Journal: Nature Communications

Article Title: Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity

doi: 10.1038/s41467-019-11325-3

Figure Lengend Snippet: Epidermal Par3 loss leads to increased DNA damage and ectopic activation of DNA damage responses. a – e Maximum projection of back-skin cross sections from P100 old epidermal Par3 A knockout ( Par3 eKO: K14Cre/+; Par3 fl/fl) and control mice (K14Cre/+) stained for γH2Ax and Keratin15 or CD34. Micrographs show ( a ) the interfollicular epidermis (IFE), Scale bar: 15 µm ( b ) infundibulum, ( c ) isthmus and junctional zone (above bulge), ( d ) bulge, ( e ) secondary hair germ, bulb, and dermal papilla (below bulge). Scale bars: 20 µm. f Quantification of γH2Ax-positive cells in the IFE, and percentage of HF bulges (labeled by CD34) with cells positive for γH2Ax. n(IFE) = 6 mice, * p = 0.0250, mean ± SD, paired two-tailed Student’s t -test; n(HF) = 5 mice; ** p = 0.0018; mean ± SD, two-way ANOVA/Sidak’s multiple comparison). g Quantification and graphical illustration of γH2Ax-positive cells in different HF compartments that were distinguished based on bulge marker expression and histological hallmarks (as shown in a – e ): Data represent pooled numbers of positive cells from five individual animals per genotype. Total numbers of γH2Ax-positive (dark gray) and γH2Ax-negative compartments (light gray) per genotype are shown. * p = 0.0455, *** p < 0.0001; two-sided Fisher’s exact test (2 × 2 contingency table). h Immunoblot for p53 in whole cell keratinocyte lysates. GAPDH served as loading control. i Quantification of h . p53 levels were normalized to GAPDH and then expressed as relative values. n = 6 independent experiments, paired two-tailed Student’s t -test, ** p = 0.0058, mean ± SD. j Immunoblot analysis of pATR and pChk1 in keratinocytes either non-treated or UV-B-treated (100 mJ/cm 2 ). GAPDH served as loading control. k Quantification of j . pATR levels were normalized to GAPDH and then expressed as relative values. pChk1 levels were normalized to Chk1 and then expressed as relative values. n(pATR no UV, 1 h post-UV) = 5 independent experiments; n(pChk1 1 h post-UV) = 6 independent experiments, paired two-tailed Student’s t -test, * p = 0.0478, ** p = 0.0038, *** p < 0.0001; mean ± SD. Cropped immunoblot data are shown. Image intensity was enhanced for better visualization. HF hair follicle, Ctrl control, KO Par3 KO, IFE interfollicular epidermis, infund. infundibulum, neg negative, pos positive

Article Snippet: To obtain the Par3-CAAX expression plasmid, the HindIII-digested and NotI-digested PCR product and pcDNA5-Par3 were ligated using T4 DNA ligase (NEB).

Techniques: Activation Assay, Knock-Out, Staining, Labeling, Two Tailed Test, Marker, Expressing, Western Blot

Journal: Nature Communications

Article Title: Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity

doi: 10.1038/s41467-019-11325-3

Figure Lengend Snippet: Par3 ensures mitotic fidelity and genome integrity of keratinocytes. a Representative images of mitotic aberrations observed in time-lapse microscopy of H2B-GFP-expressing keratinocytes (H2B-GFP in gray). Scale bar: 10 µm. b Quantification of aberrant cell divisions in primary Par3 KO and control keratinocytes observed in time-lapse microscopy. n = 5 independent experiments, paired two-tailed Student’s t -test, * p = 0.0438, mean ± SD. c Quantification of mitotic duration (Ctrl/normal n = 69 cells, Par3 KO/normal n = 95 cells, Ctrl/aberrant n = 23 cells, Par3 KO/abnormal n = 51 cells; in all cases pooled from three independent experiments). For statistical analysis a nonlinear mixed model using RStudio software was employed, yielding *** p < 0.001. Bar represents mean. d Representative images of iFISH probes targeting chromosome 2. Scale bar: 10 µm. e Quantification of d . n = 3 independent experiments, ** p = 0.0028, mean ± SD; two-way ANOVA/Sidak’s multiple comparisons test. f Immunoblot analysis of ATR activation in primary Par3-deficient and control keratinocytes after treatment with Cdk1 inhibitor Purvalanol A (10 µM). GAPDH served as loading control. g Quantification of f . pATR levels were first normalized to GAPDH and then expressed as relative values. n = 3 independent experiments; mean ± SD; two-way ANOVA/Tukey’s multiple comparisons test * p = 0.0459, ** p = 0.0026 ( Par3 KO/DMSO vs. Ctrl/PurvA), ** p = 0.0032 ( Par3 KO/DMSO vs. Par3 KO/PurvA). Cropped immunoblot data are shown. iFISH interphase fluorescence in situ hybridization, Ctrl control, KO Par3 KO, PurvA Purvalanol A, ns non-significant

