EMBO/SourceData huggingface dataset:
Figure 7A Time lapse confocal images of PHcrac at the basal surface of a giant cell, which recruits RacGEF1ΔN to membrane at time 0 induced by rapamycin.B Confocal images of PHcrac (left) and PHcrac merged with LimE (right, PHcrac as red, LimE as green) in a giant cell after RacGEF1ΔN recruitment.C Kymograph of LimE (top, green) and PHcrac (bottom, red) across the dotted line in B.D Confocal image of RalGDS (left) in a giant cell after RacGEF1ΔN recruitment and the corresponding kymograph (right).E Time lapse confocal images of PHcrac in single cells, which recruit RacGEF1ΔN to membrane induced by rapamycin at time 0. White arrow points to PHcrac patches at cell cortex.F Kymographs of PHcrac at cell cortex before and after RacGEF1ΔN recruitment. Red dashed lines in F indicate rapamycin addition at time 0.G Temporal profiles of normalized PHcrac intensities at cell cortex before and after RacGEF1ΔN recruitment (mean±s.e.m., n=20 cells). Red dashed lines in F and G indicate rapamycin addition at time 0.H Distribution of different sizes of PHcrac patches at cell cortex under LatA treatment, with (solid) or without (hollow) RacGEF1ΔN recruitment (~6000 cells from 3 independent experiments).I Temporal profiles of normalized cytosolic intensities of PHcrac in response to 1 nM cAMP stimulation at time 0, with (red) or without (blue) RacGEF1ΔN recruitment (mean±s.e.m., n=25 cells each).J Confocal images of GflB (top) and RapGAP1 (bottom) in waves at the basal surface of a giant cell.K Intensity plots of GflB (blue) and RapGAP1 (orange) across the green dotted arrow in J.L Scatter plots of first peak distances, of GflB relative to LimE, RapGAP1 relative to LimE, and RapGAP1 relative to GflB in the same giant cells (red lines indicate median, n=19 cells each).M Confocal images of GflB (top) and RapGAP1 (bottom) at the basal surface of the same giant cell after RacGEF1ΔN recruitment.N kymographs of GflB (top) and RapGAP1 (bottom) across the green dotted lines in m. All scale bars represent 20 μm.
Figure 8A, B Left, confocal images of LimE (top) and kymographs of cortical LimE (bottom) in single cells with PKBA (A) or RacGEF1ΔN (B) steadily recruited (scale bars, 10 μm). Right, color coded overlays of LimE in single cells.C Box plots of roundness (left) and cell area (middle) comparing wt, cells with RacGEF1ΔN recruited, and spreading phases of oscillators after Inp54p recruitment (n=100 cells each). Right, box plots of coefficient of variance of cell area with the above conditions in a 10-minute time window (n=30 cells each). Red bars indicate median.D Phalloidin staining of cells after perturbations. Maximal intensity projections are shown.
Figure 1B The expression of mature miR-200b and miR-200a in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01; Student's t-test).C The expression of Ttll10 (the host gene of miR-200b/a) mRNA in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; p=0.3540; Student's t-test).D The locomotor activity of the miR-200b/a KD mice was similar to that of the NC mice (NC: n=13 mice, miR-200b/a knockdown: n=15 mice; data represent the mean±SEM; p=0.5030; Student's t-test).E The odorant habituation results shown as the ratio of odor sniffs/water of the miR-200b/a KD mice were significantly reduced compared to those of the NC mice. These odorants included male urine (1:50), female urine (1:50), isopentyl acetate (50 μM), citral (50 μM), 2-heptanone (50 μM) and propyl propionate (50 μM) (n=6 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01, ***p<0.001; Student's t-test).The resident/intruder experiment showed that the attack frequency (F) of the miR-200b/a KD mice were impaired compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; ****p<0.0001; Student's t-test).G The resident/intruder experiment showed that the attack duration (G) of the miR-200b/a KD mice were impaired compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; ****p<0.0001; Student's t-test).H, I The analysis of male-female mating behaviors showed that the mating frequency (H) and duration (I) of the miR-200b/a KD mice were significantly reduced compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; **p<0.01, ***p<0.001; Student's t-test).
