bioimagesUnknownEmanuela FrittoliThe Journal of Cell Biologyhttps://www.ebi.ac.uk/biostudies/studies/S-JCBD-201403127JCB

Doxycycline-inducible RAB5AS34N-expressing MDA-MB-231 cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Three weeks after injection, mice were fed with doxycyline to induce transgene expression. Primary tumors were allowed to develop for 4–6 weeks before removal by mastectomy. Metastases were analyzed 4 weeks after removal of the primary tumor.

Serum-starved HeLa cells were transfected with dominant-negative GFP-RAB11S22N, plated onto fluorescently-conjugated gelatin (red), and stimulated with HGF for 3 hrs. Cells were stained with phalloidin (green).

Control MCF10.DCIS.com cells were injected subcutaneously into NOD SCID/IL2Rgamma null (NSG) mice. Four days after injection, mice were fed with doxycycline to induce transgene expression. Haematoxylin and eosin staining of xenograft sections was performed at 3 weeks after initiation of doxycycline treatment.

GFP-LifeAct-RAB5A-overexpressing HeLa cells were grown for 14 days on an organotypic collagen matrix culture that had been pre-conditioned with U937 macrophages in the presence of the broad-spectrum metalloprotease inhibitor GM6001.

RAB5A-expressing HeLa cells were processed for immunofluorescence and analyzed by TIRF microscopy. F-actin (white) was detected with phalloidin.

Control MCF10.DCIS.com cells were injected subcutaneously into NOD SCID/IL2Rgamma null (NSG) mice. Four days after injection, mice were fed with doxycycline to induce transgene expression. Haematoxylin and eosin staining of xenograft sections was performed at 1 week after initiation of doxycycline treatment.

Serum-starved HeLa cells were transfected with siRNA against MT1-MMP, plated onto fluorescently-conjugated gelatin (red), and stimulated with HGF for 3 hrs. Cells were stained with phalloidin (green).

Control MCF10.DCIS.com cells were injected subcutaneously into NOD SCID/IL2Rgamma null (NSG) mice. Four days after injection, mice were fed with doxycycline to induce transgene expression. Immunohistochemical analysis for SMA expression was performed on xenograft sections at 1 week after initiation of doxycycline treatment.

GFP-LifeAct-RAB5A-expressing HeLa cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Primary tumors were allowed to develop for 4 weeks before removal by mastectomy. Formalin-fixed, paraffin-embedded lung sections were analyzed by immunohistochemical analysis for GFP expression 4 weeks after removal of the primary tumor.

RAB5A-expressing HeLa cells were plated onto fluorescently-conjugated gelatin overnight under serum-starved conditions and then stimulated with suboptimal doses of HGF (1 ng/ml). RAB5A (white) was detected with anti-RAB5A antibody.

Doxycycline-induced RAB5AS34N-expressing MDA-MB-231 cells plated onto fluorescently conjugated gelatin were stimulated with HGF (100 ng/ml) for 3 hrs. Shown is a merged image of cell staining for F-actin using phalloidin (green) and of the signal from the fluorescently conjugated gelatin (red).

Intratumoral motion analysis of control HeLa cells migrating within a primary tumor was obtained by overlaying 10 differentially colored, consecutive frames of time-lapse intravital recording. Coloring indicates motile cells. See also Video 2 of the associated paper.

Control MDC10.DCIS.com cells were grown on a thick 1:1 Matrigel:type I collagen mixture and overlaid with diluted Matrigel (2 mg/mm). After 1 week, cells were treated with doxycycline to induce transgene expression and mock-treated for HGF.

Control MCF10.DCIS.com cells were injected subcutaneously into NOD SCID/IL2Rgamma null (NSG) mice. Four days after injection, mice were fed with doxycycline to induce transgene expression. Immunohistochemical analysis for SMA expression was performed on xenograft sections at 3 weeks after initiation of doxycycline treatment.

Serum-starved HeLa cells were transfected with siRNA against RAB4A and B, plated onto fluorescently-conjugated gelatin (red), and stimulated with HGF for 3 hrs. Cells were stained with phalloidin (green).

