barrier function was assessed by TER measurement after 7 days culture of NHU cells seeded at 0.5×10⁶ cells/cm² onto SnapWell™ membranes and treated with or without ABS/Ca²⁺ in the presence of DMSO (0.1%), TGFβ1 (2 ng/ml) or SB431542 (3 µM). Bars represent mean ± SD of 3 independent replicate cultures. (c–g) After 7 days pre-culture with or without ABS/Ca²⁺ to induce differentiation, confluent cultures were incubated with DMSO (0.1%, vehicle control), TGFβ1 (2 ng/ml), SB431542 (10 µM) or PD153035 (1 µM) for 3 h and then scratch-wounded. Cultures were maintained in respective treatments in an environmental chamber and images were taken every 10 minutes by time-lapse microscopy until the wounds healed. The experiment was repeated on 3 independent NHU cell lines with the same results. Differential interference contrast micrographs are shown for (c) non-differentiated (6 h) and (d) differentiated cultures (8 h) post-wounding; scale bar 500 µm. The time-course of wound repair was quantified from timelapse micrographs at 2 hour intervals and expressed against the original wound as the percentage healed for confluent (e) non-differentiated and (f) differentiated cultures. Points represent means of triplicate cultures ± SD. Phase contrast micrographs (g) of repaired scratch wounds in differentiated NHU cell cultures after 16 h in the presence of DMSO (0.1%), TGFβ1 (2 ng/ml), PD153035 (1 µM) or SB431542 (3 µM). Scale bar: 500 µm.