Three-dimensional Culture Regulates Raf-1 Expression to Modulate Fibronectin Matrix Assembly -- Winters et al. 17 (8): 3386 Data Supplement - Supplemental Material -- Molecular Biology of the Cell

Three-dimensional Culture Regulates Raf-1 Expression to Modulate Fibronectin Matrix Assembly
Mol. Biol. Cell Winters et al. 17: 3386

Supplemental Material

This article contains the following supporting material:

Figure 1 - Fibronectin (FN) matrix assembly by HT-1080 cell aggregates.
HT-1080 cells were trypsinized from near-confluent plates and re-suspended at a concentration of 1.25 X 10⁶ cells/ml in FN-depleted medium. Medium was supplemented with 30 μg/ml of rhodamine-labeled FN. 15 μl hanging drops were then incubated for 24 hours under tissue culture conditions. Hanging drops were transferred to cover slips and visualized by fluorescence microscopy. Note the presence of FN fibrils extending between the cells. Scale bar = 20 μ.
Figure 2 - Induction of fibronectin (FN) matrix assembly in 3D culture does not correlate with changes in surface α4β1 or αvβ3 integrin expression.
Surface expression of αvβ3 integrin (B and E) or of α4β1 (C and F) was determined by staining with monoclonal antibodies recognizing the α4 integrin subunit or αvβ3, respectively. Control cells were incubated in a pre-immune IgG to establish baseline mean fluorescence (A and C). No significant change in surface integrin expression was detected when cells culture in 2D (A, B, and C) and 3D (D, E, and F) were compared.
Figure 3 - Raf-B expression is similar in 2D and 3D culture.
Immunoblot analysis of B-Raf expression in lysates generated from cells cultured in 3D show no change when compared to cells cultured as monolayers.
Figure 4 - Induction of FN matrix assembly by MEK inhibition, dexamethasone treatment or serum starvation does not correlate with changes in Raf-1 expression.
To explore potential mechanisms associated with Raf-1 down regulation, HT-1080 cells in monolayer culture were incubated in the presence or absence of the MEK inhibitor, PD98059 (10 μM), or dexamethasone (10^-7 M). Alternately, cells were serum starved for 24 hours. Raf-1 expression was detected by immunoblotting.
Figure 5 - The effect of proteasome inhibition on Raf-1 expression in 3D culture.
To explore potential mechanisms associated with Raf-1 down regulation, aggregates of HT-1080 cells were incubated in the presence or absence of the proteosome inhibitors MG-132 (10 μM), Lactacystin (20 μM), or LLnL (40 μM). Raf-1 expression was detected by immunoblotting and a representative experiment is presented graphically. Proteasomal inhibition resulted in a 15-18% increase in Raf-1 expression compared to untreated controls.