Journal: Biomedicines

Article Title: Calpain Small Subunit Mediated Secretion of Galectin-3 Regulates Traction Stress

doi: 10.3390/biomedicines12061247

Figure Lengend Snippet: Extracellular Gal3 rescues the defect in traction stress in Gal3 silenced or capn4 −/− MEF cells. ( A ) Representative vector plots depict the magnitude and direction of traction stress exerted by a capn4 −/− MEF cell ( capn4 −/− ; left) and a capn4 −/− MEF cell with recombinant Gal3 added exogenously ( capn4 −/− + rGal3; right). The vectors indicate the direction and magnitude of traction stress. The color map illustrates magnitude. ( B ) The box and whisker graphs of average traction stress exerted by wild-type MEF cells, MEF cells treated with either control siRNA or Gal3 siRNA (siGal3), siGal3 rescued by expressing Gal3 plasmid (siGal3 rescued), siGal3 with recombinant Gal3 added exogenously (siGal3 + rGal3), capn4 −/− and capn4 −/− + rGal3. Data from 8–10 cells were collected for each cell type, typically 2 cells per chamber dish for 5 independent trials were collected. ( C ) Western blot of Gal3 from MEF-conditioned media, siControl, siGal3, siGal3 rescued, capn4 −/− , and control media (top), overexpressed Gal3 and endogenous Gal3 from cell lysates of siControl, siGal3 and siGal3 rescued (two middle), and GAPDH served as the loading control (bottom). A graph indicates endogenous Gal3 levels upon siRNA treatment. Data represent one of three independent trials. Whiskers represent variation in data about the mean. Student’s t -test * p < 0.05; *** p < 0.005; **** p < 0.001.

Article Snippet: Lyophilized recombinant Gal3 (rGal3) was purchased from R&D Systems and reconstituted at 250 μg/mL following the manufacturer’s protocol.

Techniques: Plasmid Preparation, Recombinant, Whisker Assay, Control, Expressing, Western Blot

Journal: Biomedicines

Article Title: Calpain Small Subunit Mediated Secretion of Galectin-3 Regulates Traction Stress

doi: 10.3390/biomedicines12061247

Figure Lengend Snippet: Gal3 mediates changes in size and localization of focal adhesions and enhances adhesion strength. ( A ) Anti-vinculin antibody (magenta) illustrates adhesion locations in wild type MEF cells (top row), capn4 −/− MEF cells (middle row), and capn4 −/− MEF cells treated with rGal3 (bottom row). Similarly, stress fibers are visualized by the staining of actin using fluorescent phalloidin (blue) in MEF, capn4 −/− and capn4 −/− + rGal3 cells (scale bar, 10 µm). The white, blue, or yellow arrows in Merge images indicate representative adhesion in different sizes: >1.5 µm 2 , <0.5 µm 2 , or 0.5–1.5 µm 2 , respectively. ( B ) Box and whisker graphs represent the average number of adhesions within three size ranges (<0.5 µm 2 , 0.5–1.5 µm 2 , or >1.5 µm 2 ) in each of the three cell lines. The number of focal adhesions was obtained from 25 to 30 cells for each cell line in three independent staining of duplicate chamber dishes per line, a total of >4000 adhesions. ( C ) Adhesion strength is expressed as a percentage of the number of cells that remain adhered after centrifugation. Data represent three independent trials, each performed in replicate, for each cell line. Whiskers represent the variation in data about the mean. Student’s t -test * p < 0.05; *** p < 0.005.

Article Snippet: Lyophilized recombinant Gal3 (rGal3) was purchased from R&D Systems and reconstituted at 250 μg/mL following the manufacturer’s protocol.

Techniques: Staining, Whisker Assay, Centrifugation

Journal: Biomedicines

Article Title: Calpain Small Subunit Mediated Secretion of Galectin-3 Regulates Traction Stress

doi: 10.3390/biomedicines12061247

Figure Lengend Snippet: Extracellular Gal3 influences migration speed and persistence. ( A ) Linear speed of wild-type MEF cells (MEF), cells treated with control siRNA (siControl) or Gal3 siRNA (siGal3), capn4 −/− MEF cells ( capn4 −/− ) and capn4 −/− MEF cells with rGal3 exogenously added ( capn4 −/− + rGal3) on fibronectin-coated glass coverslips. ( B ) Persistence of MEF, siControl, siGal3, capn4 −/− , and capn4 −/− + rGal3. An average of 20 cells were observed for each cell type. Each cell was observed for 2 h, and each group of cell types represents an independent trial (4 trials). Whiskers represent the variation in data. Student’s t -test; *** p < 0.005; n.s., not significant.