Shown at 40x magnification is immunohistochemical analysis for RAB5 on a formalin-fixed, paraffin-embedded samples of a human lymph node metastasis arising from the primary infiltrating breast tumor also shown in this figure.

Mixtures (0.5x10e6 each) of eFluor®670-labeled scramble siRNA-transfected cells (red) and CFSE-labeled RAB5A,B,C-depleted MDA-MB-231 cells (green) were co-injected into the tail vein of NOD SCID/IL2Rgamma null (NSG) mice. Labeled cells present in sections of the lungs such as the one shown here were counted 96 hours after injection.

Serum-starved HeLa cells were transfected with dominant-negative RAB4S22N, plated onto fluorescently-conjugated gelatin (red), and stimulated with HGF for 3 hrs. Cells were stained with phalloidin (green).

Serum-starved HeLa cells were transfected with dominant-negative RAB4S22N and stimulated with HGF for 3 hrs. Cells were immunolabeled for RAB4 (white).

Control HeLa cells were processed for immunofluorescence, and F-actin was detected with phalloidin. Shown is a confocal XZ-section of a cell plated onto fluorescently-conjugated gelatin (red) and stained with phalloidin (green).

RAB5A-expressing HeLa cells were processed for immunofluorescence and analyzed by TIRF microscopy. RAB5A (white) was detected with anti-RAB5 antibody.

Control serum-starved HeLa cells were transfected with scrambled siRNA, plated onto fluorescently-conjugated gelatin (red), and left in serum-free conditions for 3 hrs. Cells were stained then with phalloidin (green).

Control GFP-LifeAct-expressing HeLa cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Primary tumors were allowed to develop for 4 weeks before removal by mastectomy. Formalin-fixed, paraffin-embedded lung sections were analyzed by immunohistochemical analysis for GFP expression 4 weeks after removal of the primary tumor. Shown is a magnified view of a metastasis.

GFP-LifeAct-control HeLa cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Four weeks after injection, the invasive front (green = GFP-LifeAct) and the collagen structure (grey = SHG) of the resulting tumor were analyzed by two-photon microscopy. Shown is a projection of ~40 serial Z sections.

Intratumoral motion analysis of RAB5-expressing HeLa cells within a primary tumor was obtained by overlaying 10 differentially colored, consecutive frames of time-lapse intravital recording. Coloring indicates motile cells. See also Videos 1 and 2 of the associated paper.

Doxycycline-induced RAB5AS34N-expressing MCF10.DCIS.com cells plated onto fluorescently conjugated gelatin were stimulated with HGF (100 ng/ml) for 3 hrs. Shown is a merged image of cell staining for F-actin using phalloidin (green) and of the signal from the fluorescently conjugated gelatin (red).

Serum-starved HeLa cells were transfected with dominant-negative GFP-RAB11S22N, plated onto fluorescently-conjugated gelatin (red), and stimulated with HGF for 3 hrs. Cells were processed for epifluorescence to detect GFP-RAB11S22N.

Control HeLa cells were plated onto fluorescently-conjugated gelatin (red) overnight under serum-starved conditions and then stimulated with suboptimal doses of HGF (1 ng/ml). F-actin (green) was detected with phalloidin.

Mixtures (0.5x10e6 each) of eFluor®670-labeled scramble siRNA-transfected cells (red) and CFSE-labeled RAB5A,B,C-depleted MDA-MB-231 cells (green) were co-injected into the tail vein of NOD SCID/IL2Rgamma null (NSG) mice. Labeled cells present in sections of the lungs such as the one shown here were counted 2 hours after injection.

GFP-LifeAct-control (left) or GFP-LifeAct-RAB5A-overexpressing (right) HeLa cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Four weeks after injection, the invasive fronts (green = GFP-LifeAct) and the collagen structure (bottom row, grey = SHG) of the resulting tumors were analyzed by two-photon microscopy.