Figure 2B Representative IF staining with Ki67 antibody in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. C Quantification of the numbers of Ki67+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; **p<0.01, ****p<0.0001; Student's t-test). D Representative IF costaining with both EdU and Ki67 antibody in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the EdU+ cells and the yellow arrows indicate the cells positive for both EdU and Ki67. D' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). D: Scale bars, 20 μm; D': Scale bars, 2 μm. E Quantification of the numbers of costained Ki67+ and EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=3 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test). F The proportion of GBCs that exited the cell cycle was significantly reduced in the miR-200b/a KD mice compared with their NC controls [the cell cycle exit index was calculated using the following formula: (EdU+ Ki67− cells/total EdU+ cells)×100; n=3 mice each group; data represent the mean±SEM; **p<0.01; Student's t-test]. G Representative IF costaining with both EdU and BrdU antibody in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the BrdU+ cells, and the yellow arrows indicate the cells positive for both BrdU and EdU. G' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). G: Scale bars, 20 μm; G': Scale bars, 2 μm.H The S phase length of the GBCs in the MOE of the NC and miR-200b/a KD mice (the S phase length was calculated using the following formula: 3 h×BrdU+/BrdU+EdU-; n=3 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test).I Total cell cycle length of the GBCs in the MOE of the NC and miR-200b/a KD mice (total cell cycle length was calculated using the following formula: 20 h+(Ts×EdU+BrdU-/Edu+); n=3 mice each group; p=0.9785; data represent the mean±SEM; Student's t-test).
Figure 3B The mRNA levels of CK14, Trp63, Sox2 and Mash1 in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; CK14: p=0.2135, Trp63: p=0.1930, Sox2: p=0.5190, *p<0.05; Student's t-test).C Representative IF staining for CK14, Sox2, and Mash1 in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. D Quantification of the number of CK14+, basal Sox2+, apical Sox2+, and Mash1+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; CK14: p=0.8998, basal Sox2: p=0.7056, apical Sox2: p=0.4455, **p<0.01; Student's t-test). E Western Blot analysis of the Sox2 and Mash1 protein levels in the MOE of the NC and miR-200b/a KD mice (n=2 mice each group). GAPDH and Tubulin served as loading controls. The molecular weight of each band is indicated at the right. F Representative IF costaining with Sox2 antibody and EdU in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both Sox2 and EdU. F' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (right images). F: Scale bars, 20 μm; F': Scale bars, 2 μm. G Quantification of the number of Sox2+EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; p=0.7988; Student's t-test). H Representative IF costaining with Mash1 antibody and EdU in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both Mash1 and EdU. H' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (right images). H: Scale bars, 20 μm; H': Scale bars, 2 μm. I Quantification of the number of Mash1+EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test).