Article Snippet: To obtain the Par3-CAAX expression plasmid, the HindIII-digested and NotI-digested PCR product and pcDNA5-Par3 were ligated using T4 DNA ligase (NEB).

Techniques: Time-lapse Microscopy, Expressing, Two Tailed Test, Software, Western Blot, Activation Assay, Fluorescence, In Situ Hybridization

Journal: Nature Communications

Article Title: Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity

doi: 10.1038/s41467-019-11325-3

Figure Lengend Snippet: Par3 safeguards keratinocyte dynamics, and promotes RhoA activity and actomyosin contractility. a Snapshots from live cell-imaging videos monitoring H2B-GFP (green) fluorescence and DIC, smoothened strain rate maps from particle image velocimetry analyses (physics look-up table) and pseudo-color scale. Scale bar: 10 µm. b Quantification of mean velocity magnitude ( n = 3 independent experiments) and mean strain rate over time ( n = 30 mitotic cells pooled from three experiments). Dot plot (right panel) shows overall mean strain rate, Mann–Whitney U- test, *** p < 0.0001 (mitotic cells), mean. c Immunofluorescence micrographs of MLC2 phosphorylation (Ser19) (gray) in primary murine keratinocytes. DAPI is shown in blue. Scale bar: 25 µm. d Quantification of pMLC2 (Ser19) immunoreactivity at cell–cell junctions, intensity in arbitrary units; n(Ctrl) = 526 cells, n( Par3 KO) = 519 cells pooled from three independent experiments; *** p = 0.0002, two-sided Mann–Whitney U -test, bar represents mean. e Immunofluorescence micrographs of control keratinocytes transfected with siCtrl or siPar3 and stained for Par3 (green) and pMLC2 (red). DAPI is shown in blue. Individual channels are displayed in gray. Scale bar: 40 µm. f , g Quantification of pMLC2 f and Par3 g immunoreactivity at cell–cell junctions, intensity in arbitrary units; n = 450 cells pooled from five independent experiments; *** p < 0.0001, two-sided Mann–Whitney U -test, bar represents mean. h Immunoblot analysis of ppMLC2 (Thr18/Ser19) and pERM in whole cell keratinocyte lysates. Total MLC2 and GAPDH served as loading control, respectively. i Quantification of h . ppMLC2 (Thr18/Ser19) levels were normalized to total MLC and then expressed as relative values. n = 6 biologically independent samples, paired two-tailed Student’s t -test, *** p < 0.0001, mean ± SD. pERM levels were first normalized to GAPDH and then expressed as relative values. n = 6 biologically independent samples, paired two-tailed Student’s t -test, *** p = 0.0002, mean ± SD. j Quantification of RhoA G-LISA ® activation assay from keratinocyte lysates. Absorbance was normalized to total RhoA level determined by western blot. n = 4 biologically independent samples, Mann–Whitney U- test, * p = 0.0286, mean. k Immunoblot analysis of p190-RhoGAP. GAPDH was used as loading control. l Quantification of k , p190-RhoGAP levels were first normalized to RhoA and then expressed as relative values. n = 5 biologically independent samples, paired two-tailed Student’s t -test, ** p = 0.0068, mean ± SD. Cropped immunoblot data are shown. Ctrl control, KO Par3 KO, abs absorbance, DIC differential interference contrast

Article Snippet: To obtain the Par3-CAAX expression plasmid, the HindIII-digested and NotI-digested PCR product and pcDNA5-Par3 were ligated using T4 DNA ligase (NEB).