Control MDA-MB-231 cells plated onto fluorescently conjugated gelatin were stimulated with HGF (100 ng/ml) for 3 hrs. Shown is a merged image of cell staining for F-actin using phalloidin (green) and of the signal from the fluorescently conjugated gelatin (red).

Doxycycline-inducible RAB5AS34N-expressing MDA-MB-231 cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Three weeks after injection, mice were fed with doxycyline to induce transgene expression. Primary tumors were allowed to develop for 4–6 weeks before removal by mastectomy. Lungs were analyzed 4 weeks after removal of primary tumor.

Control GFP-LifeAct HeLa cells were grown for 14 days on an organotypic collagen matrix culture that had been pre-conditioned with U937 macrophages in the presence of the broad-spectrum metalloprotease inhibitor GM6001.

Control MCF10.DCIS.com cells plated onto fluorescently conjugated gelatin were stimulated with HGF (100 ng/ml) for 3 hrs. Shown is a merged image of cell staining for F-actin using phalloidin (green) and of the signal from the fluorescently conjugated gelatin (red).

Shown at 10x magnification is immunohistochemical analysis for RAB5 on a formalin-fixed, paraffin-embedded sample of a human primary infiltrating breast tumor.

Mixtures (0.5x10e6 each) of eFluor®670-labeled scramble siRNA-transfected cells (red) and CFSE-labeled RAB5A,B,C-depleted MDA-MB-231 cells (green) were co-injected into the tail vein of NOD SCID/IL2Rgamma null (NSG) mice. Labeled cells present in sections of the lungs such as the one shown here were counted 24 hours after injection.

Control MDA-MB-231 cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Three weeks after injection, mice were fed with doxycyline to induce transgene expression. Primary tumors were allowed to develop for 4–6 weeks before removal by mastectomy. Metastases were analyzed 4 weeks after removal of the primary tumor.

Doxycycline-inducible RAB5AS34N-expressing MDC10.DCIS.com cells were grown on a thick 1:1 Matrigel:type I collagen mixture and overlaid with diluted Matrigel (2 mg/mm). After 1 week, cells were treated with doxycycline to induce transgene expression and treated with HGF (20 ng/ml).

Control serum-starved HeLa cells were transfected with scrambled siRNA, plated onto fluorescently-conjugated gelatin (red), and stimulated with HGF for 3 hrs. Cells were stained then with phalloidin (green).

Control GFP-LifeAct HeLa cells were grown for 14 days on an organotypic collagen matrix culture that had been pre-conditioned with U937 macrophages in the presence of a vehicle control.

Doxycycline-inducible RAB5AS34N-expressing MCF10.DCIS.com cells were injected subcutaneously into NOD SCID/IL2Rgamma null (NSG) mice. Four days after injection, mice were fed with doxycycline to induce transgene expression. Haematoxylin and eosin staining of xenograft sections was performed at 3 weeks after initiation of doxycycline treatment.

RAB5A-expressing HeLa cells were processed for immunofluorescence, and F-actin was detected with phalloidin. Shown is a confocal XZ-section of a cell plated onto fluorescently-conjugated gelatin (red) and stained with phalloidin (green).

Doxycycline-inducible control MDA-MB-231 cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Three weeks after injection, mice were fed with doxycyline to induce transgene expression. Primary tumors were allowed to develop for 4–6 weeks before removal by mastectomy. Lungs were analyzed 4 weeks after removal of the primary tumor.

RAB5AAS34N-expressing HeLa cells were plated onto fluorescently-conjugated gelatin (red) overnight under serum-starved conditions and then stimulated with suboptimal doses of HGF (1 ng/ml). F-actin (green) was detected with phalloidin.

Control GFP-LifeAct-expressing HeLa cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Primary tumors were allowed to develop for 4 weeks before removal by mastectomy. Formalin-fixed, paraffin-embedded lung sections were analyzed by haematoxylin and eosin staining 4 weeks after removal of the primary tumor.

