FSP is expressed by variable numbers of cells within LAM nodules.

<p>There is heterogeneity between different LAM lung donors in the number of anti-FSP immunoreactive cells in LAM nodules. Panels (a) and (b) at x10 magnification, (c) at x20, (d) at x40. (e) and (f) show anti-tuberin reactive and non-reactive cells (arrowed) within nodules from two donors, x20.</p

Fibroblasts protect 621–101 cells against apoptosis.

<p>(a) Culture of either 621–101 cells or fibroblasts on glass in serum free conditions results in cell death. Co-culture of the two cell types under the same conditions results in formation of viable cell colonies. (b) Under serum-free conditions, 621–101 cells have a basal rate of apoptosis of 8% assessed by TUNEL positive nuclear staining (arrowed). TUNEL positivity is reduced by half in the presence of fibroblast conditioned medium (*p = 0.032).</p

LAM nodules express fibroblast markers.

<p>LAM nodules react with antibodies against alpha-Smooth Muscle Actin, Fibroblast Surface Protein (FSP), Fibroblast Activation Protein (FAP) and S100A4. Left panels are x20 magnification and right are inset area at x40 taken from serial sections of a representative donor. Fibroblast markers display different expression patterns within LAM tissue, with anti-FSP reacting with 30–70% of cells within a nodule, anti-FAP detecting the majority of cells and S100A4 detecting only small nests of cells within nodules.</p

Characteristics of wild-type fibroblast-like cells obtained from LAM lungs.

<p>(a) Explant culture of lung tissue from patients with LAM yields a fibroblast-like population with spindle shaped morphology under phase contrast microscopy (phase) and immunoreactivity to anti-alpha Smooth Muscle Actin, and fibroblast markers FSP, S100A4 and TE-7 (these cells are also DAPI stained for clarity). (b) Western blot of lysates of fibroblast like-cells from two donors with LAM compared with TSC-2 mutation bearing 621–101 cells. Unlike 621–101 cells fibroblast-like cells have full length tuberin, and suppression of phospho-S6 in the absence of serum.</p

TSC1 and TSC2 differentially regulate migration and invasiveness.

<p>A: Representative membrane showing migration of serum-deprived <i>Tsc2^+/+, Tsc2^−/−, Tsc1^+/+,</i> and <i>Tsc1^−/−</i> MEFs. Serum-deprived cells were placed on collagen-saturated membranes in serum-free medium, and allowed to migrate in the Boyden chamber for 4 h in the absence of any stimuli. Then membranes were fixed, stained with Hemacolor stain set, and analyzed using Gel Pro software. B: Statistical analysis of migration experiments. Data represent mean values ± SE from measurements performed in triplicate from six separate experiments by ANOVA (Bonferroni-Dunn test). Basal migration of <i>Tsc2^+/+</i> cells was taken as 1 fold. C: <i>Tsc2^−/−</i> MEFs have increased invasiveness. Invasiveness of serum-deprived <i>Tsc2^+/+, Tsc2^−/−, Tsc1^+/+,</i> and <i>Tsc1^−/−</i> MEFs was analyzed using the Cultrex 96 Well BME Cell Invasion Assay kit according to manufacturer’s protocol. Data represent the percentage of invaded cells per total number of cells taken as 100%. Data represent mean values ± SE from two independent experiments by ANOVA (Bonferroni-Dunn test).</p

Morphology and dynamics of <i>Tsc1^−/−</i> and <i>Tsc2^−/−</i> MEFs during wound closure.

<p>Phase-contrast micrographs of cell motility during wound closure at 4 h after wound scraping. Arrows indicate direction of cell movement. Images were taken using a Nikon Eclipse TE2000-E microscope at 100X magnification in the phase contrast channel. Images are representative from three independent experiments. Scale bar, 100 µm.</p

mTOR and Rictor mediate cell migration.

