Fig 1: Kindlin-3 expression in human tumorsA. Downregulation in Kindlin-3 mRNA observed in human tumors from several origins compared to normal corresponding tissues Relative expression of Kindlin-3 mRNA to the reference gene PPIA assessed by qRT-PCR (a) Melanoma tissues compared to benign melanocytic naevi and normal skin tissues (n=71, n=11 and n= 17 respectively); (b) Primary breast tumors compared to normal breast tissues (n=129 and n= 23 respectively); (c) Kidney cancers compared to normal kidney (n=15 and n= 11 respectively); (d) 18 matched pairs of lung cancer versus normal lung tissues from the same patients. P values were calculated using two-sided Student t test. Means and 95% confidence intervals are shown;*, P <0.05. B. Immunohistochemical staining of Kindlin-3 in sections of human cancer and normal tissues (a) Expression of Kindlin-3 in endothelial cells within blood vessels, as positive control; (b and c) naevi and melanoma; (d and e) normal and breast cancer (f and g) normal and kidney cancer respectively. Higher magnification images are shown below each image for breast and kidney tissues. The illustrated immunostaining shown was performed with the Genosphere anti-Kindlin-3 Ab. Images are representative of 6 tumor specimens for each cancer type. Similar staining was obtained using the Abcam anti-Kindlin-3 Ab. Note that staining is always lower in the corresponding cancer tissues. Scale bar = 20μm. C. Specificity of the anti-Kindlin-3 antibodies used Western blot analyses of N-terminal GST-tagged recombinant Kindlin-1 (300ng), Kindlin-2 (300ng) or Kindlin-3 (150ng) proteins (H00055612-P01, H00010979-P01, H00083706-P01; Novus Biologicals Cambridge, UK). Blots were probed with 2 different kindlin-3 antibodies (upper panels) or GST antibody; Millipore) (lower panels). A single band corresponding to GST-tagged Kindlin-3 was obtained with both antibodies used, ab68040 (Abcam, Cambridge MA, USA) and the custom made anti-Kindlin-3 antibody produced against a human Kindlin-3 specific peptide corresponding to amino acids 19 –31 (RVFVGEEDPEAES) and affinity purified on a sepharose matrix (Genosphere, France). Importantly, neither of the two antibodies recognized GST tagged Kindlin-1 or Kindlin-2 even when 2x quantity of these recombinant proteins was loaded.
Fig 2: Kindlin-3 silencing enhances malignant properties of tumor cells AKindlin-3 silencing decreases cell adhesion and increases cell migration and invasion a. Melan-a (normal melanocytes) were silenced for Kindlin-3 (Kindlin-3 siRNA) or transfected with scrambled siRNA (control) and subjected to cell adhesion assay on fibronectin (Fn), collagen I (Col1) or collagen III (Col3), to cell migration and to invasion assays. Bars represent means from three independent experiments carried out in triplicate; error bars refer to 95% confidence intervals; *, P <0.05. b-e. Tumor cells, breast (MDA-MB-231) and melanoma (SKMEL-28) were silenced by siRNA transfection for Kindlin-3 expression. Kindlin-3 expression was analyzed by qRT-PCR and Immunofluorescence. They were then analysed for (c) adhesion, (d) migration and (e) invasion. Bars represent means from three independent experiments carried out in triplicate; error bars refer to 95% confidence intervals; *, P <0.05. B. Time-lapse pictures of representative adhering/migrating live cells SKMEL28 cells (Kindlin-3 siRNA and control siRNA transfected) transduced by CellLight® Actin-RFP were tracked for 24h (3min interval). Imaging was performed using 40x objective on a Nikon BioStation IM Live Cell Recorder. Time-lapse montages show representative images over a period of 11hr. Scale bar = 50 μm. Migrating cells were more often seen in Kindlin-3 siRNA cells with densely packed actin stress fibers and induction of filopodia formation in the direction of migration (arrows). Note that the filopodia seen on the left side of the cell at 2hr is no longer seen at 4hr (arrows), and that the cell moved to the right of the field at 11hr. Also, stress fibers appear disorganized, in particular at the later time points. By contrast, in control, more cells were engaged in adhesion process displaying a normal distribution of filamentous actin (actin in red). See corresponding supplemental movies 1 (control siRNA) and 2 (Kindlin-3 siRNA). C. Kindlin-3 silencing enhances tumor cell growth and clonogenicity of tumor cells a. The proliferation of MDA-MB-231 and SKMEL28 Kindlin-3 or control siRNA transfected cells was measured by MTS assay. Results are presented as the mean of three independent experiments carried out in triplicate; bars refer to 95% confidence intervals; *, P <0.05. b. Kindlin-3 siRNA increased the number of colonies formed with MDA-MB-231 and SKMEL-28 cells. Representative images of three independent experiments carried out in triplicate are shown. c. Kindlin-1 and Kindlin-2 expression analyzed by qRT-PCR using specific primer and probe sets were not altered by Kindlin-3 siRNA transfection. Columns, means of three independent experiments carried out in triplicate; bars refer to 95% confidence intervals.