Article Snippet: Lyophilized recombinant Gal3 (rGal3) was purchased from R&D Systems and reconstituted at 250 μg/mL following the manufacturer’s protocol.

Techniques: Migration, Control

Journal: Biomedicines

Article Title: Calpain Small Subunit Mediated Secretion of Galectin-3 Regulates Traction Stress

doi: 10.3390/biomedicines12061247

Figure Lengend Snippet: Extracellular Gal3 is not required for sensing the homeostatic tension of the underlying substrate. ( A ) Representative images (10×) of MEF, siControl, siGal3, capn4 −/− , and capn4 −/− + rGal3 show cell morphology of the cells on fibronectin-coated hard and soft polyacrylamide substrates. siRNA-treated cells have been co-nucleofected with a GFP plasmid to ensure that only nucleofected cells are considered during counting (scale bar, 50 µm). ( B ) Bar graphs represent the percentage of round cells when seeded on hard or soft polyacrylamide substrates coated with fibronectin (at least 80 cells were examined for each condition, replicates of 3 trials). Error bars represent mean ± SEM. Student’s t -test * p < 0.05; *** p < 0.005; n.s., not significant.

Article Snippet: Lyophilized recombinant Gal3 (rGal3) was purchased from R&D Systems and reconstituted at 250 μg/mL following the manufacturer’s protocol.

Techniques: Plasmid Preparation

Journal: Biomedicines

Article Title: Calpain Small Subunit Mediated Secretion of Galectin-3 Regulates Traction Stress

doi: 10.3390/biomedicines12061247

Figure Lengend Snippet: Gal3 is not involved in sensing a locally applied mechanical stimulus. ( A ) Representative time-lapse images display cellular responses of MEF, siControl, siGal3, capn4 −/− , and capn4 −/− + rGal3 in response to an externally applied local mechanical stimulus. The migration trajectories prior to stimulation are indicated by red arrows. The yellow arrows in the second column denote the orientation in which the blunted needle is pushed onto the substrate (scale bar, 10 µm). ( B ) The table summarizes the rate of positive responses (rounding up of the cell or migrating away from the stimulus) observed for each cell type. The number of cells that showed a positive response is indicated, and collection of data from 1 of each cell type was attempted in each of the 10 trials.

Article Snippet: Lyophilized recombinant Gal3 (rGal3) was purchased from R&D Systems and reconstituted at 250 μg/mL following the manufacturer’s protocol.

Techniques: Migration

Journal: Biomedicines

Article Title: Calpain Small Subunit Mediated Secretion of Galectin-3 Regulates Traction Stress

doi: 10.3390/biomedicines12061247

Figure Lengend Snippet: c-Abl kinase, but not Arg kinase, regulates Gal3 secretion and traction force production, which is recovered by extracellular Gal3. ( A ) Western blot of Gal3 from conditioned media of MEF cells treated with either vehicle (DMSO) or Abl kinase inhibitor (STI571). ( B ) Quantification of secreted Gal3 levels of MEF cells with DMSO or STI571. Normalized intensity expressed in arbitrary units in the bar graphs is an average of three independent experiments. ( C ) Bar graph of average traction stress exerted by MEF cells treated with either DMSO (n = 19) or STI571 (n = 37), and MEF cells treated with STI571 with conditioned media from wild-type MEF cells (STI571 + MEF conditioned media; n = 15) or with rGal3 (STI571 + rGal3; n = 18) exogenously added to the media. ( D ) Western blot of Arg from cell lysates of wild-type MEF cells (MEF) and cells nucleofected with control siRNA (siControl) or Arg siRNA (siArg). GAPDH served as the loading control. ( E ) Quantification of Arg levels of MEF cells and cells with siControl or siArg. Normalized intensity expressed in arbitrary units in the bar graphs is an average of three independent experiments. ( F ) Western blot of Gal3 from conditioned media of MEF cells and cells treated with siControl or siArg. ( G ) Quantification of secreted Gal3 levels of MEF cells and cells with siControl or siArg. Normalized intensity expressed in arbitrary units in the bar graphs is an average of three independent experiments. Total protein level by Coomassie Blue staining is used to normalize the intensity of secreted Gal3. ( H ) Bar graph of average traction stress exerted by MEF cells (n = 14), and cells treated with siControl (n = 22) or siArg (n = 21). Error bars represent mean ± SEM. Student’s t -test *** p < 0.005; **** p < 0.001; n.s., not significant.