Figure 5A The number of differentially expressed olfrs from RNA sequencing in the MOE of the NC and miR-200b/a KD mice.C TET 3' UTR-dependent expression of a luciferase reporter gene was suppressed by miR-200a overexpression. Luciferase reporter gene under the control of the wild-type (WT) or mutant (Mut) TET3 3' UTR was transfected along with the negative control (nc) RNA or miR-200a mimic duplexes into HeLa cells (n=6 from six independent experiments; data represent the mean±SEM; Mut-nc vs. Mut-mimic: p=0.3697, *p<0.05; Student's t-test).D, E The expression of mature miR-200a (D) and TET3 (E) mRNA in the 3T3-L1 cells treated with miR-200a inhibitor, miR-200a mimic or control, as revealed by qPCR analysis (n=4 from four independent experiments; data represent the mean±SEM; D: F=66.37, E: F=16.39, NC vs. mimic: p=0.2597, *p<0.05, ***p<0.001; one-way ANOVA and Bonferroni pairwise comparisons).F Western Blot analysis of the TET3 protein level in the 3T3-L1 cells treated with miR-200a inhibitor, miR-200a mimic or control (n=3 from three independent experiments). Actin served as a loading control. The molecular weight of each band is indicated at the right.Western Blot analysis of the TET3 protein level in the MOE of the mice intranasally injected with miR-200a (G) NC mimic (n=3 mice each group). Actin served as a loading control. The molecular weight of each band is indicated at the right.H Western Blot analysis of the TET3 protein level in the MOE of the mice intranasally injected with miR-200b (H) or NC mimic (n=3 mice each group). Actin served as a loading control. The molecular weight of each band is indicated at the right.I Western Blot analysis of the TET3 protein level in the MOE of the NC and miR-200b/a KD mice (n=3 mice each group). Actin served as a loading control. The molecular weight of each band is indicated at the right. The blue number shows the ratio of the average intensity of TET3/Actin.J Representative IF staining of TET3 in the MOE of the NC and miR-200b/a KD mice. Scale bars: 20 μm.K Quantification of the number of TET3+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test).L Representative IF staining of 5mC in the MOE of the NC and miR-200b/a knockdown mice. L' is shown at higher magnification of the boxed region. L: Scale bars, 20 μm; L': Scale bars, 2 μm.M Quantification of the number of 5mC+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test).N Representative IF staining of 5hmC in the MOE of the NC and miR-200b/a knockdown mice. N' is shown at higher magnification of the boxed region. N: Scale bars, 20 μm; N': Scale bars, 2 μm.Quantification of the number of 5hmC+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; **p<0.01; Student's t-test).
Figure 6A Representative IF staining for costaining with TET3 and Mash1 antibodies in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both TET3 and Mash1. A' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). A: Scale bars, 20 μm; A': Scale bars, 2 μm. B Quantification of the number of costained TET3+ and Mash1+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test). C, D The resident/intruder experiment showed that the attack frequency (C) and duration (D) of the miR-200b/a+TET3 DKD mice were significantly improved compared with those of the miR-200b/a KD controls; [200 KD: miR-200b/a KD; (200+T) DKD: (miR-200b/a+TET3) DKD; (NC: n=18 mice, miR-200b/a KD: n=14 mice, miR-200b/a+TET3 DKD: n=16 mice; data represent the mean±SEM; C: F=17.84, D: F=12.06, *p<0.05, ***p<0.001, ****p<0.0001; One-way ANOVA and Bonferroni pairwise comparisons)].E, F The analysis of male-female mating behaviors showed that the frequency (E) and duration (F) of mating to females of the miR-200b/a+TET3 DKD mice were significantly reduced compared with those of their NC controls and were not dramatically different compared with those of the miR-200b/a KD mice (NC: n=18 mice, miR-200b/a KD: n=14 mice, miR-200b/a+TET3 DKD: n=16 mice; data represent the mean ±SEM; E: F=15.62, F: F=15.56, ***p<0.001, ****p<0.0001; one-way ANOVA and Bonferroni pairwise comparisons).G Representative IF staining for Mash1, Tuj1, Cas3 and Ki67 in the MOE of the NC, miR-200b/a KD, and miR-200b/a+TET3 DKD mice. Scale bars: 20 μm. H-K Quantification of the numbers of Mash1+(H), Tuj1+(I), Cas3+(J) and Ki67+(K) cells in the MOE of the NC, miR-200b/a KD, and miR-200b/a+TET3 DKD mice (n=4 mice each group; data represent the mean±SEM; Mash1: F=41.45, Tuj1: F=113.9, Cas3: F=92.51, Ki67: F=61.35, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; one-way ANOVA and Bonferroni pairwise comparisons).