Doxycycline-inducible RAB5AS34N-expressing MCF10.DCIS.com cells were injected subcutaneously into NOD SCID/IL2Rgamma null (NSG) mice. Four days after injection, mice were fed with doxycycline to induce transgene expression. Immunohistochemical analysis for SMA expression was performed on xenograft sections at 1 week after initiation of doxycycline treatment.

Doxycycline-inducible RAB5AS34N-expressing MDC10.DCIS.com cells were grown on a thick 1:1 Matrigel:type I collagen mixture and overlaid with diluted Matrigel (2 mg/mm). After 1 week, cells were treated with doxycycline to induce transgene expression and mock-treated for HGF.

GFP-LifeAct-RAB5A-overexpressing HeLa cells were grown for 14 days on an organotypic collagen matrix culture that had been pre-conditioned with U937 macrophages in the presence of a vehicle control.

Serum-starved HeLa cells were transfected with siRNA against RABENOSYN-5, plated onto fluorescently-conjugated gelatin (red), and stimulated with HGF for 3 hrs. Cells were stained with phalloidin (green).

GFP-LifeAct-RAB5A-expressing HeLa cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Primary tumors were allowed to develop for 4 weeks before removal by mastectomy. Formalin-fixed, paraffin-embedded lung sections were analyzed by immunohistochemical analysis for GFP expression 4 weeks after removal of the primary tumor. Shown is a magnified view of metastases.

Shown at 40x magnification is immunohistochemical analysis for RAB5 on a formalin-fixed, paraffin-embedded sample of a human primary infiltrating breast tumor.

Control GFP-LifeAct-expressing HeLa cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Primary tumors were allowed to develop for 4 weeks before removal by mastectomy. Formalin-fixed, paraffin-embedded lung sections were analyzed by immunohistochemical analysis for GFP expression 4 weeks after removal of the primary tumor.

GFP-LifeAct-RAB5A-expressing HeLa cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Primary tumors were allowed to develop for 4 weeks before removal by mastectomy. Formalin-fixed, paraffin-embedded lung sections were analyzed by haematoxylin and eosin staining 4 weeks after removal of the primary tumor.

GFP-LifeAct-RAB5A-expressing HeLa cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Primary tumors were allowed to develop for 4 weeks before removal by mastectomy. Metastases were analyzed 4 weeks after removal of the primary tumor.

Control MDC10.DCIS.com cells were grown on a thick 1:1 Matrigel:type I collagen mixture and overlaid with diluted Matrigel (2 mg/mm). After 1 week, cells were treated with doxycycline to induce transgene expression and treated with HGF (20 ng/ml).

Doxycycline-inducible RAB5AS34N-expressing MCF10.DCIS.com cells were injected subcutaneously into NOD SCID/IL2Rgamma null (NSG) mice. Four days after injection, mice were fed with doxycycline to induce transgene expression. Immunohistochemical analysis for SMA expression was performed on xenograft sections at 3 weeks after initiation of doxycycline treatment.

Doxycycline-inducible RAB5AS34N-expressing MCF10.DCIS.com cells were injected subcutaneously into NOD SCID/IL2Rgamma null (NSG) mice. Four days after injection, mice were fed with doxycycline to induce transgene expression. Haematoxylin and eosin staining of xenograft sections was performed at 1 week after initiation of doxycycline treatment.

Control GFP-LifeAct HeLa cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Primary tumors were allowed to develop for 4 weeks before removal by mastectomy. Metastases were analyzed 4 weeks after removal of the primary tumor.

GFP-LifeAct-RAB5A-overexpressing HeLa cells were injected into the mammary fat pads of NOD SCID/IL2Rgamma null (NSG) mice. Four weeks after injection, the invasive front (green = GFP-LifeAct) and the collagen structure (grey = SHG) of the resulting tumor were analyzed by two-photon microscopy. Shown is a projection of ~40 serial Z sections.

Shown at 10x magnification is immunohistochemical analysis for RAB5 on a formalin-fixed, paraffin-embedded sample of a human lymph node metastasis arising from the primary infiltrating breast tumor also shown in this figure.

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