Middle-aged and aged serum induces some senescent cells.

<p><b>A</b> Representative images of SA-β of control cells, cells treated with 100μM H₂O₂, 10% v/v young, middle-aged and aged serum. Graph shows the quantification of SA-βgal positive cells versus total cells stained with Mayer´s Hematoxylin. Arrow head indicated positive cell for SA-β. <b>B</b> Immunofluorescence of histone H2A.X phosphorylated at Ser139 and DNA stained by Hoechst. All assays were performed in quadruplicate (n = 8 young, n = 8 middle-aged, n = 8 aged).</p

Middle-aged and aged serum do not induces GF proliferation.

<p><b>A</b> Representative images of Immunofluorescence for Ki67 and Hoechst, GF treated with young serum (18 yo), middle-aged serum (30 yo) and aged serum (65 yo). <b>B</b> Graph indicated the quantification of ki67(+)cells/total cells. <b>C</b> Flow cytometry analysis showed that young serum promotes the proliferation of GF, graph of PI versus count of cells, control (green), young serum (red), middle-aged serum (gray) and aged serum (black). Asterisks indicate statistically significant differences between control and young serum treatment p = 0.001. Bars indicate standard error. All assays were performed in quadruplicate. (n = 8 young, n = 8 middle-aged, n = 8 aged).</p

Old rat serum present higher levels of pro-inflammatory cytokine.

<p><b>A</b> Representative image of the cytokine proteome array from young and old rat serum <b>B</b> Graph indicated the quantification of the pixel mean density of each cytokine. <b>C</b> TNF distribution in wound from young and old rats after 7 days of repair 5X, 40X and inset of 100X.<b>D</b> TNF distribution in non wound gingiva 5X (scale bar 150 um) and 40X (scale bar 50um). <b>E</b> Negative control 5X and 40X.</p

Old rats present impaired wound healing compared to young rats.

<p>Full-thickness wounds were made in the palatal gingiva. <b>A.</b> Sections of wounded gingiva from young and aged rats were stained with Masson Trichrome at day 7 post wounding. Images were scanned with nanozoomer at 2,5X, 5X and 20X. Scale bar:400μm. <b>B</b> Quantification of area granulation tissue in wounded area. <b>C</b> Immunostained for Ki67 (green) and vimentin (red). Scale bar 50μm <b>C</b> Quantification of positive cells for Ki67/total cells in epithelial and the connective tissue in the wound area. Arrowhead indicated focus of collagen. Bars indicate standard error. All assays were performed in quadruplicate. Asterisks indicate statistically significant differences (p = 0.0285 for Epithelial Tissue) and p = 0.00286 for Connective Tissue. n = 5.</p

TRPM4 promotes cellular contractility and wound healing.

<p>A) Three-dimensional (3D) invasion assay of TREx293-TRPM4 cells. TRPM4 expression was induced by adding 1 μg/mL Tetracycline to the media (n = 3, p<0.05 compared to control). *, significant difference (p<0.05) versus non-stimulated cell control. Statistical analysis was performed using a Mann-Whitney test. B) Fibroblasts migration from skin grafts (E) treated with DMSO or 20 μM 9-phenanthrol. Grafts were fixed 5 days after explant and labeled with Hoechst (blue). Scale bar: 1 mm. C) Quantification of the experiment shown in (B). The data correspond to cell counts from 3 independent experiments (13 and 12 explants for DMSO and 9-phenanthrol treatments, respectively). *, significant difference (p<0.05) versus DMSO controls. Statistical analysis was performed using a Mann-Whitney test. D) Three dimensional contraction assay of MEFs transfected with shRNA^Scramble and shRNA^TRPM4. Contraction was induced by incubating the immersed cells with 10% v/v serum for 48 h. The upper graph represents the collected data for 4 independent assays. *, significant difference (p<0.05) versus shRNA^Scramble controls. **, significant difference (p<0.05) versus shRNA^TRPM4. Statistical analysis was performed using a two-way ANOVA test. E) Frames (t = 0 and 14 min) from time lapse of untreated, DMSO and 9-phenanthrol treated wounds in zebrafish tails. Scale bar: 1 mm. F) Quantification of the wound closure experiments from (E) (n = 10 larvae per condition). Statistical analysis was performed using a two-way ANOVA test. G) Excisional cutaneous wounds were created using a 3 mm biopsy punch. Images from the time course of wound closure in the presence of DMSO (control) and 9-phenanthrol (n = 5 mice). Scale bar: 1.5 mm. H) Wound closure was monitored measuring the area of the wound on the indicated days post-wounding. Statistical analysis was performed using a two-way ANOVA test. I) Images of skin wounds at 3 days post-wounding. Bottom panels show magnifications of the areas marked in the upper panels. Arrowheads mark the epithelial tissue. Scale bar: 50 μm. J) Images of wounds at 5 days post-wounding. The dashed lines indicate the limit of the wound area. Scale bar: 500 μm.</p

TRPM4 controls cellular migration <i>via</i> Rac1 GTPase activity.