Figure 7A The REST mRNA expression in the MOE of the NC, miR-200b/a KD and miR-200b/a+TET3 DKD mice, as revealed by qPCR analysis [200 KD: miR-200b/a KD; (200+T) DKD: (miR-200b/a+TET3) DKD; n=4 mice each group; data represent the mean±SEM; F=9.409, NC vs. 200: p=0.1367, 200 vs. 200+T: p=0.1896, **p<0.01; one-way ANOVA and Bonferroni pairwise comparisonsB Western Blot analysis of the REST protein level in the MOE of the NC, miR-200b/a KD and miR-200b/a+TET3 DKD mice (n=3 mice each group). Actin served as a loading control.C Representative IF staining for REST in the MOE of the NC, miR-200b/a KD, and miR-200b/a and TET3 DKD mice. Scale bars: 20 μm.D Quantification of the number of REST+ cells in the MOE of the NC, miR-200b/a KD, and miR-200b/a+TET3 DKD mice (n=4 mice each group; data represent the mean±SEM; F=12.11, *p<0.05; one-way ANOVA and Bonferroni pairwise comparisons).Representative IF costaining with REST and Mash1 antibodies in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both REST and Mash1. E' are shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). E: Scale bars, 20 μm; E': Scale bars, 2 μm.F Quantification of the number of costained REST+ and Mash1+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; **p<0.01; Student's t-test).G Mouse MOE proteins were immunoprecipitated with polyclonal antibodies against TET3. Input protein (100 ug) and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies. IgG antibodies were used as the negative control.H Mouse MOE proteins were immunoprecipitated with polyclonal antibodies against REST. Input protein (100 ug) and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies. IgG antibodies were used as the negative control.
Figure 8A, B The resident/intruder experiment showed that the attack frequency (A) and duration (B) of the miR-200b/a+REST DKD mice were dramatically improved compared with those of the miR-200b/a KD mice [200 KD: miR-200b/a KD; (200+R) DKD: (miR-200b/a+REST) DKD; NC: n=10 mice, miR-200b/a KD: n=9 mice, miR-200b/a+REST DKD: n=13 mice; data represent the mean±SEM; A: F=10.62, B: F=10.54, ***p<0.001; one-way ANOVA and Bonferroni pairwise comparisons].C, D The analysis of male-female mating behaviors showed that the frequency (C) and duration (D) of mating to females of the miR-200b/a+REST DKD mice were significantly improved compared with those of the miR-200b/a KD mice (NC: n=10 mice, miR-200b/a KD: n=9 mice, miR-200b/a+REST DKD: n=13 mice; data represent the mean±SEM; C: F=6.162, D: F=6.360, *p<0.05, **p<0.01; one-way ANOVA and Bonferroni pairwise comparisons).Representative IF staining for Mash1, Tuj1, Cas3 and Ki67 in the MOE of the NC, miR-200b/a KD, and miR-200b/a+REST DKD mice. Scale bars: 20 μm. F-I Quantification of the number of Mash1+(F), Tuj1+(G), Cas3+(H) and Ki67+(I) cells in the MOE of the NC, miR-200b/a KD, and miR-200b/a+REST DKD mice (n=4 mice each group; data represent the mean±SEM; Mash1: F=35.53, Tuj1: F=62.28, Cas3: F=115.7, Ki67: F=77.43, **p<0.01, ***p<0.001, ****p<0.0001; one-way ANOVA and Bonferroni pairwise comparisons).
Figure 1A) oxLDL plasma concentration in ACM patients and HC (n=36; Mann-Whitney test). B) oxLDL plasma concentration in mutated ACM (n=7) and NON ACM relatives, carriers of the same causative mutation (n=9; Mann-Whitney test). C) oxLDL plasma concentration in ACM patients carriers of a PKP2 mutation and ACM patients carriers of other desmosomal or non desmosomal mutations, or gene elusive (n=10 vs. n=26; One-Way ANOVA). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.D) Representative images of MDA immunostaining (green) on ACM and HC ventricular tissue sections and relative quantification (n=4 biological replicates; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). E) Representative images of CD36 immunostaining on ACM and HC ventricular tissue sections and relative quantification (n=4 biological replicates; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.