Techniques: Activity Assay, Live Cell Imaging, Fluorescence, MANN-WHITNEY, Immunofluorescence, Transfection, Staining, Western Blot, Two Tailed Test, Activation Assay

Journal: Nature Communications

Article Title: Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity

doi: 10.1038/s41467-019-11325-3

Figure Lengend Snippet: Reduced phosphorylation of MLC2 and ERM in Par3 eKO mice. a Immunohistochemistry of P100 murine back-skin paraffin cross-sections stained for pMLC2 (Ser19) (gray). Left panels: overview micrographs (×20 objective, scale bar: 100 µm). Middle and right panel: higher magnification micrographs referring to signals within the IFE (middle panel) and HF (right panel). ×40 objective, scale bar: 40 µm (IFE), 20 µm (HF). b Quantification of pMLC immunoreactivity a . Intensity in arbitrary units. n = 4 mice, Mann–Whitney U- test, * p = 0.0286, mean ± SD. c Immunohistochemistry of P100 murine back-skin paraffin cross-sections stained for ppMLC2 (Thr18/Ser19) (gray) (Cell Signaling Technologies #3674). ×63 objective, scale bar: 20 µm. d Quantification of ppMLC immunoreactivity c . Intensity in arbitrary units, n = 6 mice, Mann–Whitney U -test, * p = 0.0411, mean ± SD. e Immunohistochemistry of P100 murine back-skin paraffin cross-sections stained for pERM (gray). Left panels: overview micrographs (×20 objective, scale bar: 100 µm). Middle and right panel: higher magnification micrographs referring to signals within the IFE (middle panel) and HF (right panel). ×63 objective, scale bar: 20 µm (IFE and HF). Image intensity was enhanced for better visualization (×63). f Quantification of pERM immunoreactivity e . Intensity in arbitrary units. n = 4 mice, Mann–Whitney U -test, mean ± SD. In all micrographs DAPI is shown in blue. Ctrl control, IFE interfollicular epidermis, HF hair follicle, ns non-significant

Article Snippet: To obtain the Par3-CAAX expression plasmid, the HindIII-digested and NotI-digested PCR product and pcDNA5-Par3 were ligated using T4 DNA ligase (NEB).

Techniques: Immunohistochemistry, Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity

doi: 10.1038/s41467-019-11325-3

Figure Lengend Snippet: Re-establishment of actomyosin contractility rescues viscoelastic properties in Par3-deficient cells. a Schematic representation of dispase sheet contraction assay. b Representative images of dispase sheet contraction assay 24 h after 1 nM CalA treatment using control and Par3 KO keratinocytes. Scale bar: 15 mm. c Quantification of keratinocyte sheet area after dispase sheet contraction assay 24 h following treatment with CalA (1 nM). n = 4 independent experiments; ** p = 0.0029, *** p = 0.0004 (Ctrl/DMSO vs Par3 KO/DMSO), *** p = 0.0002 ( Par3 KO/DMSO vs. Ctrl/CalA) mean ± SD; one-way ANOVA/Tukey’s multiple comparisons test. d Quantification of keratinocyte sheet area after dispase sheet contraction assay 24 h following treatment with the Rho activator CN03 (5 µg/ml). n = 3 independent experiments; ** p = 0.0024 (Ctrl/NT vs. Par3 KO/NT), ** p = 0.0011 ( Par3 KO/NT vs. Ctrl/CN03), *** p = 0.0008, one-way ANOVA/Tukey’s multiple comparisons test. Mean ± SD. e Young Modulus box-plot based on force indentation spectroscopy ( n = 2000 measurements on primary keratinocytes, pooled from three independent experiments; * p = 0.0368, *** p < 0.0002 (Ctrl/DMSO vs. Ctrl/CalA), *** p < 0.0001 (Ctrl/DMSO vs. Par3 KO/DMSO), *** p < 0.0001 ( Par3 KO/DMSO vs. Ctrl/CalA), *** p < 0.0001 ( Par3 KO/DMSO vs. Par3 KO/CalA), Kruskal–Wallis/Dunn’s multiple comparison test, box plots show minimum (boundary of lower whisker), 25th percentile (lower boundary of box), median (center line), 75th percentile (upper boundary of box), and maximum (boundary of upper whisker). f , g Distribution histogram of Young Modulus upon DMSO f and 24 h CalA (1 nM) g treatment of experiments shown in e . Ctrl control, CalA Calyculin A, NT non-treated, ns non-significant

Article Snippet: To obtain the Par3-CAAX expression plasmid, the HindIII-digested and NotI-digested PCR product and pcDNA5-Par3 were ligated using T4 DNA ligase (NEB).