Article Snippet: Lyophilized recombinant Gal3 (rGal3) was purchased from R&D Systems and reconstituted at 250 μg/mL following the manufacturer’s protocol.

Techniques: Western Blot, Control, Staining

Journal: Biomedicines

Article Title: Calpain Small Subunit Mediated Secretion of Galectin-3 Regulates Traction Stress

doi: 10.3390/biomedicines12061247

Figure Lengend Snippet: Capn4-mediated Y107 phosphorylation of Gal3 is required for its secretion and subsequent generation of traction forces ( A ) Bar graph of average traction stress exerted by MEF cells nucleofected with pEGFP-N3 (empty vector; n = 25), pECFP-Gal3 (w.t. Gal3; n = 19), or pEGFP-Y107F-Gal3 (Y107F-Gal3; n = 25). ( B ) Western blot of Gal3 from conditioned media of MEF cells with empty vector, w.t. Gal3 or Y107F-Gal3. ( C ) Quantification of secreted Gal3 levels of MEF cells with empty vector, w.t. Gal3 or Y107F-Gal3. Total protein level by Coomassie Blue staining is used to normalize the intensity of secreted Gal3 ( D ) Western blot of overexpressed and endogenous Gal3 from cell lysates of empty vector, w.t. Gal3 or Y107F-Gal3 in MEF cells. GAPDH served as the loading control. ( E ) Quantification of overexpressed Gal3 levels of MEF cells with empty vector, w.t. Gal3 or Y107F-Gal3. Normalized intensity expressed in arbitrary units in the bar graphs is an average of three independent experiments. Error bars represent mean ± SEM. Student’s t -test * p < 0.05; n.s., not significant. Extracellular Gal3 does not alter FAK autophosphorylation. ( F ) Western blot of active and total β1 integrin from cell lysates of MEF cells with empty vector, w.t. Gal3 or Y107F-Gal3. GAPDH served as the loading control. ( G ) Quantification of active β1 integrin levels of MEF cells with empty vector, w.t. Gal3 or Y107F-Gal3. ( H ) Western blot of FAK Y397 phosphorylation from cell lysates of w.t. MEF cells, cells nucleofected with siControl or siGal3, capn4 −/− MEF cells, and capn4 −/− MEF cells with recombinant Gal3 added exogenously. GAPDH served as the loading control. ( I ) Quantification of FAK Y397 phosphorylation levels of MEF cells in C. Normalized intensity expressed in arbitrary units in the bar graphs is an average of three independent experiments. Error bars represent mean ± SEM. Student’s t -test * p < 0.05; n.s., not significant.

Article Snippet: Lyophilized recombinant Gal3 (rGal3) was purchased from R&D Systems and reconstituted at 250 μg/mL following the manufacturer’s protocol.

Techniques: Plasmid Preparation, Western Blot, Staining, Control, Recombinant

Journal: Biomedicines

Article Title: Calpain Small Subunit Mediated Secretion of Galectin-3 Regulates Traction Stress

doi: 10.3390/biomedicines12061247

Figure Lengend Snippet: Postulated pathway for Capn4-triggered Gal3 phosphorylation, secretion, and consequent traction force production. All direct interactions, phosphorylation, secretion, and clustering have been formulated based on experimental or published results. Capn4, together with large catalytic subunits of Capn1 and 2 holoenzymes, modulates proteolytic and mechanosensing pathways. On the other hand, Capn4 governs the traction force pathway, independently from large subunits. Capn4 may help in stimulating c-Abl kinase activation resulting in tyrosine phosphorylation of Gal3. c-Abl kinase phosphorylates Gal3 on three tyrosine residues: Y79, Y107, and Y118. Tyrosine-phosphorylated Gal3, on Y107, could be secreted through exosomes or by other non-classical pathways. Secreted Gal3 can form oligomers through its N-terminal domain in the ECM and can link the cell to the ECM through clustering and/or activating cell surface receptors including integrins. As a result, active β1 integrin-signaling may occur in an FAK-independent, myosin-II-mediated manner to produce traction forces during cell migration.

Article Snippet: Lyophilized recombinant Gal3 (rGal3) was purchased from R&D Systems and reconstituted at 250 μg/mL following the manufacturer’s protocol.

Techniques: Activation Assay, Migration