<p>A) MEFs were incubated with DMSO (0.1% v/v final) vehicle or 10 μM 9-phenanthrol for 16 h. F-actin was labeled with the F-actin stain Alexa 555 phalloidin (red) and Hoechst (blue). B) Quantified results from A. The percentage of wound closure is expressed as mean ± standard deviation; s.d. (n = 3, p<0.05). *, significant difference (p<0.05) versus DMSO controls. Statistical analysis was performed using a Mann-Whitney test. C) Graph from Transwell Boyden chamber migration assays of MEFs transfected with shRNA^Scramble and shRNA^TRPM4. Cells were stimulated with 10% v/v serum for 16 h. The bars represent the mean ± s.d. (n = 3; p<0.05 compared to control). *, significant difference (p<0.05) versus shRNA^Scramble controls. Statistical analysis was performed using a two-way ANOVA test. D) Graph from Transwell Boyden chamber migration assays of MEFs transfected with shRNA^Scramble and shRNA^TRPM4 and coexpressing Rac1(Q61L). Cells were stimulated with 10% v/v serum for 16 h. The bars represent the mean ± s.d. (n = 3; p<0.05 compared to control). *, significant difference (p<0.05) versus shRNA^Scramble/Mock controls. **, significant difference (p<0.05) versus shRNA^TRPM4/Mock controls. Statistical analysis was performed using a two-way ANOVA test.</p

TRPM4 regulates the number and size of focal adhesions.

<p>A) Representative immunoblot from MEFs subjected to shRNA-mediated TRPM4 knock down. Membranes were incubated with mouse anti-TRPM4 mAb, and anti-Grp75 mAb as a loading control. B) Graph of the densitometric analyses of three independent immunoblot experiments. Statistical analysis was performed using a Mann Whitney test. C) Immunofluorescence labeling of MEFs transfected with shRNA^Scramble, and shRNA^TRPM4. Cells were labeled with Hoechst (blue), and mouse anti-TRPM4 mAb (green); tRFP (red) was used as transfection marker. Scale bar corresponds to 10 μm. D) Immunofluorescence labeling of MEFs transfected with shRNA^Scramble and shRNA^TRPM4. Cells were labeled with Hoechst (blue), and mouse anti-vinculin mAb (green); tRFP (red) was used as transfection marker. Scale bar corresponds to 10 μm. Quantification of FA number (E) and areas (F) from the shRNA-transfected cells (n = 15 cells for shRNA^Scramble and n = 15 cells for shRNA^TRPM4 from 7 independent experiments). G) Immunofluorescence labeling of MEFs treated with DMSO (0.1% v/v) and 10 μM 9-phenanthrol. Cells were labeled with Hoechst (blue) and mouse anti-vinculin mAb (green). Scale bar corresponds to 10 μm. Graphs of FA number (H) and areas (I) are shown (20 cells per condition, n = 3, p<0.05). The number and areas of the FAs were analyzed using NIH/ImageJ software. The graphs correspond to mean ± standard deviation. Statistical analysis was performed using a Mann-Whitney test.</p

TRPM4 localizes to focal adhesions.

<p>A) Classification of putative TRPM4-associated proteins identified by LC-MS/MS. B) FA-related proteins identified as putative TRPM4 interacting proteins. C) TRPM4 localizes to FAs in MEFs. Cells were labeled with Hoechst (blue), mouse anti-TRPM4 mAb (green), and tRFP (red). D) Magnification of the section from C. E) TRPM4 (green) colocalizes with Focal Adhesion Kinase (FAK, magenta) in MEFs. F) Amplification of the region marked in E. G) Biochemical isolation of FA complexes (FA) from MEFs. The cell body fraction is labeled as CB. TRPM4 localizes to FAs in mouse pancreatic (H) and skin fibroblasts (I); Human Umbilical Vein Endothelial Cells (HUVEC) (J) and astrocytes (K). Scale bar: 5 μm.</p

TRPM4 regulates FA turnover.

<p>(A) Representative time-lapse imaging from MEFs coexpressing shRNA^Scramble or shRNA^TRPM4 with EGFP-Paxillin. Assembling FAs are marked with arrows. Arrowheads mark FAs undergoing turnover. Scale bar: 5 μm. Quantification of assembly (B) and disassembly rates (C) of FAs from live-cell time-lapse recordings of EGFP-Paxillin transfected MEFs. Statistical analysis was performed using a Mann-Whitney test from 7 independent experiments.</p