Techniques: Contraction Assay, Spectroscopy, Whisker Assay

Journal: Nature Communications

Article Title: Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity

doi: 10.1038/s41467-019-11325-3

Figure Lengend Snippet: Restoration of Rho activity or actomyosin contractility rescues mitotic fidelity and p53 stabilization in Par3 KO cells. a Immunofluorescence micrographs of mitotic keratinocytes stained for pericentrin (green) and alpha-tubulin (magenta), scale bar: 10 µm. Intensity was enhanced for better spindle visualization. b Schematic representation of analysis for mitotic spindle geometry. c Quantification of coherency index (Ctrl/DMSO-treated: n = 73 cells, Par3 KO/DMSO-treated: n = 58 cells; Ctrl/CalA-treated: n = 41 cells, Par3 KO/CalA-treated: n = 55 cells; in all cases pooled from four independent experiments), * p = 0.0207, bar represents mean, one-way ANOVA/Dunnett’s multiple comparisons test. d Quantification of aberrant cell divisions in primary Par3 KO and control keratinocytes observed in time-lapse microscopy upon DMSO and CalA (1 nM) treatment (relative values). n = 3 independent experiments, mean ± SD, * p = 0.0154 (Ctrl/DMSO vs. Par3 KO/DMSO), ** p = 0.0078 ( Par3 KO/DMSO vs. Ctrl/CalA), ** p = 0.0076 ( Par3 KO/DMSO vs. Par3 KO/CalA) one-way ANOVA/Tukey’s comparison test. e Immunoblot analysis of p53 upon DMSO and CalA (1 nM) treatment. GAPDH served as loading control. f Quantification of e . p53 levels were first normalized to GAPDH and then expressed as relative values. n = 6 biological independent samples; ** p = 0.0022 (DMSO-treated Ctrl vs. Par3 KO); *** p < 0.0001 ( Par3 KO DMSO-treated vs. CalA-treated); *** p < 0.0001 (DMSO-treated Par3 KO vs. CalA-treated Ctrl); * p = 0.0261 (Ctrl/DMSO vs. Ctrl/CalA-treated); mean ± SD; two-way Anova/Tukey’s multiple comparison test. g Quantification of aberrant cell divisions in primary Par3 KO and control keratinocytes observed in time-lapse microscopy either non-treated (NT) or treated with 0.5 µg/ml CN03 (relative values). n = 4 biological independent samples, mean ± SD, * p = 0.0236 (Ctrl/NT vs. Par3 KO/NT), * p = 0.0349 ( Par3 KO/NT vs. Ctrl/CN03), ** p = 0.0016 ( Par3 KO/NT vs. Par3 KO/CN03), one-way ANOVA/Tukey’s comparison test. h Immunoblot analysis of p53 from NT or CN03 treated primary Par3 KO and control keratinocytes. GAPDH was used as loading control. i Quantification of h . p53 level were first normalized to GAPDH and then expressed as relative values. n = 6 biological independent samples; *** p = 0.0003 (Ctrl/NT vs. Par3 KO/NT), *** p < 0.0001, mean ± SD; two-way ANOVA/Tukey’s multiple comparison test. Cropped immunoblot data are shown. Ctrl control, KO Par3 KO, CalA Calyculin A, NT non-treated, ns non-significant

Article Snippet: To obtain the Par3-CAAX expression plasmid, the HindIII-digested and NotI-digested PCR product and pcDNA5-Par3 were ligated using T4 DNA ligase (NEB).