Figure 2B) Left panels: representative images of three cases of ACM in Steady State Free Procession sequences at cardiac MRI. On the left a case with RV dilation, regional bulging of the RV wall without fat infiltration, and oxLDL levels below the cut-off; in the center and on the right, two cases with fat infiltration in the right or left ventricle wall (white arrow), respectively, and oxLDL levels above the cut-off. Right panel: quantification of the myocardial fat mass of the two ACM subpopulations (above or below the cut-off) whose MRI was available for re-analysis (n=14 oxLDL<86ng/ml, n=25 oxLDL>86ng/ml; Two-tailed Student's t-test).G) Kaplan-Meier analysis of actual MAE free survival of patient belonging to the two ACM cohort subgroups in the first 5-year follow-up (n=26 oxLDL<86ng/ml vs. n=41oxLDL>86ng/ml; Log-Rank p<0.0001; HR=0.223[0.116-0.428]).
Figure 3B) Left panels: representative images of MDA immunostaining (green) on ACM and HC C-MSC in GM. Nuclei are counterstained with Hoechst33342 (blue). Right panel: image quantification (n=4 biological replicates ACM, n=5 biological replicates HC; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05, ** p<0.01.D) Top panel: representative images of Western Blot analysis of proteins extracted from ACM and HC C-MSC cultured in GM, hybridized with anti-CD36 and anti-PPARγ antibodies. Immunostaining of the housekeeping GAPDH is shown for normalization. Bottom panel: d.a. of PPARγ (n=18 biological replicates) and CD36 (n=17 vs. n=16 biological replicates) levels, normalized on GAPDH (Mann-Whitney test). Data information: mean ± SEM. * p<0.05, ** p<0.01.F) Top panels: representative images of internalization of oxLDL (red) in HC and ACM cells, cultured either in GM or in AM, and subjected to 10μg/ml DiI-oxLDL treatment. Nuclei are counterstained with Hoechst33342 (blue). Bottom panel: quantification of the relative mean DiI fluorescence for each sample, measured by FACS analysis (n=3 biological replicates; Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01.
Figure 4A) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 150µg/ml oxLDL. Middle panel: image quantification (n=11 biological replicates; Two-Way Anova). Right panels: representative images of Western Blot of CD36, PPARγ and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 150µg/ml oxLDL (n=5 biological replicates) and d.a. normalized on the house-keeping GAPDH (Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.B) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both. Right panel: image quantification (n=13 biological replicates; Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.C) Left panel: representative images of Western Blot of CD36, PPARγ and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both (n=8 biological replicates). Right panels: d.a. normalized on the house-keeping GAPDH (Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.
Figure 5D) Left panel: representative images of internalization of oxLDL (red) in ACM C-MSC treated with scramble siRNA or CD36 siRNA, cultured in AM and subjected to 10μg/ml DiI oxLDL treatment. Right panel: quantification of the DiI fluorescence normalized on nuclei number (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.E) Left panel: representative images of internalization of oxLDL (red) in ACM C-MSC cultured in AM or AM+5μM GW9662 and subjected to 10μg/ml DiI oxLDL treatment. Nuclei are counterstained with Hoechst33342 (blue). Right panel: quantification of the DiI fluorescence normalized on nuclei number for each sample (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.F) Left panel: representative images of Western Blot of CD36 and GAPDH expression of protein extracts of ACM C-MSC cultured in AM or AM+5μM GW9662. Right panels: d.a. normalized on the house-keeping GAPDH (n=7 biological replicates; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.
Figure 6C) Left panels: representative images of ORO staining of HFD-fed WT and Pkp2+/- cardiac sections. Right panel: quantification of ORO positive area percentage (n=10). For comparison, quantification of ORO positive area of cardiac sections of CD-fed WT and Pkp2+/- mice (n=9; Two-Way Anova) is shown (Appendix Figure S3). D) Representative images of PPARγ (green) immunostaining on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Quantification of the PPARγ staining in HFD is shown relative to the values of CD (n=9). E) Representative images of MDA (green) immunostaining on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Quantification of the MDA staining in HFD is shown relative to the values of CD (n=9). F) Representative images of CD36 immunostaining (green) on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Nuclei are counterstained with Hoechst33342 (blue). Quantification of the staining in HFD is shown relative to the values of CD (n=9). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.