Fig 3: Kindlin-3 knockdown promotes metastasis formation through Integrin mediated effects AStable Kindlin-3 knockdown enhances in vitro invasion and in vivo tumor growth a. Transcript quantification by qRT-PCR of Kindlin-1, Kindlin-2 and Kindlin-3 in melanoma SKMEL28 cells stably transfected with Kindlin-3 shRNA (top); Columns, means of three independent experiments carried out in triplicate; bars refer to 95% confidence intervals; *, P <0.05. Cell invasion on matrigel coated filters using a modified Boyden chamber (bottom). Stably transfected SKMEL28 cells were plated at a density of 2.0 × 104 per insert. Medium with 10% FBS was in the lower chamber as a chemo-attractant. After 24h of incubation, invasive cells on the lower surface were counted after fixing and staining. Shown are representative data of three independent experiments. b. Subcutaneous xenograft mouse model with Kindlin-3 inactivation. Stable Kindlin-3 silencing by shRNA in SKMEL28 cells enhanced tumor growth. Representative photos of tumors 5 weeks after injection. Results represent two independent experiments with 5 mice in each group. Columns, means of tumor volume; bars refer to 95% confidence intervals; *, P=0.036. c. Immunohistochemical (IF) analysis of Kindlin-3 in xenograft tumors from control or Kindlin-3 shRNA SKMEL28 cells 5 weeks after injection, confirms Kindlin-3 silencing in cells with Kindlin-3 shRNA. Representative IF images of 20 primary tumors analyzed are shown. d. Transcript quantification of Kindlin1, Kindlin-2 and Kindlin-3 in the xenograft tumors by qRT-PCR using human specific primer and probe sets. Columns, means of 20 primary tumors analyzed; bars refer to 95% confidence intervals; *, P<0.05. B. Kindlin-3 knockdown promotes lung metastasis formation in SKMEL28 melanoma model a. Number of metastatic lung foci in Kindlin-3 shRNA-GFP and Control shRNA-GFP injected mice. Columns, means of 20 analyzed lungs; bars refer to 95% CI; *, P<0.05. b. Fluorescence analyses of lung tissue sections from GFP-control shRNA and GFP-Kindlin-3 shRNA inoculated SKMEL28 tumor cells showing GFP-positive micrometastases in mice bearing Kindlin-3-shRNA. Representative IF images of 20 lung tissues analyzed are shown. C. Kindlin-3 knockdown promotes metastasis formation in MDA-MB-231 breast cancer model a. Tumor growth analysis of Kindlin-3 shRNA vs control xenografts. Results represent two independent experiments with 5 mice in each group. Columns, means of tumor volume; bars refer to 95% confidence intervals. b. Number of metastatic liver foci in Kindlin-3 shRNA-GFP and Control shRNA-GFP injected mice. Columns, means of 20 livers analyzed; bars refer to 95% confidence intervals; *, P<0.05. c. Fluorescence analyses of liver, lung and lymph node tissue sections from GFP-control shRNA and GFP-Kindlin-3 shRNA inoculated MDA-MB-231 tumor cells showing larger GFP-positive micrometastases in mice bearing Kindlin-3-shRNA. Representative IF images are shown from 20 mice. D. Kindlin-3 silencing reduced Integrin-β3 phosphorylation (p-Tyr785) and talin-Integrin interaction in vivo shown by Immunofluorescence analysis (left) and In situ PLA (right). Nuclei were stained with DAPI (blue). Representative images of 10 primary tumors analyzed are shown.
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