Techniques: Activity Assay, Immunofluorescence, Staining, Time-lapse Microscopy, Western Blot

Journal: Nature Communications

Article Title: Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity

doi: 10.1038/s41467-019-11325-3

Figure Lengend Snippet: Reduced actomyosin contractility following Par3 loss drives ectopic differentiation and suprabasal fate. a Immunoblot analysis for Keratin1 expression in keratinocyte lysates following 48 h CalA (1 nM) treatment. GAPDH served as loading control. b Quantification of a . Keratin1 levels were first normalized to GAPDH and then expressed as relative values. n = 5 biological independent samples; *** p = 0.001 (Ctrl vs. Par3 KO, DMSO-treated); *** p = 0.0003 ( Par3 KO, DMSO-treated vs. CalA-treated), *** p < 0.0001 ( Par3 KO/DMSO-treated vs. Ctrl/CalA-treated), mean ± SD, two-way ANOVA/Tukey’s multiple comparisons test. c Immunoblot analysis for Keratin1 expression in keratinocyte lysates following siCtrl or sip53 transfection. GAPDH served as loading control. Relative p53 protein levels are shown below the immunoblot. d Quantification of c . Keratin1 levels were first normalized to GAPDH and then expressed as relative values. n = 3; *** p = 0.0009 (Ctrl vs. Par3 KO, siCtrl-treated); *** p = 0.0001 ( Par3 KO siCtrl-treated vs. Ctrl sip53-treated); ** p = 0.008 ( Par3 KO siCtrl-treated vs. sip53-treated); ** p = 0.0049 (Ctrl sip53-treated vs. Par3 KO sip53-treated); mean ± SD; two-way ANOVA/Tukey’s multiple comparisons test. e Schematic representation of the Transwell filter culture system and experimental setup. Par3 KO keratinocytes were positive for H2B-GFP, control keratinocytes isolated from littermates were negative for H2B-GFP. f Immunofluorescence micrographs of stratified cultures incubated for 10 days either in DMSO or in 1 nM CalA. Par3 KO keratinocytes were H2B-GFP (green) labeled, DAPI is shown in magenta. Bottom panels show zx-projection. Scale bar: 50 µm. g Pie graphs showing cell distribution according to basal or suprabasal position following a 10-day culture period of mixed control and Par3 KO keratinocyte cultures on Transwell filters. DMSO-treated samples: n = 180 cells (basal/DMSO-treated: n = 171 cells; suprabasal/DMSO-treated: n = 9 cells); CalA-treated samples: n = 170 cells (basal/CalA-treated: n = 143 cells; suprabasal/CalA-treated: n = 27 cells). An experimental representaton of three independent experiments is shown. Cropped immunoblot data are shown. Image intensity was enhanced for better visualization. Ctrl control, KO Par3 KO, CalA Calyculin A, ns non-significant, SE short exposure, LE long exposure, siCtrl small interfering non-targeting RNAs, sip53 small interfering RNAs targeting p53

Article Snippet: To obtain the Par3-CAAX expression plasmid, the HindIII-digested and NotI-digested PCR product and pcDNA5-Par3 were ligated using T4 DNA ligase (NEB).

Techniques: Western Blot, Expressing, Transfection, Isolation, Immunofluorescence, Incubation, Labeling

Journal: Nature Communications

Article Title: Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity

doi: 10.1038/s41467-019-11325-3

Figure Lengend Snippet: Par3 directs epidermal fate decisions through coupling Rho/actomyosin contractility to mitotic fidelity. Model. Par3 regulates actomyosin contractility in a Rho-dependent manner. Loss of Par3 alters keratinocyte mechanics resulting in mitotic infidelity and accumulation of DNA damage and p53, which in turn fuels differentiation and disturbs epithelial homeostasis. Restoring myosin activity in Par3-deficient keratinocytes is sufficient to normalize mitosis, and to prevent ectopic DNA damage, premature differentiation, and suprabasal fate. These data establish a role of Par3 in keratinocyte mechanics and mitotic fidelity. Rather than promoting asymmetric/perpendicular mitotic spindle orientation, altered polarity protein function in adult epithelia causes ectopic differentiated fate due to impaired cortical contractility that yields mitosis-related DNA damage and as consequence lower fitness

Article Snippet: To obtain the Par3-CAAX expression plasmid, the HindIII-digested and NotI-digested PCR product and pcDNA5-Par3 were ligated using T4 DNA ligase (NEB).

Techniques: Activity Assay