Partial Blindness to Submicron Topography in <b><i>NF1</i></b> Haploinsufficient Cultured Fibroblasts Indicates a New Function of Neurofibromin in Regulation.

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Partial Blindness to Submicron Topography in <b><i>NF1</i></b> Haploinsufficient Cultured Fibroblasts Indicates a New Function of Neurofibromin in Regulation of Mechanosensoric Mol Syndromol 2012;3:169–179 - DOI:10.1159/000342698 Fig. 1. Principle of soft lithography and generation of microstructured PDMS substrates for cell cultures. Grooves: d = width, h = height. © 2012 S. Karger AG, Basel

Partial Blindness to Submicron Topography in <b><i>NF1</i></b> Haploinsufficient Cultured Fibroblasts Indicates a New Function of Neurofibromin in Regulation of Mechanosensoric Mol Syndromol 2012;3:169–179 - DOI:10.1159/000342698 Fig. 2. Cell orientation in respect to the orientation of grooves. The angle α was measured. To characterize the mean cell orientation, the order parameter S = <cos(2α)> was calculated: random orientation: S = 0, perfect orientation along the grooves: S = 1. © 2012 S. Karger AG, Basel

Partial Blindness to Submicron Topography in <b><i>NF1</i></b> Haploinsufficient Cultured Fibroblasts Indicates a New Function of Neurofibromin in Regulation of Mechanosensoric Mol Syndromol 2012;3:169–179 - DOI:10.1159/000342698 Fig. 3. Highest orientation of control fibroblasts was found along the grooves in PDMS substrates with height of 200 nm and width of 2 µm. a Phase contrast microscopy images of human fibroblasts of a control (K14) cultured for 2 days on PDMS substrates (1) without grooves, (2) with grooves of d = 10 µm and h = 200 nm and (3) with d = 2 µm (h = 200 nm). Arrow: orientation of the grooves. b The average orientation (S = <cos(2α)>) of the control fibroblasts (K14) was determined by measuring the angle α. The cells were cultured on substrates with groove heights h = 200 nm (circles) and h = 100 nm (squares) and groove widths d of 2, 3, 5, and 10 µm; N = 100–145. © 2012 S. Karger AG, Basel

Partial Blindness to Submicron Topography in <b><i>NF1</i></b> Haploinsufficient Cultured Fibroblasts Indicates a New Function of Neurofibromin in Regulation of Mechanosensoric Mol Syndromol 2012;3:169–179 - DOI:10.1159/000342698 Fig. 4. Fibroblasts of a control and NF1 differ in shape. The age-matched cell pair was cultured for 2 days on PDMS substrates (grooves d = 2 µm, h = 200 nm). Phase contrast microscopy images of (a) fibroblasts derived from the skin of control (K14) or (b) a NF1 patient (NF82b). Arrow: orientation of the grooves. Scale bar: 50 µm. © 2012 S. Karger AG, Basel

Partial Blindness to Submicron Topography in <b><i>NF1</i></b> Haploinsufficient Cultured Fibroblasts Indicates a New Function of Neurofibromin in Regulation of Mechanosensoric Mol Syndromol 2012;3:169–179 - DOI:10.1159/000342698 Fig. 5. Higher relative orientation of control fibroblasts as NF1+/– fibroblasts on grooves with a width of 2µm. Age-matched pairs of control und NF1 fibroblasts (K19/NF213) were cultured for 2 days on substrates with grooves with different widths (h = 200 nm, d = 2, 3, 5, and 10 µm). The cellular orientation S (S = <cos(2α)>) was determined between control and NF1 fibroblasts by measuring the angle α. N = 141–201; P(C) ≠ S(NF1). ***p < 0.001. © 2012 S. Karger AG, Basel

Partial Blindness to Submicron Topography in <b><i>NF1</i></b> Haploinsufficient Cultured Fibroblasts Indicates a New Function of Neurofibromin in Regulation of Mechanosensoric Mol Syndromol 2012;3:169–179 - DOI:10.1159/000342698 Fig. 6. Control and NF1+/– fibroblasts differ in orientation in time. The cell pair (K19 and NF213) was cultured for 1, 2 and 3 days on grooves (d = 2 µm, h = 200 nm). The average orientation was determined. N = 138–348. ***p < 0.001, *p < 0.05. © 2012 S. Karger AG, Basel

Partial Blindness to Submicron Topography in <b><i>NF1</i></b> Haploinsufficient Cultured Fibroblasts Indicates a New Function of Neurofibromin in Regulation of Mechanosensoric Mol Syndromol 2012;3:169–179 - DOI:10.1159/000342698 Fig. 7.NF1+/– fibrobasts are less orientated on defined topographies than NF1+/+ fibroblasts. Cells were cultured for 2 days on structured PDMS substrates (d = 2 µm, h = 200 nm). The pairs are aged and passage (p) matched. Pair 1: K20/NF183 (p7/p7); pair 2: K8/NF329 (p6/p5); pair 3: K17/NF329 (p5/p5); pair 4: K19/NF213 (p5/p5); pair 5: K19/NF213 (p7/p8); pair 6: K5/NF82c (p5/5); pair 7:K8/NF324 (p5/p5); pair 8: K14/NF82b (p5/p5); pair 9: K23/NF84a (p7/p8); pair 10: K24/NF196 (p8/p8); pair 11:K9/NF180 (p8/p8). N = 110–436; p for S(C) ≠ S(NF1). ****p < 0.0001, ***p < 0.001, **p < 0.01. © 2012 S. Karger AG, Basel

Partial Blindness to Submicron Topography in <b><i>NF1</i></b> Haploinsufficient Cultured Fibroblasts Indicates a New Function of Neurofibromin in Regulation of Mechanosensoric Mol Syndromol 2012;3:169–179 - DOI:10.1159/000342698 Fig. 8. Inhibitors of pathways dysregulated by NF1 haploinsufficiency ameliorate the mean orientation (S) in NF1+/– fibroblast. NF329 fibroblasts were treated for 2 days with inhibitors (I) of neurofibromin regulated pathways. I 1: Ras (30 µm FTI-277); I 2: Rac (50 µm NSC23766); I 3: MAPK (25 µm PD98059); I 4: mTOR (5 µm Rapamycin); I 5: ROCK (10 µm Y27632). K8: fibroblasts of an age matched control. S = <cos(2α)>: orientation of the cells on the grooves (h = 200 nm, d = 2 µm). N = 122–173 cells; p: S(NF1 untreated) ≠ S(NF1 treated). ***p < 0.001, **p < 0.01, *p < 0.05. © 2012 S. Karger AG, Basel