<p>A: siRNA-induced mTOR depletion inhibits <i>Tsc2^−/−</i> MEF migration. <i>Tsc2^−/−</i> MEFs were transfected with siRNA mTOR or control siRNA followed by migration assay. Migration of control siRNA-transfected cells was taken as 100%. B: Rictor is required for serum-induced MEF migration. <i>Tsc2^−/−</i> MEFs were transfected with siRNA Rictor,siRNA Raptor, or control siRNA, and migration assays were subsequently performed. Migration of <i>Tsc2^−/−</i> MEFs transfected with control siRNA was taken as 1 fold. C: Rictor is required for cell migration under nutrient-replete conditions but is not sufficient to modulate all migration. Migration assays were performed with <i>Rictor^+/+</i> and <i>Rictor^−/−</i> MEFs under basal (unstimulated) and serum-stimulated (10% FBS) conditions. Migration of <i>Rictor^+/+</i> MEFs under basal conditions was taken as 100%.</p

mTOR and Rictor, but not Raptor, modulate stress fiber formation in TSC2-null ELT3 cells.

<p>A: Cells were co-microinjected with siRNA mTOR, siRNA Rictor, siRNA Raptor, or control siRNA, and GFP to detect microinjected cells, serum-deprived followed by F-actin staining. Images were taken using a Nikon Eclipse TE-2000-E Microscope at 400X magnification. Scale bar, 20 µm. B: Statistical analysis. Data represent the percentage of cells without stress fibers per total number of microinjected cells taken as 100%. Data represent mean values ± SE by ANOVA (Bonferroni Dunn). C: mTOR activity is required for stress fiber formation in TSC2-null cells. Cells transfected with HA-tagged mTOR-KD were serum-deprived followed by staining with rhodamine phalloidin and immunostaining with anti-HA antibody to detect transfected cells. Data represent the percentage of cells without stress fibers per total number of transfected cells taken as 100%. Data represent mean values ± SE by ANOVA (Bonferroni Dunn). Scale bar, 20 µm.</p

siRNATSC1 and siRNATSC2 induce opposite effects on NIH 3T3 fibroblast migration.

<p>Cells were transfected with siRNA TSC1 (A), siRNA TSC2 (B), or control siRNA. 48 h post-transfection, protein levels were detected by immunoblot analysis with anti-TSC1 or anti-TSC2 antibodies. C, Upper panel: Representative image of hemacolor-stained membrane with migrated NIH 3T3 fibroblasts for 4 h. 3T3 fibroblasts were transfected with siRNA TSC1, siRNA TSC2, and siGLO RISC-Free siRNA as control cells, serum-deprived followed by migration assay in the presence or absence of 10 ng/ml PDGF performed in triplicate for each experimental condition. C, Lower panel: Statistical analysis of NIH 3T3 cell migration. Data represent mean values ± SE from two independent experiments, six repetitions in each experiment. *P<0.01 for siRNA TSC1 vs. control siRNA, **P<0.001 for siRNA TSC2 vs. control siRNA by ANOVA (Bonferroni-Dunn).</p

TSC1 and TSC2 re-expression or siRNA-induced knock-out validate their differential role in regulating cell migration.

<p>A: Re-expression of TSC1 rescues <i>Tsc1^−/−</i> MEFs migration. MEFs were transiently transfected with TSC1 for 48 h, serum deprived followed by migration assay. B: Re-expression of TSC2 inhibits <i>Tsc2^−/−</i> MEF migration. <i>Tsc2^+/+</i> and <i>Tsc2^−/−</i> MEFs were transiently transfected with TSC2, serum deprived followed by migration assays. TSC1 and TSC2 re-expression were confirmed by immunoblot analysis of equalized in protein content samples. Migration of <i>Tsc1^−/−</i> (A) and <i>Tsc2^−/−</i> MEFs (B) transfected with control plasmid was taken as 1 fold. Data represent mean values ± SE from two different experiments with three replicates for each condition by ANOVA (Bonferroni-Dunn test). C: Downregulation of TSC2, but not TSC1, promotes migration of wild type MEFs. <i>Tsc2^+/+</i> and <i>Tsc1^+/+</i> MEFs were transfected with siRNA TSC1, siRNA TSC2, and control siRNA. 48 h post-transfection, migration assays were performed. Protein levels were detected by immunoblot analysis with specific anti-TSC1 and anti-TSC2 antibodies under the same experimental conditions. Migration of wild type <i>Tsc1^+/+</i> (right) or <i>Tsc2^+/+</i> MEFs (left) transfected with siGLO RISC-Free siRNA was taken as 1 fold. Data represent mean values ± SE from measurements performed in triplicate from two separate experiments.</p